U.S. patent application number 15/635801 was filed with the patent office on 2018-01-04 for transcription factor decoys, compositions and methods.
The applicant listed for this patent is PROCARTA BIOSYSTEMS LTD. Invention is credited to Michael McArthur.
Application Number | 20180002740 15/635801 |
Document ID | / |
Family ID | 38739101 |
Filed Date | 2018-01-04 |
United States Patent
Application |
20180002740 |
Kind Code |
A1 |
McArthur; Michael |
January 4, 2018 |
TRANSCRIPTION FACTOR DECOYS, COMPOSITIONS AND METHODS
Abstract
Compositions and methods for identifying and using
cis-regulatory and decoy sequences are disclosed.
Inventors: |
McArthur; Michael;
(Rocklands All Saints, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PROCARTA BIOSYSTEMS LTD |
Norwich |
|
GB |
|
|
Family ID: |
38739101 |
Appl. No.: |
15/635801 |
Filed: |
June 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12681588 |
Aug 20, 2010 |
9702012 |
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PCT/GB2008/003353 |
Oct 3, 2008 |
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15635801 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/10 20180101;
C12Q 1/6897 20130101; C12Q 1/689 20130101; C12N 15/1048 20130101;
A61P 43/00 20180101; A61P 31/04 20180101; C12N 15/1086 20130101;
C12N 15/1048 20130101; C12Q 1/6811 20130101; C12Q 2525/179
20130101; C12N 15/1079 20130101; C12Q 1/6811 20130101; C12Q 1/6897
20130101; C12Q 2600/136 20130101; C12Q 2600/158 20130101; C12Q
2522/101 20130101; C12Q 2537/1373 20130101; C12Q 2522/101 20130101;
C12Q 2537/1373 20130101; C12Q 2525/179 20130101; C12Q 2525/179
20130101; C12Q 2537/1373 20130101; C12Q 2522/101 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 15/10 20060101 C12N015/10 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 2007 |
GB |
0719367.5 |
Claims
1. A method of treating a bacterial infection, the method
comprising administering a decoy polynucleotide comprising a
binding site for a transcription factor, wherein the transcription
factor binding site in the decoy polynucleotide is not operably
linked to a gene; and wherein the decoy polynucleotide reduces
binding of the transcription factor to a cis-regulatory sequence in
a prokaryotic cell and causes an alteration in expression of gene
or genes operably linked to the cis-regulatory sequence in the
cell.
2. The method according to claim 1, wherein the transcription
factor is a regulator of expression of one or more genes that
encode resistance to an antibiotic.
3. The method according to claim 2, wherein the antibiotic is
selected from one or more: the aminoglycosides; the carbapenems;
the cephalosporins; the glycopeptides; the penicillins; the
polypeptide antibiotics; the quinolines; the sulfonamides; or the
tetracyclines.
4. The method according to claim 3, wherein the antibiotic is
selected from one or more of: kanamycin, meropenem, cefepime,
vancomycin, daptomycin, ampicillin, carbenicillin, penicillin,
polymixcin B, levaquin, Bactrim, tetracycline, chloramphenical,
rifampicin and Zyvox.
5. The method according to claim 1, wherein the gene or genes
operably linked to the cis-regulatory sequence encode resistance to
vancomycin.
6. The method according to claim 5, wherein the transcription
factor is the VanR transcription factor.
7. The method according to claim 1 wherein the prokaryotic cell is:
(a) a gram positive bacterium of a genus selected from:
Actinomyces; Streptomyces; Mycobacteria; Clostridium; Bacillus;
Listeria; Staphylococcus; Streptococcus; Enterococcus; or (b) a
gram negative bacterium of a genus selected from:
Enterobacteriaceae; Pseudomonas; Moraxella; Helicobacter;
Stenotrophomonas; Bdellovibio, Acetic acid bacteria; Legionella;
Cyanobacteria; Spirochaetes; Green Sulphur bacteria; and Green
Non-sulphur bacteria.
8. The method according to claim 1, wherein the prokaryotic cell is
a pathogen selected from: Mycobacterium tuberculosis; Mycobacterium
bovis; Mycobacterium africanum; Mycobacterium microti;
Mycobacterium leprae; Clostridium difficile; Clostridium botulinum;
Clostridium perfingens; Clostridium tetani; Salmonella sp.;
Escherichia coli; Enterococcus faecium; Enterococcus faecalis;
Neisseria gonorrhoeae; Nerisseria meningitides; Moraxella
catarrhalis; Hemophilus influenza; Kebsiella pneumoniae; Legionella
pneumophila; Pseudomonas aeruginosa; Proteus mirabilis;
Enterobacter cloacae; Serratia marcescens; Helicobacter pylori;
Salmonella enteritidis; and Salmonella typhi.
9. The method according to claim 1, wherein the prokaryotic cell is
a vancomycin resistant pathogen.
10. The method according to claim 1, wherein the decoy
polynucleotide comprises: circular double stranded DNA or a linear
oligonucleotide; and/or at least one element of secondary
structure; and/or more than one copy of the transcription factor
binding site; and/or additional sequence to the binding site(s);
and/or modified bases or sugars to increase nuclease resistance of
the polynucleotide; and/or a plasmid or plasmid library.
11. The method according to claim 10, wherein the decoy
polynucleotide comprises a circular dumbbell.
12. The method according to claim 10, wherein the decoy
polynucleotide comprises multiple direct repeats of the binding
site.
13. The method according to claim 10, wherein the decoy
polynucleotide comprises a linear oligonucleotide having at least
one 5' cholesterol modification.
14. The method according to claim 10, wherein the decoy
polynucleotide comprises a plasmid and wherein the plasmid has one
or more copies of a monomer sequence comprising a snare sequence,
the snare sequence comprising the transcription factor binding site
wherein the binding site is not operably linked to a gene.
15. The method according to claim 14, wherein the plasmid
comprises: (a) two or more or at least 10, 20 or 30 copies of the
monomer sequence; and/or (b) tandem repeats of the monomer
sequence.
16. The method according to claim 14, wherein the monomer
additionally comprises: (a) a binding site for a primer wherein the
primer is suitable for use in rolling circle amplification; and/or
(b) a recognition and/or cutting site for one or more restriction
enzymes.
17. The method according to claim 14, wherein the monomer comprises
from 10-1000 or from 10-500 nucleotides.
18. The method according to claim 14, wherein the snare sequence
comprises from 10 to 30 nucleotides or from 15 to 20
nucleotides.
19. The method according to claim 14, wherein the plasmid is a
broad host range or shuttle vector and/or is a high copy number
plasmid.
20. The method according to claim 1, wherein the transcription
factor binding site in the decoy polynucleotide comprises the
sequence of SEQ ID NO: 21 or SEQ ID NO: 26.
21. The method according to claim 1, wherein treating the bacterial
infection further comprises use of one or more antibiotics.
22. A method for modulating antibiotic resistance of a prokaryotic
cell, the method comprising administering a decoy polynucleotide
comprising a binding site for a transcription factor; wherein the
transcription factor binding site in the decoy polynucleotide is
not operably linked to a gene; and wherein the decoy polynucleotide
reduces binding of the transcription factor to a cis-regulatory
sequence in the prokaryotic cell and causes an alteration in
expression of the operably linked gene or genes in the cell,
thereby modulating antibiotic resistance of the cell.
Description
FIELD OF THE INVENTION
[0001] The invention provides methods and compositions for
identifying cis-regulatory sequences.
[0002] The invention also relates to methods and compositions for
specifically modulating cell phenotypes, such as prokaryotic
phenotypes, by altering the association of transcription factors to
cis-regulatory elements in vive with a concomitant alteration in
the pattern of gene expression.
BACKGROUND TO THE INVENTION
[0003] Gene expression is a major determinant of a cell's
phenotype, and in turn, DNA-protein interactions largely determine
the patterns of gene expression. Modification of gene expression so
as to affect phenotype is a major aim of biology and medicine, be
it in industrial microbes, experimental models, pathogens or to
tackle human disease. In this context the cis-regulatory sequences
within the genome, the DNA component of the transcriptional
machinery, are attractive targets for intervention. In comparison
to tackling the proteins, working with DNA is far easier:
sequencing is highly automated and relatively inexpensive, and DNA
can be easily manipulated and readily amplified or synthesized.
DNA-based therapies have also emerged as an exciting new class of
therapeutic agents. In comparison to traditional pharmaceuticals,
natural products or small molecules, DNA is an attractive type of
therapeutic agent as it can be: [0004] designed by a rational
process, most simply by examination of sequence data; [0005]
cheaply manufactured at scale, by chemical synthesis of
oligonucleotides or biological replication; [0006] predicted to
have low toxicity, as DNA is a `natural` compound and does not, in
and of itself, typically induce immunogenic responses, and
specificity can be controlled by sequence of the DNA-based therapy;
[0007] greatly reduce R&D expenditure, as all stages of
conventional drug development (target identification, lead compound
discovery, medicinal chemistry) are truncated.
[0008] The challenge then becomes how to identify the key
cis-regulatory elements. The technologies and expertise that have
developed in parallel with the genome sequencing projects, such as
massively parallel gene expression analysis using DNA microarrays
and the use of bioinformatics to annotate the genome databases, are
not sufficient to either identify all of the cis-regulatory
elements or ascribe function to those that are known. In 2003, the
National Institutes of Health in the US launched the ENCODE project
to catalogue 1% of the cis-regulatory elements in the human genome
(Science (2004) 306: 636-640; Nature (2007) 447: 799-816) and to
develop high-throughput technologies as discovery platforms. The
procedures developed included use of chromatin immunoprecipitation,
probing hypersensitivity sensitivity to in vive digestion by DNaseI
(with DNA microarrays, high-throughput quantitative PCR and genomic
libraries) and the development of new algorithms for
bioinformatical detection. While these techniques have the
potential to greatly accelerate the rate of discovery of
cis-regulatory elements, they will not necessarily lead to their
functional characterization: the output of the project is a
comprehensive catalogue of these elements. Even though, from such
work, it is likely that the tissue-specificity of the majority of
elements will be known, and their distance from a gene and
classification according to what type of trans-acting factor binds
to them, this will not be sufficient to determine what the
biological function of the element actually is. A further drawback
common to all of these procedures is that they rely on the genome
of the organism being sequenced. Furthermore, the approach using
hypersensitivity to DNaseI digestion has the extra disadvantage
that it is specific for eukaryotic cells, as, in this context,
DNaseI is a probe of chromatin structure, and no comparable
structure exists in prokaryotes.
[0009] Further, as yet there is no means for rapidly screening a
large number of sequences for potential cis-regulatory
sequences.
SUMMARY OF THE INVENTION
[0010] The inventors addressed these problems in the art by
providing new methods for identifying and characterising
cis-regulatory sequences in both prokaryotic and eukaryotic
organisms.
[0011] One way to identify and characterize the function of
individual cis-regulatory elements is to use transcription factor
decoys (TFDs). Decoy oligonucleotides are designed to mimic the
binding sites of transcription factors and prevent the latter from
binding to their cognate genomic targets, with a consequent
modification of gene expression. As such they represent a simple
and generic tool for manipulating the DNA-protein interactions that
regulate specific genes and that consequently determine phenotypes.
Their utility has been demonstrated primarily in eukaryotic
systems, where a spur to their development was their potential to
function as novel classes of therapeutic agents (Mann & Dzau
(2000) J. Clin. Investigation 106: 1071-1075). To this end, decoy
oligonucleotides have been used to demonstrate that transcription
factor EF2 represses smooth muscle proliferation in rats (Morishita
et al. (1995) Proc. Natl. Acad. Sci. USA 95: 5855-5859); to block
STAT3-mediated proliferation of carcinomas (Leong et al. (2003)
Proc. Natl. Acad. Sci. USA 100: 4138-4143); and to show that
targeting of the cAMP response element can control cancer
proliferation in vivo (Park et al. (1999) J. Biol. Chem. 274:
1573-1580).
[0012] However a system has not been developed to use TFDs, on a
large scale, that is capable of querying every occurrence of a
sequence within a genomic fragment or entire genome. As it stands,
that would require the synthesis of very large numbers of decoy
oligonucleotides and an onerous experimental programme involving
their transfection and screening for phenotypic change.
[0013] The invention described here addresses this need by
developing a plasmid-borne library based system capable of
systematically testing large numbers of sequences to determine
whether they act in the genome as cis-regulators and associate them
with a specific phenotypic effect. We refer to this system herein
as "n[snare]".
[0014] Furthermore, once the relevant cis-regulatory sequences have
been found, those sequences can be used to create TFDs that can be
used to modify gene expression and gain control over phenotype. For
several reasons, though decoys were developed for use in mammalian
cells, they are better suited to use in bacteria. Getting decoys to
work in eukaryotes can be problematic as they can be rapidly
degraded in serum and nuclear extracts (Chu & Orgel (1992)
Nucl. Acids. Res. 20: 5857-5858), cellular uptake of the decoy and
its transition across the nuclear membrane can be inefficient
(Griesenbach et al. (2002) Gene Therapy 9: 1109-1115), and some
treatments can trigger non-specific or toxic effects. Using decoys
in prokaryotes should circumvent many of these problems and as such
they might prove to be an effective tool for the rapid
identification of cis-acting regulatory sequences, such as
transcription factor binding sites controlling both specific genes
and larger regulatory networks. A successful demonstration of the
approach was the use of an AT-rich decoy to alter the expression of
CO.sub.2-responsive genes in Cyanobacterium (Onizuka et al. (2003)
FEBS Lett. 542: 42-46). In that system, the complimentary
oligonucleotides with modified backbones (containing
phosphorothioate to slow degradation by nucleases) were annealed to
form double stranded decoy oligonucleotides that incorporated
previously identified binding sites for a transcription factor.
These were added directly to the medium from where it efficiently
entered the cells. That report is, however, the only example we
know of in which this approach has been successfully attempted to
modify a particular prokaryotic trait. Accordingly, there remains a
need in the art to extend the decoy methodology into the field of
prokaryotic transcription factors to thereby alter a broad range of
prokaryotic phenotypes. In addition, there remains a need in the
art for high-throughput methods for identifying cis-acting
regulatory factors, whether in prokaryotic or eukaryotic systems.
This patent disclosure provides solutions to the above-noted
limitations and needs in the art.
[0015] In one particular aspect the invention provides new means
for increasing susceptibility of cells, e.g. bacterial cells, to
antibiotics.
[0016] Accordingly, in one aspect the invention provides use of a
decoy polynucleotide in a method for modulating antibiotic
resistance of a cell, the method comprising:
[0017] (a) providing a decoy polynucleotide comprising a binding
site for a transcription factor (a decoy sequence);
[0018] and
[0019] (b) introducing the polynucleotide into the cell, wherein
the cell comprises a gene or genes operably linked to a
cis-regulatory sequence comprising a binding site for the
transcription factor;
[0020] wherein introduction of the polynucleotide reduces binding
of the transcription factor to the cis-regulatory sequence in the
cell and causes an alteration in expression of the operably linked
gene or genes in the cell, thereby modulating antibiotic resistance
of the cell.
[0021] The invention also provides: [0022] a decoy polynucleotide
comprising a binding site for a transcription factor, wherein the
transcription factor is a regulator of expression of one or more
antibiotic resistance genes in a prokaryote or eukaryote and
wherein the binding site in the decoy polynucleotide is not
operably linked to a gene. [0023] a plasmid comprising one or more
copies of a monomer sequence, wherein the monomer sequence
comprises a snare sequence that comprises a transcription factor
binding site, and wherein the binding site is not operably linked
to a gene. [0024] a plasmid library comprising two or more plasmids
of the invention wherein the snare sequences in the library
together comprise all or substantially all of the cis-regulatory
sequence in the genomic DNA or in a fragment of the genomic DNA of
a prokaryote or eukaryote; [0025] a plasmid library comprising two
or more plasmids of the invention wherein the snare sequence in
each plasmid comprises a sequence of randomised nucleotides of
length n nucleotides, wherein substantially all nucleotide
sequences of length n nucleotides are represented in the library;
[0026] a method of preparing a plasmid comprising two or more
copies of a monomer sequence, the method comprising:
[0027] (1) providing a circular oligonucleotide comprising: [0028]
(i) a test sequence of interest; and [0029] (ii) a binding site for
a primer suitable for use in rolling circle amplification;
[0030] wherein the monomer sequence comprises (i) and (ii);
[0031] (2) performing rolling circle amplification using the
circular oligonucleotide as a template, thereby providing a
polynucleotide comprising repeats of the monomer sequence; and
[0032] (3) cloning the polynucleotide into a plasmid vector; [0033]
a method for preparing a plasmid library from a sample of genomic
DNA wherein each plasmid comprises one or more copies of a monomer
sequence, and wherein each monomer sequence comprises a snare
sequence that is derived from the genomic DNA; the method
comprising:
[0034] (1) providing a sample of double stranded genomic DNA;
[0035] (2) fragmenting the genomic DNA;
[0036] (3) ligating an adaptor to each end of the DNA fragments
from (2), wherein each adaptor comprises: [0037] (iii) a means for
immobilisation of the DNA fragment; [0038] (iv) a recognition site
for a first restriction enzyme that cuts at a distance from the
recognition site; and [0039] (v) a recognition and cutting site for
a second restriction enzyme;
[0040] (4) optionally removing unligated adaptor;
[0041] (5) digesting the fragment bearing the adaptors with the
first restriction enzyme, thereby producing two adaptored
fragments, wherein each adaptored fragment comprises: [0042] (vi)
an adaptor, and [0043] (vii) a DNA fragment comprising: a shorter
strand: and a longer strand, wherein the longer strand comprises
the snare sequence;
[0044] (6) immobilising the adaptored fragments produced in
(5);
[0045] (7) denaturing the fragments to provide single stranded
fragments;
[0046] (8) recreating the recognition site for the second
restriction enzyme by ligating a complementary oligonucleotide to
the adaptor and digesting the adaptored fragment with the second
restriction enzyme, thereby producing an adaptor-snare fragment
comprising: [0047] (viii) an adaptor fragment; and [0048] (ix) a
single stranded snare sequence; and
[0049] (9) releasing the adaptor-snare fragment produced in (8)
from immobilisation; and
[0050] (10) cloning the adaptor-snare fragment into a plasmid
vector; [0051] a method for preparing a plasmid library wherein
each plasmid comprises two or more copies of a monomer sequence,
and wherein each monomer sequence comprises a sequence of
randomised nucleotides of length n nucleotides, the method
comprising:
[0052] (1) providing a circular oligonucleotide comprising: [0053]
(i) a randomised sequence of length n nucleotides; and [0054] (ii)
a binding site for a primer suitable for use in rolling circle
amplification;
[0055] wherein the monomer sequence comprises (i) and (ii);
[0056] (2) performing rolling circle amplification using the
circular oligonucleotide as a template, thereby providing a
polynucleotide comprising repeats of the monomer sequence; and
[0057] (3) cloning the polynucleotide into a plasmid vector, [0058]
a plasmid or plasmid library prepared according to a method of the
invention; [0059] a cell comprising an exogeneous decoy
polynucleotide, the polynucleotide comprising a binding site for a
transcription factor (a decoy sequence) which is not operably
linked to a gene; wherein the cell comprises a gene or genes
operably linked to a cis-regulatory sequence comprising a binding
site for the transcription factor; and wherein the decoy
polynucleotide causes an alteration in antibiotic resistance of the
cell. [0060] a host cell or cells comprising a plasmid or plasmid
library of the invention; [0061] a method for identifying the
boundaries of one or more protein binding site(s) in a protein-DNA
complex, the method comprising: [0062] 1. providing a protein-DNA
complex; [0063] 2. carrying out a digestion with [0064] a. an
enzyme having non specific DNA nicking ability; and [0065] b. a
5'-3' exonuclease; and [0066] 3. determining the position of the 5'
deletions generated in each DNA strand in (2) relative to a known
fixed point on the DNA strand; [0067] a method for identifying a
cis-acting regulator of gene expression of a prokaryotic or
eukaryotic gene comprising: [0068] 1. providing a plasmid library
of the invention; [0069] 2. introducing the plasmid library into a
host cell(s) wherein the host cell comprises the cis-regulatory
sequence of interest operably linked to a gene or genes, the
expression of which can be determined directly or indirectly; and
[0070] 3. determining directly or indirectly the expression of the
gene (or genes) in the presence and absence of the plasmid library;
[0071] a method for altering expression of a gene or genes in a
prokaryotic cell, the method comprising:
[0072] (a) providing a polynucleotide comprising a binding site for
a prokaryotic transcription factor wherein the binding site is not
operably linked to a gene in the polynucleotide; and
[0073] (b) introducing the polynucleotide into the cell;
[0074] wherein the cell comprises the gene or genes operably linked
to a cis-regulatory sequence which comprises the transcription
binding site or which competes with the transcription factor
binding site for binding of transcription factor;
[0075] and wherein:
[0076] (i) the polynucleotide comprises a plasmid or a plasmid
library according to the invention;
[0077] (ii) the polynucleotide comprises more than one copy of the
binding site;
[0078] (iii) the polynucleotide comprise multiple direct repeats of
the binding site;
[0079] (iv) the polynucleotide comprises additional sequence to the
binding site;
[0080] (v) the polynucleotide comprises at least one element of
secondary structure;
[0081] (vi) the polynucleotide comprises circular double stranded
DNA; [0082] a method for altering expression of a gene or genes in
a prokaryotic or eukaryotic cell, the method comprising:
[0083] (a) providing a polynucleotide comprising a binding site for
a prokaryotic or eukaryotic transcription factor wherein the
binding site is not operably linked to a gene in the
polynucleotide; and
[0084] (b) introducing the polynucleotide into the cell;
[0085] wherein the cell comprises the gene or genes operably linked
to a cis-regulatory sequence which comprises the transcription
binding site or which competes with the transcription factor
binding site for binding of transcription factor;
[0086] and wherein the cis-regulatory sequence is one identified by
the method of the invention; [0087] a method for altering
expression of a gene or genes in a eukaryotic cell, the method
comprising:
[0088] (a) providing a polynucleotide comprising a binding site for
a eukaryotic transcription factor wherein the binding site is not
operably linked to a gene in the polynucleotide; and
[0089] (b) introducing the polynucleotide into the cell;
[0090] wherein the cell comprises the gene or genes operably linked
to a cis-regulatory sequence which comprises the transcription
binding site or which competes with the transcription factor
binding site for binding of transcription factor;
[0091] and wherein the polynucleotide comprises a plasmid or a
plasmid library according to the invention; [0092] a cell prepared
according to a method of the invention; [0093] a decoy
polynucleotide for use in treating bacterial infection, wherein the
polynucleotide comprises a binding site for a transcription factor
and the binding site is not operably linked to a gene; [0094] use
of a decoy polynucleotide for treating bacterial infection, wherein
the polynucleotide comprises a binding site for a transcription
factor and the binding site is not operably linked to a gene;
[0095] use of a decoy polynucleotide for the manufacture of a
medicament for treating bacterial infection, wherein the
polynucleotide comprises a binding site for a transcription factor
and the binding site is not operably linked to a gene.
[0096] Thus, in one embodiment of the invention of this patent
disclosure, we provide a method which combines the decoy approach
with a simple in vivo footprinting protocol to rapidly identify
candidate cis-acting regulatory motifs. Functional validation of
these sequences was achieved by incorporating them into dumbbell
decoy oligonucleotides, whose circular format suppresses
degradation by exo- and endo-nucleases [Ahn et al. (2003)
Biochemical Biophysical Res. Comm. 310: 1048-1053.]), and by
testing their effect on phenotype in vivo.
[0097] In another embodiment according to this invention, we
demonstrate that, using decoy oligonucleotides identified via
application of the footprinting method, that we can significantly
alter the level of antibiotic production in streptomycetes.
[0098] In a further embodiment according to this invention, we
demonstrate the ability to utilize prokaryotic decoys in a
therapeutic approach whereby pathogenic bacteria which are
resistant to vancomycin are rendered, once again, susceptible to
the antibiotic in the therapeutic window.
[0099] Thus the inventors provide a generic screening system by
means of which cis-acting regulatory elements are identified in any
genome, and sequences derived from such elements identified in this
fashion are utilized to alter selected phenotypes in a fashion
analogous to that demonstrated according to that illustrated by
additional embodiments of the invention.
[0100] The inventors have also adapted and extended the decoy
oligonucleotide technique for use in prokaryotes and demonstrate
the ability to increase antibiotic production.
[0101] Furthermore the inventors have demonstrates that decoy
oligodeoxynucleotides may be utilized to advantage to overcome
antibiotic resistance in treating pathogens.
[0102] Other embodiments, utilities and details of this invention
may be appreciated by a review of the complete disclosure,
including the specific examples provided herein and the claims
appended to this disclosure, including equivalents thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0103] FIG. 1. Construction and testing of n[snare] plasmids. This
protocol can also be used to produce `generic` n[snare] libraries,
made from random oligonucleotides, or species-specific libraries,
from fragmented genomes.
[0104] FIG. 2. Provides a graphic representation of how n[snare]
plasmids can be used to affect gene expression. The n[snare]
plasmid is introduced into the targeted cell (e.g. prokaryote), by
standard means, and its stable propagation is ensured by selecting
for its marker, usually a gene encoding resistance to an
antibiotic. When thus introduced into a cell the n[snare]plasmid is
able to affect expression of the targeted gene by titrating off the
transcription factor "B" from the genomic promoter to relieve
transcriptional repression of the downstream gene (shown as
horizontal box "D", top right).
[0105] FIG. 3. Demonstration of n[snare] approach. n[snare]
plasmids are capable of modifying control of gene expression in a
predictable way with a concomitant change in phenotype. These
plasmids are cheaper to produce and easier to transform into the
cells than decoys, give a more sustained effect and crucially, they
allow a library approach to discovering key regulatory
elements.
[0106] FIG. 4. Part of the process for creating custom n[snare]
plasmid libraries: Converting genomic fragments to short single
stranded nucleotides. n[snare] plasmid libraries are created from
large pieces of DNA in such a fashion that every cis-regulatory
sequence within that DNA should be represented in the library.
[0107] FIG. 5. n[snare] plasmid libraries can be used to gain
control of targeted phenotypes, such as antibiotic production.
Following introduction of members of the library by conjugation,
clones are selected on the basis of increased production of
cinnamycin (right).
[0108] FIG. 6. Further details for application of n[snare] to
combating antibiotic resistance. The schematic in this figure shows
how a library of n[snare] plasmids is used to identify
cis-regulatory sequences controlling antibiotic resistance when
nothing is known about the genetic pathway of the mechanism.
[0109] FIG. 7. Reporter constructs developed to identify
cis-regulatory elements within an n[snare] plasmid library. In this
figure the targeted promoter of the cin7 gene from the cinnamycin
biosynthetic cluster of the producing strain of S. cinnamoneous is
used to drive expression of the neo gene encoding resistance to the
antibiotic kanamycin.
[0110] FIG. 8. Detecting negative cis-regulators of cinnamycin
production using a generic reporter system and a custom n[snare]
library. A simple adaptation makes the reporter system capable of
detecting positive regulation by creating a cassette consisting of
a chimeric reporter gene incorporating the coding sequence of the
glucose kinase gene (glkA) driven by the targeted promoter.
[0111] FIG. 9. Library hybridization to identify candidate clones.
To distinguish `background` members that form a considerable part
of the total signal from n[snare] plasmids genuinely able to
interfere with transcription, a library hybridization strategy was
developed using standard methods.
[0112] FIG. 10. Hybridization screens to detect candidates from
independent repeats of the procedure to detect cis-regulators
capable of regulating cinnamycin production using a generic
reporter system and a custom n[snare] library. In this example two
independent S. cinnamoneous libraries were tested with reporter
strains and 96 of the most kanamycin resistant clones from each
used to make a filter containing the n[snare] plasmids for each
clone (F1 and F2), and two probe sets (P1 and P2). All of the
clones identified by cross-hybridization (F1 versus P2 and F2
versus P1) were considered strong candidates, and priority was
given to those common to both cross-hybridization experiments
(circled in red).
[0113] FIG. 11. Restriction digest analysis of the Escherichia coli
K12 genomic n[snare] library confirms it has the expected sequence
properties. This mixture of plasmids was digested with EcoRI to
regenerate the fragments used to create the n[snare] libraries, and
these were subjected to MmeI digestion to confirm the molecular
biology of construction had worked: as expected the inserts were of
large size and these collapsed to a 30 bp monomer on digestion to
completion with MmeI.
[0114] FIG. 12. n[snare] libraries are used to engineer phenotypes
without prior knowledge of the genetic network. This process was
repeated four times and the cell viability measured each time.
[0115] FIG. 13. Schematic overview of the protocol for in vive T7
exonuclease/DNaseI mapping procedure. The novel combination of T7
exonuclease and DNaseI in a footprinting protocol allows detection
of all the boundaries of DNA-protein complexes within a promoter
region, and not just those closest to a chosen restriction
site.
[0116] FIG. 14. T7 exonuclease/DNaseI mapping of candidate
regulatory motifs within the promoter of actII-orf4. (A) Cells of
S. coelicolor M145 were harvested from a culture grown in rich (R5)
media at a time point (indicated by arrow) preceding visible
actinorhodin production. (B) Boundaries were mapped on both
strands, as described in FIG. 13, and their positions determined
following size analysis by 12% non-denaturing PAGE, followed by
chemo-luminescent detection of DIG-labeled products. (C) The
sequence of the actII-orf4 promoter showing the positions of the
putative cis-regulatory elements (relative to the primers used in
the mapping protocol).
[0117] FIG. 15. Plate assays demonstrating that decoy
oligonucleotides can influence antibiotic production in S.
coelicolor. Filter discs were saturated with solutions of decoys
or, as control, buffer (as shown) and applied to a lawn of S.
coelicolor M145 overlaid with SNA medium.
[0118] FIG. 16. Uptake and stability of decoy oligonucleotide.
Actively growing cells were transfected with a solution containing
decoy and the uptake of the oligonucleotide and its stability
estimated by quantitative real-time PCR (qrt-PCR).
[0119] FIG. 17. Decoy-mediated increase in actinorhodin production
in cultures grown in R5 liquid medium. S. coelicolor M145 was grown
for 20 hours before transfection (indicated by arrows) with (A) a
no-decoy control or with (B) the A24.5 decoy.
[0120] FIG. 18. Decoy-mediated increase in actinorhodin production
in cultures grown in SMM liquid medium. Comparison of the data
obtained with (A) a mock-transfected control and (B) a decoy
A24.5-treated culture revealed that the decoy oligonucleotide
caused a pronounced increase in actinorhodin production.
[0121] FIG. 19. Deletion of SCO5812 leads to underproduction of
actinorhodin on R5 agar and overproduction of undecylprodigiosin on
SMMS agar. M145 (left side of plates) and M145 ASCOS812 (right side
of plates) were streaked on (A) R5 agar medium or (B) SMMS agar
medium and incubated for 72 hr and 96 hr, respectively.
[0122] FIG. 20 provides application of the transcription factor
decoy (TFD) approach to combating antibiotic resistance. The
schematic demonstrates how TFDs are used to counter known
resistance mechanisms in pathogenic bacteria. Resistance to the
prescribed antibiotic vancomycin is used as an example.
[0123] FIG. 21. The structure of the vancomycin gene. From Hong et
al. 2004 Molecular Microbiology 52: 1107-1121.
[0124] FIG. 22. Evidence of cyclization of the oligonucleotide
decoy vanH5. The oligonucleotide was resuspended at a final
concentration of 100 pmol/ul in a T4 DNA ligase buffer (as supplied
by the manufacturer of the enzyme, New England Biolabs) and 400 U
of T4 DNA ligase and incubated at 16 degrees Centigrade for various
times.
[0125] FIG. 23. Shows the effect of incubating a vancomycin
resistant bacterium with and without increasing levels of the vanH5
decoy. S. coelicolor strain M600 was grown in liquid MMCGT medium
(Molecular Microbiology 52: 1107-1121), growth was measured by
recording the absorbance of the culture at 430 nm (Cell Density)
and plotted as a function of time of incubation.
[0126] FIG. 24. Shows the amplification product obtained using the
primers in SEQ ID NOS: 24 & 25 and an appropriate vector
substrate (Example 7.2).
[0127] FIGS. 25 & 26. Show growth curves for E. faecium grown
in the presence of vancoymcin after treatment with either the VAN
transcription factor decoy sequence or a negative control (CON), as
described in Examples 8(a) and 8(b) respectively.
[0128] FIG. 27. A copy of Tables 1 and 2 from Poole (2005) J.
Antimicrobial Chemother. 56, 22-24 listing the efflux-mediated
resistance to non-fluorquinoline antibiotics and fluoroquinoline
antibiotics respectively. Reference numbers in the right hand
column refer to those given in the paper.
BRIEF DESCRIPTION OF THE SEQUENCES
[0129] SEQ ID NO: 1--a decoy oligonucleotide containing the AfsR
binding site (Example 1).
[0130] SEQ ID NO: 2--a joining polynucleotide R-T7, which contains
a partial complement of a commonly used primer, T7 (Example 1).
[0131] SEQ ID NO: 3--an oligonucleotide containing the AfsR binding
site and use to create a cyclised decoy sequence in Example 1.
[0132] SEQ ID NOs: 4 & 5--sequences of each strand of a double
stranded adaptor molecule (Example 2) SEQ ID NO: 6--a Bbv
complementary oligonucleotide (Example 2).
[0133] SEQ ID NO: 7--an oligonucleotide comprising a NotI site and
a randomised nucleotide sequence, where each randomised nucleotide
is represented as "n" (Example 4).
[0134] SEQ ID NOs: 8-12--decoy oligonucleotides designed based on
cis-regulatory sequences A24.1, A24.2, A24.3, A24.4 and A24.5
respectively (Example 5).
[0135] SEQ ID NO: 13--a decoy oligonucleotide designed based on a
scrambled A24.5 sequence (Example 5).
[0136] SEQ ID NOs: 14-20--PCR primers used in quantitative PCR as
in Example 5.
[0137] SEQ ID NO: 21--a vanH5 decoy oligonucleotide containing a
binding site for phosphorylated VanR (Example 6).
[0138] SEQ ID NOS: 22 & 23--oligonucleotide primers used for
amplification of a target sequence from the pGEMT-Easy vector as in
Example 7.1.
[0139] SEQ ID NOS: 24 & 25--oligonucleotide primers used for
amplification of a target sequence from the pGEMT-Easy vector in
the production of dumbbell decoys as in Example 7.2.
[0140] SEQ ID NO: 26--the VAN decoy sequence used in Examples 8(a)
and 8(b), and containing the regulatory element controlling
induction of VanA type resistance in E. faecium.
DETAILED DESCRIPTION OF THE INVENTION
[0141] As described in more detail herein, the present inventors
have devised methods and compositions for identifying,
characterising and targeting cis-regulatory sequences in
prokaryotes and eukaryotes.
[0142] A cis-regulatory sequence or element generally refers to a
nucleotide sequence which occurs upstream (5') or downstream (3')
of a gene or genes and which functions to modulate expression of
the gene or genes. Typically, a cis-regulatory sequence comprises a
binding site for a protein (transcription factor) which regulates
transcription of the given gene(s). Binding of the protein to the
sequence results directly or indirectly in modulation of expression
of the gene(s). For example, the bound protein may interact with
another protein bound to a nearby region which is needed for
transcription and anchor the protein in the correct position, or
may inhibit binding of another protein which is necessary for
transcription. Typically the cis-regulatory sequence or element
occurs in the promoter region of a gene, but it is not unusual in
prokaryotes for cis-regulatory sequences to be positioned hundreds
of base pairs upstream or downstream of the genes they affect. In
eukaryotes, cis-regulatory sequences can act at great distances to
influence expression of a gene, typically on the order of 1-2 kb,
but it is not unknown for sequences to act over 100 kb to 1 Mb.
[0143] A cis-regulatory sequence may be repressive (inhibits or
reduces transcription of the gene(s) when bound by a transcription
factor) or activatory (activates or increases transcription of the
gene(s) when bound by a transcription factor). Thus a transcription
factor which binds a cis-regulatory sequence may be a negative
effector (repressor protein) or a positive effector
(activator).
[0144] The expression of certain genes will also be modified by
indirect effects where, for example, the impact of a cis-regulatory
sequence is on a separate gene which, in turn, influences the
expression of the targeted gene. This may occur, for example, by
causing changes to a regulatory network of which the targeted gene
is a part, or due to a more global effect, such as a
shock-response, which causes modification of the expression of the
targeted gene.
[0145] Modulation of protein binding at cis-regulatory sequences
can provide a useful means for modulating gene expression in a
cell. One way of doing this is to provide a decoy nucleotide
sequence. The decoy sequence comprises a transcription factor
binding site which comprises of competes with the native or
endogenous cis-regulatory sequence in the cell for binding to the
cognate transcription factor. By reducing binding of the
transcription factor to the native sequence, the decoy alters
expression of the gene(s) whose expression is normally regulated by
the cis-regulatory sequence in the cell. Such alteration can
provide a useful alteration in phenotype, e.g. in metabolite
production or antibiotic resistance of a prokaryotic cell. Decoy
function as used herein refers to the capability of a sequence to
compete with a cis-regulatory sequence for binding to a cognate
transcription factor in this way.
[0146] The inventors have developed new means for identifying
sequences which compete with cellular transcription factor binding
sites for transcription factor binding, to alter cellular gene
expression, and which may therefore provide new cis-regulatory
sequences and decoy sequences.
[0147] n[Snare] Plasmids
[0148] The approach described herein is to make important
adaptations of the known application in eukaryotes of the
technology of `decoy` oligonucleotides, which has been used to
modulate expression in vivo. The logic of decoy oligonucleotides is
simple and relevant to all biological systems: assert genetic
control by perturbing the binding of transcription factors to their
cognate sites. We improve on this technology by providing a generic
and high-throughput tool for analysis of genetic regulation. A
first modification involves making the approach plasmid-borne by
developing a method to clone homopolymers of decoy sequences, to
create high-copy `n[snare]` plasmids. These are easily introduced
into all bacteria and maintained by positive selection,
circumventing the major deficiencies of the decoy approach, that
the oligonucleotides can be difficult to introduce into the cell
and can be sensitive to exonucleases, giving a relatively short
half-life.
[0149] An overview of the molecular biology protocol used to create
an n[snare] plasmid is given in FIG. 1. An overview of how the
n[snare] plasmids affects gene regulation is given in FIG. 2. A
demonstration of the efficacy of the n[snare] plasmid is given in
FIG. 3 where it is used to ablate the production of antibiotic in
S. coelicolor by the introduction of a known cis-regulatory
sequence from a positive pleiotropic regulator of antibiotic
synthesis, AfsR.
[0150] 1. Design of n[Snare] Plasmids
[0151] The advantages of creating plasmid-borne versions of decoy
oligonucleotides rather than decoys themselves for use in
identifying the biological activity of cis-regulatory include:
[0152] 1. Cost of manufacture: The molecular biology protocol
described below is simple and robust and can be applied either to
synthesized oligonucleotides or fragments of DNA from diverse
sources. As the plasmids self-replicate there is no need to produce
large quantities of the plasmids. This compares favourably with a
potential need to create substantial amounts of decoy
oligonucleotides with potentially expensive modifications of the
nucleotide backbone; [0153] 2. Resistance to degradation: In
comparison to decoys, plasmids show little susceptibility to in
vivo degradation by nucleases; [0154] 3. Plasmid concentration is
maintained: As plasmids are self-replicating (and can have
mechanisms to control their intracellular concentrations or copy
number) and can be subjected to positive selection if necessary,
the half life of an n[snare] plasmid is theoretically indefinite;
[0155] 4. Broad range of hosts: In Example 1 a `shuttle plasmid` is
used which can be readily transformed and propagated in E. coli
(for ease of genetic manipulation) and S. coelicolor. Many plasmids
are known to those skilled in the art which allow transformation of
a broad range of bacterial hosts; [0156] 5. Combinations of
cis-regulatory sequences can be tested using distinct n[snare]
plasmids: Compatible plasmids can potentially be used to determine
whether or not simultaneous treatment with two or more n[snare]
plasmids have synergenistic effects.
[0157] In one aspect therefore the invention provides a plasmid
which is suitable for testing possible decoy function of a known or
putative cis-regulatory sequence, or for screening for new
cis-regulatory sequences (which may act as decoy sequences). The
plasmid is designated an n[snare] plasmid.
[0158] The principle of use of an n[snare] plasmid is illustrated
in FIG. 2. The plasmid comprises a "snare" sequence (shown in
multiple copies in the Figure). If the "snare" comprises a
transcription factor binding site which competes with a cellular
cis-regulatory sequence for binding to a transcription factor,
introduction of the n[snare] plasmid to the cell will result in a
titration of transcription factor off the cellular cis-regulatory
sequence and onto the snare.
[0159] This can be detected as a change in expression of a gene or
gene(s) whose expression is regulated in the cell by that
cis-regulatory sequence, or by alteration in the phenotype of the
cell. A change in gene expression or phenotypic output thus
indicates that the snare comprises a sequence with decoy function,
and the plasmid can be used to identify or confirm the function of
a cis-regulatory sequence.
[0160] In general an n[snare] plasmid comprises a plasmid vector
and an insert sequence (the insert comprising the snare).
Incorporating the snare sequence in a plasmid addresses the
problems of decoy degradation in the art, and allows stable
maintenance of the decoy (and any affect on gene expression) in the
cell.
[0161] The insert sequence comprises one or more copies of a
monomer sequence (which comprises the snare). Thus the insert may
comprise (for example) from 1 to 200 monomer sequences. Typically
there are two or more copies, for example, 2-200 copies, e.g. at
least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
150, 160, 170, 180, or 190 copies. For example, there may be 5-200,
5-150, 5-100, 10-150, 10-50, 5-50, 5-40, 10-40, 5-30 copies. For
example, there may be 30 copies of the monomer sequence. The
plasmid typically comprises a homopolymer of the monomer. Typically
they are multiple copies of the monomer, for example, multiple
direct repeats of the monomer sequence. Providing multiple copies
of the monomer (and thus of the snare) increases the titrating
power of the decoy.
[0162] The monomer sequence comprises the snare sequence. The snare
comprises a nucleotide sequence which is to be tested for or used
for cis-regulatory or decoy function as described herein.
[0163] For example, a snare may comprise a known or putative
cis-regulatory sequence, a decoy sequence, a fragment of sequence
to be tested for decoy function, e.g. a genomic fragment, such as a
promoter fragment, or a randomised nucleotide sequence, or a
combination of cis-regulatory sequences (e.g. 2, 3, 4 or more
cis-regulatory sequences). n[snare] plasmid libraries may be
prepared, comprising snare sequences derived from and covering,
substantially all of the sequence (or cis-regulatory sequence) of a
genome or genomic fragment. n[snare] plasmid libraries may also be
prepared, in which the snare sequences comprise randomised
nucleotide sequences of a given length ("n" nucleotides in length).
Preferably all or substantially all possible sequences of length n
are represented in the library. These libraries can be useful for
screening to identify sequences that comprise and/or compete with
cellular cis-regulatory sequence for transcription factor binding,
and therefore to identify new cis-regulatory and decoy
sequences.
[0164] Typically a transcription factor binding site in a snare is
not operably linked to a gene, e.g. in the snare or snare plasmid.
In that sense the binding site is isolated from its cognate gene or
genes. A binding site in a snare may also be isolated from other
elements in its cognate promoter. In one instance, the monomer
sequence does not comprise a gene.
[0165] A monomer may comprise additional sequence in addition to
the snare. Often such additional sequence derives from the method
used to produce the snare and/or the plasmid insert. For example, a
monomer may comprise an adaptor sequence, such as the adaptor
sequence which typically results when "custom" snares are produced
for a custom n[snare] library according to the methods herein. An
adaptor sequence may comprise, for example, recognition and/or
cutting sites for one or more restriction enzymes.
[0166] A monomer may comprise nucleotide sequence which provides a
binding site for a primer, e.g. a primer used in production of the
monomer or of an insert comprising multiple monomers.
[0167] For example, when a plasmid insert is prepared using a
rolling circle amplification method as described herein, a monomer
typically comprises a segment which corresponds to the binding site
for the primer used in rolling circle replication, e.g. a T7
primer.
[0168] A monomer comprising a randomised snare sequence typically
also comprises a region or regions of constant sequence. For
example, a randomised snare sequence of n nucleotides may be
flanked by regions of constant sequence. Alternatively, a central
core of constant sequence may be flanked by regions of randomised
nucleotide sequence.
[0169] The length of a monomer can be, for example, up to 1000
nucleotides, for example, up to 900, 800, 700, 600, 500, 400, 300,
200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 nucleotides.
Typically the length of the monomer is in the range of, for example
10-100, 10-50, 20-75, 30-60, 30-50, 35-55 such as 35-54
nucleotides. For example, the length of a monomer may be 30, 40 or
50 nucleotides.
[0170] A snare portion of a monomer may typically range in size
from 10-30 nucleotides, for example, 10-25, 10-20, 15-20, such as
15, 16, 17, 18, 19 or 20, for example 19 nucleotides.
[0171] An adaptor sequence may comprise, for example, 5-30
nucleotides, for example, 5-25, 5-20, 5-15 or 5-10 nucleotides such
as 10, 11, 12, 13, 14 or 15 nucleotides.
[0172] Typically, an insert in the n[snare] plasmid comprises one
or more copies of a monomer as described herein. Where the insert
comprise multiple repeats of a monomer, these may be tandem
repeats.
[0173] An insert in the plasmid vector may comprise, for example
about 1.5 kb, for example 1-2 kb, for example 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7 or 1.8 kb. However, any suitable insert size, which
allows stable maintenance of the plasmid in a suitable host cell
and efficient use in the present methods may be used.
[0174] Typically the sequences of all monomers in a single plasmid
are the same.
[0175] The plasmid vector for use in the n[snare] plasmid may be
suitable for use in a prokaryotic or eukaryotic host. For example,
the vector may be for use in a prokaryotic such as a bacterial,
e.g. actinomycete host. For example, the plasmid vector may be
suitable for use in a Streptomycete or E. coli strain, e.g. one or
more of Streptomyces coelicolor e.g. A3(2) (or strain M145 or
M600), E. coli, Streptomyces lividans or Streptomyces cinnamoneous.
Suitable hosts are described further herein.
[0176] Typically, the vector is a broad host range and/or shuttle
vector and can therefore be maintained and propagated in more than
one host. The plasmid may a conjugative plasmid. This allows easy
transfer from one cell to another by conjugation.
[0177] Preferably the plasmid is self-replicating. Typically the
plasmid is a high copy number plasmid. For example, the plasmid may
be maintained at, for example, 20-100 copies per cell, for example
20, 30, 40, 50, 60, 70, 80, 90 or 95 or 100 copies per cell. High
copy number increases titrating power of a decoy sequence in the
snare.
[0178] Typically an n[snare] plasmid comprises an origin of
replication. Suitable origins are known in the art. Typically the
plasmid additionally comprises one or more detectable marker genes,
for example, one or more genes encoding antibiotic resistance, e.g.
the aac gene encoding apramycin resistance. Expression of the
marker gene(s) allows screening for maintenance of the plasmid in a
host cell.
[0179] Examples of suitable plasmid vectors include, for example,
pIJ86. Suitable vectors are known in the art.
[0180] A monomer or snare sequence may comprise a cis-regulatory
sequence or decoy sequence described herein and/or identified
according to the methods described herein.
[0181] For example, a transcription factor binding site in a
plasmid may comprise or compete for transcription factor binding
with, a cis-regulatory sequence which regulates expression of a
gene or genes which have a role in metabolite production. The
metabolite may be, e.g. an antibiotic, enzyme or pharmaceutical. An
antibiotic may be, e.g. actinorhodin, undecylprodigiosin or
cinnamycin.
[0182] For example, a snare may comprise a binding site for AfsR
protein (a pleitropic regulator of antibiotic synthesis in S.
coelicolor). An AfsrR binding site is shown in bold in SEQ ID NO: 1
(Example 1). A monomer may thus comprise SEQ ID NO: 1. Thus a
monomer may be the double stranded version of SEQ ID NO: 1 as in
Example 1. In one example there may be 30 repeats of the monomer
and/or the plasmid may comprise the shuttle vector pIJ86. Thus in
one aspect, the invention relate to an n[snare] plasmid(s) prepared
according to the method in Example 1.
[0183] In another example a snare may comprise a cis-regulatory
sequence in the S. coelicolor actII-orf4 promoter, for example a
repressor cis-regulatory sequence, or a sequence that competes with
such a sequence for transcription factor binding, as described
herein. For example a cis-regulatory sequence may comprise sequence
A24.1, A24.2, A24.3, A24.4. or A24.5 identified herein. In one
aspect a monomer may comprise one of SEQ ID NOs: 8-12 described
herein or SEQ ID No: 13.
[0184] A transcription factor binding site in a plasmid may
comprise, or compete for transcription factor binding with, a
cis-regulatory sequence which regulates expression of a gene or
genes which have a role in determining antibiotic resistance, such
as any one or more of the genes listed herein.
[0185] For example, a snare may comprise a binding site for the
VanR transcription factor, for example, a VanR binding site located
in the vanH promoter of, for example, Entercoccus faecium or S.
coelicolor, or a sequence which competes with such a site. An
example of a 30 bp VanR binding site is shown (capitalised) in SEQ
ID NO: 21 and a further example is provided in SEQ ID NO:26 which
contains the VanR binding site for Enterococci. In one example a
monomer may comprise SEQ ID NO: 21 or SEQ ID NO:26 or a variant
thereof which competes for binding of VanR transcription factor
with a native VanR binding site in a cell of interest. For example,
a variant may comprise the native VanR binding site in another
species or strain.
[0186] A transcription factor binding site in a plasmid may
comprise, or compete for transcription factor binding with, a
cis-regulatory sequence which regulates expression of a gene or
genes which have a role in determining solvent tolerance, e.g.
butanol tolerance.
[0187] A snare may comprise a sequence derived or isolated from a
genome or genomic fragment. A genomic fragment may comprise a gene
or genes encoding a particular function or phenotype of interest,
e.g. production of a metabolite(s) such as an antibiotic (e.g.
cinnamycin, actinorhodin, undecylprodigiosin), tolerance to a
particular solvent(s) e.g. butanol, or toxin(s), resistance to a
particular antibiotic(s), or any other function or phenotype of
interest. A snare may comprise, for example, a sequence derived or
isolated from the promoter region of such gene(s) or surrounding
sequences, e.g. sequence at a greater distance from a gene(s) than
the promoter, e.g. at up to 200 bp or more.
[0188] A snare may comprise a sequence that competes with such a
sequence for transcription factor binding, as described herein. For
example, a snare may comprise a cis-regulatory sequence or decoy
sequence derived from a fragment of the S. cinnamoneus genome
comprising the cinnamycin biosynthetic cluster of genes. Methods
for preparing such snares are described herein. A snare may
comprise a cis-regulatory sequence from the S. cinnamoneus cin7
promoter or a sequence that competes with such a sequence for
transcription factor binding, as described herein.
[0189] A snare may comprise sequence derived or isolated from the
genome or a genomic fragment of a prokarytote which displays
solvent tolerance, e.g. E. coli K12, which has butanol tolerance.
In particular, a snare may comprise a cis-regulatory sequence
derived from the promoter of a gene encoding solvent tolerance or
whose expression is associated with solvent tolerance, or a
sequence which competes with such a cis-regulatory sequence for
transcription factor binding. Methods for preparing such snares are
described herein.
[0190] A snare may comprise a sequence (e.g. a cis-regulatory
sequence or decoy sequence) derived from or isolated from the
genome or a genomic fragment of a prokaryote which displays
antibiotic resistance (typically the fragment comprises the gene(s)
encoding resistance). The genomic fragment may comprise a gene or
gene(s) encoding antibiotic resistance. A snare may comprise a
cis-regulatory sequence from the promoter of a gene(s) encoding
antibiotic resistance, or a decoy sequence which competes with such
a sequence for transcription factor binding. Any antibiotic
resistance of interest may be targeted. For example, antibiotic
resistance to: the class of antibiotics known as aminoglycosides
(such as kanamycin and gentamycin); the glycopeptides (such as
vancomycin,); the beta-lactams which include the penicillins (such
as ampicillin, carbenicillin and penicillin), the beta-lactamase
inhibitors and combinations of (such as piperacillin and
tazobactam), the cephalosporins (such as cefepime), the carbapenems
(such as meropenem), the monobactams (such as Aztreonam); the
polypeptide antibiotics (such as polymixcin B); the quinolines
(such a levaquin); the fluorquinolines (such as ciprofloxacin); the
sulfonamides (such as Bactrim); the tetracyclines (such as
tetracycline); the macrolides and ketolides (such as azithromycin);
the oxazolidinones (such as linezolid); the nitroimidazoles (such
as metronidazole); the nitrofurans (such as nitrofurantoin); the
streptogramins (such as dalfopritsin); the cyclic lipopeptides
(such as daptomycin); the lincosamides (such as clindamycin) and
variously, chloramphenicol, rifampicin, isoniazid, ethambutol,
telvancin, teicoplanin, oritavancin, dalbvancin,
trimethoprim/sulfamethoxazole, fosfomycin, nitrofurantoin and
tigecycline and Zyvox.
[0191] For example, antibiotic resistance to: the class of
antibiotics known as aminoglycosides (such a kanamycin); the
carbapenems (such as meropenem); the cephalosporins (such as
cefepime); the glycopeptides (such as vancomycin); the penicillins
such an ampicillin, carbenicillin and penicillin); the polypeptide
antibiotics (such as polymixcin B); the quinolines (such a
levaquin); the sulfonamides (such a Bactrim); the tetracyclines
(such as tetracycline); and variously, chloramphenicol, rifampicin,
Zyvox, and daptomycin.
[0192] n[Snare] Plasmid Libraries
[0193] Having described the creation of n[snare] plasmids capable
of establishing functional activity of single cis-regulatory
elements, we here generalize the methods by which these sequences
may be discovered and characterized, including via a method using
libraries of n[snare] plasmids, a methodology compatible with
high-throughput screening.
[0194] The overall aim of this methodology is to develop a new
method to rapidly identify cis-acting regulators of prokaryotic as
well as eukaryotic genes, and in doing so create tools capable of
modifying expression in vivo. The method allows for dissection of
large scale regulatory networks. The anticipated advances in our
understanding of the mechanics of genetic regulation will parallel
the breakthrough afforded by microarray transcriptomic
analysis.
[0195] This work identifies genetic modifiers and determines
pathways in a different way to existing technologies such as
creation of gene knock-outs or insertional mutagenesis. Those
approaches generally identify trans-acting regulators (such as the
transcription factors). By identifying cis-acting factors, this
technology circumvents problems associated with these traditional
approaches: redundancy--due to the inherent complexity of
transcriptional regulation, meaning that more than one
transcription factor can bind at the same site; polar
effects--mutagenesis may affect the expression of genes located 3'
of the site of mutation in the same transcription unit; high cost
and turnaround time--generating and validating a single knock-out
may take many weeks to years (depending on the species) and
performing saturation mutagenesis is a lengthy and expensive
procedure; species-specificity--the approach described herein has
potential utility in all experimental systems, and it is
anticipated that a universal `decoy` library can be applied to map
regulation in the majority of species.
[0196] In Example 1 we demonstrate a substantial improvement on
decoy technology, reporting a modification making the approach
plasmid-borne by developing a method to clone homopolymers of decoy
sequences, to create high-copy `n[snare]` plasmids. These are
easily introduced into many bacterial species and maintained by
positive selection, circumventing the major deficiencies of the
decoy approach, that the oligonucleotides can be difficult to
introduce into the cell and can be sensitive to exonucleases,
giving a relatively short half-life. In a second modification and
improvement we create n[snare] libraries, consisting of either
fragments of the entire genome, detailed in Example 2, or sequences
derived from randomized oligonucleotides, described in Example 4.
These approaches allow comprehensive collection of regulatory
elements to be screened in parallel, which is a far more powerful
approach than use of decoys for screening purposes, which is
limited to sequential testing of defined sequences. Below, in
Example 3 a reporter system, using regulation of antibiotic
production as a model, is used in one exemplary embodiment to
select for positive and negative regulators within the library.
[0197] FIG. 4 is a schematic showing part of the process of how
custom n[snare] plasmid libraries are constructed.
[0198] FIG. 5 shows an experimental approach using an n[snare]
library to detect cis-regulatory elements capable of upregulating
production of the antibiotic cinnamycin from Streptomyces
cinnamoneous.
[0199] FIG. 6 provides further details for application of n[snare]
to combating antibiotic resistance.
[0200] n[Snare] Plasmid Libraries as Discovery Tools
[0201] Gene expression, and concomitantly phenotypic effect, are
largely controlled by DNA-protein interactions. Efforts to
understand and control gene expression generally place more stress
on manipulating the protein component of the transcriptional
machinery, the trans-acting factors such as transcription factors,
rather than the DNA sequences (the cis-regulatory elements), that
they bind to. Targeting the cis-regulatory elements, as opposed to
the trans-acting factors, has the following advantages: it is
easier to transfect oligonucleotides into cells than to make
genetic deletions of targeted proteins; use of oligonucleotides is
more adaptable to high-throughput analysis; use of cis-acting
elements circumvents the problem of redundancy in regulatory
networks, where deletion of one particular transcription factor is
compensated for by the activity of another binding to the same
cis-regulatory sequence. A potential bottleneck with the decoy
approach, however, is the identification of candidate
sequences.
[0202] For this purpose the utility of n[snare] vectors is
demonstrated by creating libraries from a genomic fragment and
testing to see whether members of the resulting library can exert
control of the product synthesized by the genes contained on that
fragment. In addition, the molecular biology of library creation is
tested; to confirm that the libraries are sufficiently complex and
have the expected molecular structure of direct repeats of the same
sequence. These successes are developed further to create a
powerful screening method with broad and generic applications.
[0203] As described above therefore, the invention also relates to
n[snare] plasmid libraries. The plasmid libraries may be used
according to the methods of the invention to screen for new
cis-regulatory sequences and decoy sequences in either prokaryotes
or eukaryotes. Use of n[snare] libraries for screening allows high
throughput screening of multiple sequences for decoy function.
[0204] The snare sequences of the plasmids in the library may be
derived from a genomic fragment or a genomic DNA. The library may
then comprise snare sequences representing or covering
substantially all of the genomic fragment or genome sequence. This
type of plasmid library is referred to herein as a custom n[snare]
library. Alternatively, the snare sequences of the plasmids in the
library may comprise a randomised sequence of length "n"
nucleotides. The library may then comprise substantially every
possible nucleotide sequence of length "n" as described herein.
Such a library is described herein as a universal n[snare]
library.
[0205] Custom n[Snare] Plasmid Libraries
[0206] Typically the snares are derived from a genomic fragment or
genomic DNA (prokaryotic or eukaryotic). For example, the genome or
a genomic fragment of a prokaryote such as a bacterium, e.g
Streptomycetes, such as S. cinnamoneus, S. lividans or S.
coelicolor, or E. coli.
[0207] The genome or genomic fragment from which the library is
derived may comprise a gene or genes encoding a particular function
or phenotype of interest, e.g. production of a metabolite(s) such
as an antibiotic (e.g. cinnamycin, actinorhodin,
undecylprodigiosin), tolerance to a particular solvent(s) or
toxin(s), resistance to a particular antibiotic(s), or any other
function or phenotype of interest. The snares may comprise sequence
derived or isolated from the promoter region of such gene(s).
Suitable genes and functions/phenotypes have been described above
in relation to snare sequences.
[0208] Thus the genomic DNA or fragment may comprises a gene or
genes which have a role in metabolite production, e.g. where the
metabolite is an antibiotic, enzyme or pharmaceutical, or a gene or
genes which have a role in determining antibiotic resistance or a
gene or genes which have a role in determining solvent
tolerance.
[0209] A genomic fragment which comprises a gene or genes encoding
a particular function or phenotype may have been identified by
detecting horizontal transfer of the genes encoding the function or
phenotype into a heterologous cell.
[0210] A genomic fragment which comprises a gene or genes encoding
a particular function or phenotype may have been identified by
bioinformatical analysis. Alternatively it may have been identified
by a functional screen of a collection of such fragments.
[0211] Methods for preparing custom n[snare] libraries are
described herein.
[0212] Universal Libraries
[0213] In a universal library, a snare typically comprises a
randomised nucleotide sequence of n nucleotides in length. The
library may comprise snares representing all or substantially all
permutations of sequences of n nucleotides in length. In general n
may range from 5-50, for example 10-50, for example 20-40 e.g.
25-35 e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
nucleotides.
[0214] A monomer in such a library may additionally comprise
constant sequence. Thus the randomised sequence (variable region)
may be flanked by a constant region on one or both sides.
Alternatively, the monomer may comprise a defined core sequence
flanked by variable regions. A nucleotide bias may be introduced in
the variable region. For example, the variable region may comprise
a higher GC bias where this is appropriate, e.g. where the library
is for use in screening for cis-regulatory sequences in an organism
in which the DNA displays a high GC bias.
[0215] In one example, the snare comprises a randomised nine
nucleotide sequence (n=9). The monomer in the n[snare] plasmids may
comprise the oligonucleotide sequence of SEQ ID NO: 7. Methods for
producing a universal n[snare] library are described herein.
[0216] Methods for Preparing n[Snare] Plasmids
[0217] In general, an n[snare] plasmid as described herein may be
prepared by a method comprising: [0218] providing a polynucleotide
comprising one or more copies of a monomer sequence; and [0219]
cloning the polynucleotide into a suitable plasmid vector.
[0220] The composition of the monomer is as described herein for
the n[snare] plasmid. Suitable plasmid vectors have also been
described.
[0221] The polynucleotide comprising one or more copies of a
monomer sequence may be provided by a method comprising:
[0222] (1) providing a circular oligonucleotide comprising: [0223]
(i) a test sequence of interest (B in FIG. 1); and [0224] (ii) a
binding site for a primer suitable for use in rolling circle
amplification (A in FIG. 1);
[0225] wherein the monomer sequence comprises (i) and (ii); and
[0226] (2) performing rolling circle amplification using the
circular oligonucleotide as a template, thereby providing a
polynucleotide comprising repeats of the monomer sequence.
[0227] Step (1) of the method may further comprise amplifying the
rolling circle amplification products by PCR and isolating
polynucleotide fragments of the required size, for example,
fragments comprising 30-50 repeats of the monomer sequence. This
can be done by, e.g PAGE analysis.
[0228] A circular oligonucleotide may be prepared by a method
comprising: [0229] providing a linear single stranded
oligonucleotide comprising: the test sequence of interest (i) and
the binding site for a primer suitable for use in rolling circle
amplification (ii); [0230] circularising the oligonucleotide, e.g.
using Taq ligase, typically in the presence of a universal joining
oligonucleotide; [0231] optionally digesting remaining linear DNA
with an exonuclease; and [0232] recovering monomeric circular
oligonucleotides, e.g. using PAGE.
[0233] Primers suitable for use in rolling circle amplification are
known in the art. For example, a T7 primer may be used.
[0234] Methods for carrying out rolling circle amplification are
known in the art. For example, BstI polymerase may be used.
[0235] In one example, PCR amplification of the rolling circle
amplification products is carried out using the same primer that
was used for rolling circle amplification, e.g. T7 primer.
[0236] In general the test sequence (i) in the monomer comprises a
snare sequence as described herein.
[0237] As described herein, snare sequences may be isolated from
genomic DNA, e.g. from an entire genome, or from a genomic
fragment. A snare sequence, once isolated, may be used to form a
n[snare] plasmid by the method above. Thus a library of n[snare]
plasmids derived from a genome or genomic fragment (a custom
library as described herein), may be prepared.
[0238] A protocol for preparation of snare sequences from a genome
or genomic fragment is illustrated in FIG. 4. Typically a method
comprises:
[0239] (1) providing a sample of double stranded genomic DNA;
[0240] (2) fragmenting the genomic DNA;
[0241] (3) ligating an adaptor to each end of the DNA fragments
from (b), wherein each adaptor comprises: [0242] (iii) a means for
immobilisation of the DNA fragment; [0243] (iv) a recognition site
for a first restriction enzyme that cuts at a distance from (e.g.
downstream (3') of) the recognition site; and [0244] (v) a
recognition and cutting site for a second enzyme;
[0245] (4) optionally removing unligated adaptor;
[0246] (5) digesting the fragment bearing the adaptors with the
first restriction enzyme, thereby producing two adaptored
fragments, wherein each adaptored fragment comprises: [0247] (vi)
an adaptor; and [0248] (vii) a DNA fragment comprising: a shorter
strand: and a longer strand (e.g. with a 3' overhang), wherein the
longer strand comprises the snare sequence;
[0249] (6) immobilising the adaptored fragments produced in
(5);
[0250] (7) denaturing the fragments to provide single stranded
fragments;
[0251] (8) recreating the recognition site for the second
restriction enzyme by ligating a complementary oligonucleotide to
the adaptor and digesting the adaptored fragment with the second
restriction enzyme, thereby producing an adaptor-snare fragment
comprising: [0252] (viii) an adaptor fragment; and [0253] (ix) a
single stranded snare sequence; and
[0254] (9) releasing the adaptor-snare fragment from
immobilisation.
[0255] The adaptor-snare fragment produced in (8) comprises the
single stranded snare sequence and a fragment of the adaptor that
remains after digestion with the second restriction enzyme
(corresponding to the portion of the adaptor between the
recognition site for the second enzyme and the end of the adaptor
that is linked to the snare). The monomer in the eventual n[snare]
plasmid (and the test sequence (i) above) will comprise the
adaptor-snare fragment.
[0256] The genomic DNA sample may be derived from a prokaryotic or
eukaryotic cell. The genomic DNA sample may comprise a genomic
fragment or an entire genome. The sample may be from a cell which
displays a particular phenotype of interest, e.g. production of a
particular metabolite, resistance to a particular antibiotic, such
as a native strain (e.g. a pathogen) or clinical isolate. For
example, the sample may be from S. cinnamoneus which produces
cinnamycin, from S. coelicolor which produces actinorhodin,
undecylprodigiosin, from E. coli K12 which displays tolerance to
butanol, or from Enterococcus faecium or S. coelicolor which
display resistance to vancomycin. The sample may be from a
bacterial model, e.g. which has acquired a particular phenotype or
function by horizontal gene transfer.
[0257] Typically the fragments produced in step (2) are about 500
bp in length, but may range for example, from 100-1000 bp, such as
200-900, 300-800, 400-600 bp, such as 150, 250, 350, 450, 550, 650,
750, 850, 950 bp. Any suitable fragmentation method may be used to
produce the fragments. Preferably the method produces randomised or
unbiased fragments. For example, sonication may be used.
[0258] Fragments produced in step (2) may comprise different types
of ends--for example, blunt ends, or 5' or 3' overhangs of
different lengths. Therefore, the fragments produced in
fragmentation step (k) may be further treated to produce a
population of fragments that are homogeneous in that each fragment
has the same 3' dNTP overhang--dA or dT. This may be done, for
example, by treating with Taq polymerase and suitable dNTPs to
repair fragment ends if necessary, and add a 3' dNTP e.g. dA
overhang.
[0259] Adaptors may be attached to the fragments by any suitable
means. For example, an adaptor may comprise a 5'dNTP overhang that
is complementary to the 3' dNTP overhang of each of the fragments.
Thus, for example, if the fragments produced in step (b) comprise a
3'dA overhang, an adaptor may comprise a 5' dT overhang.
[0260] In the method set out above, the adaptors comprise a means
for immobilising the DNA fragment. Immobilisation is not essential
to the present method but has the advantage that it reduces
experimental background. An adaptor may comprise any suitable means
for immobilising the fragments. For example, an adaptor may
comprise (typically at the end distal to the DNA fragment) one
member of a pair of binding molecules, wherein the binding
molecules in the pair bind each other, and wherein the other member
of the pair may be comprised in a suitable immobilisation matrix.
For example, the binding molecules in the pair may be biotin and
streptavidin. Biotinylated adaptors may be used, and the adaptored
fragment captured on a streptavidin matrix.
[0261] The first restriction enzyme cuts at a distance from its
recognition site. For example, the enzyme may cut downstream (3')
of its recognition site. The distance at which it cuts determines
the length of the snare sequence. Suitable lengths for the snare
sequence are described herein. Examples of enzymes which may be
used are MmeI. Other examples include GsuI, BpmI and isochizomers
thereof. Further examples include members of the family of
restriction enzymes known as type IIs that cut outside their
recognition site to one side. In the current description those
enzymes that cut on the 3' side are used and those which cut at
greatest distance preferred, typically 15-25 nt (reviewed in Gene
(1991) 100; 13-26). The nature of the double stranded cut
introduced is not crucial but in the methods described here a 3'
overhang is used. The method could be adapted to use both a 5'
overhang and a blunt cut. The nature of the snare could be altered
accordingly. For example, if an enzyme was used which gave an
asymmetric product with a longer 5' overhang, it would be
advantageous to turn the 5'overhang into the snare. Also depending
on how the recognition site (which is typically asymmetric) is
positioned in the adaptor enzymes could be used that introduced
cuts 5' of its recognition site.
[0262] The second restriction enzyme cuts in the adaptor sequence.
Examples of enzymes that can be used include Nt.Bbv.CI. This is
known as a nicking endonuclease and is used in this application as
it introduces a single stranded nick in the top strand to allow the
recovery of the snare. However the method can be adapted to use the
majority of commonly available restriction enzymes. Examples of
enzymes that could be used include those that generate 3' or 5'
overhangs or generate a blunt-ended break.
[0263] Once prepared, an n[snare] plasmid library can be tested for
bias in its construction. Typically this is done by isolating the
plasmids, digesting to release the insert, and further digesting
(e.g. with the first restriction enzyme) to isolate the monomer
sequences.
[0264] Creation of custom n[snare] plasmid libraries is described
in the present Examples. A library is prepared from an S.
cinnamoneous cinnamycin biosynthetic gene cluster in Example 2, and
a library is prepared from E. coli K12 genomic DNA in Example 4.
The invention relates to the libraries as prepared in these
Examples.
[0265] As described herein, a snare sequence may comprise
randomised nucleotide sequence.
[0266] Typically the snare comprises a randomised (or variable)
nucleotide sequence of "n" nucleotides in length. In general n may
range from 5-50, for example 10-50, for example 20-40 e.g. 25-35
e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
nucleotides. A library of plasmids may be prepared in which the
snares comprise randomised sequence of length n. Such a library is
a universal n[snare] library.
[0267] An oligonucleotide comprising the randomised sequence (and
optionally constant sequence as described) may be synthesised using
methods known in the art. Typically, in preparing snares for an
n[snare] universal library, oligonucleotides comprising all or
substantially all possible nucleotide sequences of length n are
prepared.
[0268] A nucleotide bias may be introduced into randomised (or
variable) region of the oligonucleotide. For example a GC bias may
be introduced if appropriate.
[0269] The monomer or test sequence of interest (B in FIG. 1) in
the method above may comprise an oligonucleotide prepared in this
way. An n[snare] plasmid may then be prepared as described.
[0270] Example 4 describes preparation of a universal n[snare]
library in which n=9. The invention relates to the library prepared
according to the method in Example 4.
[0271] In one aspect the invention relates to an n[snare] plasmid
which comprises a monomer and/or snare comprising a cis-regulatory
sequence or decoy sequence described herein and/or identified
according to the methods described herein, including any of the
n[snare] plasmids or plasmid libraries described herein and/or
prepared according to the methods herein, including the
Examples.
[0272] Host Cells
[0273] The invention also relates to a host cell or cells
comprising an n[snare] plasmid or plasmid library as described
herein. The invention further relates to a host cell or cells
comprising a decoy polynucleotide (decoy molecule) as described
herein. In particular, the invention relates to such a host cell(s)
which displays altered gene expression and/or phenotype, due to the
presence of the plasmid, or decoy polynucleotide, e.g. increased
production of a metabolite or fermentation product such as an
antibiotic or enzyme, increased resistance to a solvent, increased
sensitivity to one or more antibiotics, compared to the cell in the
absence of the plasmid/decoy molecule.
[0274] Typically, the plasmid or plasmid library or polynucleotide
has been introduced to the cell(s) by a suitable means, for
example, transformation, transfection or conjugation.
[0275] A host cell for use in the present methods may be
prokaryotic or eukaryotic. For example, a prokaryote such as a
bacterial cell may be used. A host may be for example, an
actinomycete such as a streptomyces species, e.g. S. coelicolor,
for example S. coelicolor A3 (2), (strain M145 or M600), S.
lividans, S. cinnamoneous, or E. coli. Other examples may include
other species of gram positive bacteria, such as those from the
group Actinobacteria, for example, bacteria from the genus
Mycobacterium, such as the pathogenic bacteria Mycobacterium
tuberculosaris, M. bovis, M. africanum, and M. microti; M. leprae.
A further example of a genus of gram positive bacteria is
Clostridium, which includes pathogenic bacteria such as Clostridium
difficile (a human pathogen), C. botulinum, C. perfingens and C.
tetani, as well as bacteria of potential industrial use such as C.
acetylburylictum, C. thermocellum and C. ljungdahlii. Other genera
of gram positive bacteria may include Bacillus, Listeria,
Staphylococcus, Clostridium. Corynebacterium, Streptococcus, and
Enterococcus. Host cells may also be gram negative bacteria, which
includes the genera Enterobacteriaceae which includes human
pathogens, such as Salmonella and Escherichia coli. Other examples
of genera of gram negative bacteria may include Pseudomonas,
Bordetella, Borrelia, Brucella Campylobacter, Francisella,
Haemophillus, Klebsiella, Neisseria, Proteobacteria, Rickettsia,
Vibrio, Yersina Moraxella, Helicobacter, Stenotrophomonas,
Bdellovibrio, acetic acid bacteria, Legionella, the cyanobacteria,
spirochaetes, green sulfur and green non-sulfur bacteria and many
others. Important gram negative pathogens include the cocci species
which cause a sexually transmitted disease (Neisserla gonorrhoeae),
a meningitis (Neisseria meningitidis), and respiratory symptoms
(Moraxella catarrhalis). Other Medically relevant Gram-negative
bacilli include a multitude of species. Some of them primarily
cause respiratory problems (Hemophilus influenzae, Klebsiella
pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa),
primarily urinary problems (Escherichia coli, Proteus mirabilis,
Enterobacter cloacae, Serratia marcescens), and primarily
gastrointestinal problems (Helicobacter pylori, Salmonella
enteritidis, Salmonella typhi). Host cells could also include
eukaryotes such as yeasts (examples of which may include: those
used for industrial production such as the genera Saccharomyces
(such as S. cerevisiae), Schizosacharromyces (S. pombe) and the
methyltrophic yeast genera Pichia [such as P. pastoris] and
Candida, also Hansensda polymorpha; pathogenic yeasts such as the
Candida genera [such as C. albicans and C. tropicalis], also the
Cryptococci genera [such as C. neoformans), fungi (which may
include: pathogenic fungi such as the genera Candida, Aspergillus
[such as A. fumigatus and A. flavus], Crptococcus, Histoplasma
[such as H. capsulatum], Pneumocystis [such as P. jirovecii] and
Stachybotyrus [such as S. chartarum]; fungi used in industrial
production such as the genera Aspergillus [in particular A. niger
and A. oryzae]) and members of the Neurospora genus, such as N.
crassa, plant cells and mammalian cells, avian cells, either in
cell culture or part of a tissue.
[0276] In general, a host cell is compatible with the plasmid
vector. For example, the cell is compatible with the plasmid origin
of replication. The cell is typically one in which the plasmid can
be stably maintained and replicated. The host cell is also
typically compatible with any detectible marker gene in the plasmid
so that the gene can be expressed in the cell and the cell can
thereby be screened for successful introduction and maintenance of
the plasmid.
[0277] A host cell may simply be intended for manipulation of a
plasmid or polynucleotide. Preferably such a strain is a laboratory
strain which can be easily manipulated and maintained under
laboratory conditions, e.g. E. coli.
[0278] Alternatively or additionally, a host cell may be used to
screen for cis-regulatory sequences using an n[snare] plasmid
library according to the invention and/or to test for decoy
function of a putative cis-regulatory sequence using an n[snare]
plasmid as described herein. The host cell may also be one in which
it is desired to alter gene expression using an n[snare] plasmid or
other decoy molecule as described herein.
[0279] In general a host cell comprises the cis-regulatory sequence
of interest, i.e. the cis-regulatory sequence that is being
screened for, or with which the decoy sequence introduced into the
cell is intended to compete. Typically, in the cell the
cis-regulatory sequence is operably linked to a gene or genes, the
expression of which can be detected, directly or indirectly.
Typically the cell comprises a promoter containing the
cis-regulatory sequence, operably linked to the gene(s). By
operably linked is meant that the cis-regulatory sequence and/or
promoter is linked to the gene or genes in such a way that the
sequence and/or promoter can function (under appropriate
conditions, e.g. presence of the requisite transcription factor(s))
to regulate expression of the gene(s). Thus, when bound by the
cognate transcription factor, the cis-regulatory sequence functions
to regulate (repress or activate) expression of the gene or genes.
The promoter may be for example, one which regulates a gene(s)
which encodes a phenotype of interest, such as any of the genes or
phenotypes described herein.
[0280] Functioning of the cis-regulatory sequence in the cell can
be determined by monitoring for expression of the linked gene(s).
This may be done by monitoring for expression of the gene directly,
or by monitoring for expression of a particular phenotype which is
associated with expression of the gene(s). For example, screening
for function may comprise screening for production of a given
metabolite or fermentation product, e.g. an antibiotic such as
cinnamycin, actinorhodin and/or undecylprodigiosin; for resistance
to a particular antibiotic e.g. vancomycin; or for tolerance to a
solvent such as butanol. Methods for screening are described
herein.
[0281] In one aspect the plasmid causes a change gene expression
and/or phenotype of a host cell, e.g. an increase in antibiotic
synthesis, a decrease in antibiotic resistance, or an increase in
solvent tolerance in the cell.
[0282] A host cell may comprise a cis-regulatory sequence (e.g. a
promoter containing the sequence) operably linked to its native
(cognate) gene(s), i.e. linked to the gene or genes the expression
of which the sequence (or promoter) regulates in its native
occurrence. Thus, for example, where a cis-regulatory sequence of
interest (or its native promoter) regulates genes(s) encoding
metabolite (e.g. antibiotic or enzyme) production, a host cell may
comprise a native producing cell. Where a cis-regulatory sequence
of interest (or its native promoter) regulates a gene or genes
encoding antibiotic resistance, a host cell may comprise a native
antibiotic resistant strain, e.g. a pathogen, or a clinical
isolate. Thus the cis-regulatory sequence in the cell may be
operably linked to a gene or genes, the expression of which is or
are regulated by that sequence in its natural context.
[0283] For example, as described in the present Examples, S.
coelicolor is a native producer of actinorhodin and
undecylprodigiosin. It has been reported that production of these
antibiotics requires binding of AsfR protein to a binding site
upstream of the asfS gene. S. coelicolor may therefore be used as a
host cell when screening snares for the presence of a sequence
comprising the AsfR binding site (or when introducing a decoy
sequence which competes with such sequence). Competition with the
native cellular AsfR binding site will result in reduced antibiotic
production.
[0284] Also as described in the Examples, S. cinnamoneous produces
cinnamycin antibiotic. S. cinnamoneous may be used as a host to
screen snares for the presence of cis-regulatory sequences in the
promoters of genes in the S. cinnamoneous cinnamycin biosynthetic
cluster (or when introducing a decoy which competes with such
sequence). Competition with the native cis-regulatory sequences can
be screened for by monitoring the production of cinnamycin in
transformed strains, as described herein.
[0285] Also as described, E. coli K12 comprises genes encoding
butanol tolerance. E. coli K12 cells may be used to screen snares
for the presence of cis-regulatory sequences in the promoters of
genes encoding the tolerance (or when using a decoy which competes
with such sequence). The presence of a competing sequence in the
transformed cells may be assayed for by determining viability of
cells in the presence of varying concentrations of butanol.
[0286] Alternatively, a host cell may comprise a model, e.g. a
bacterial model, of a given cellular phenotype, such as metabolite
production or antibiotic resistance. Such a model comprises the
cis-regulatory sequence of interest (e.g. a promoter containing the
sequence) operably linked to the native gene(s) but in a
heterologous cell. In other words, the cis-regulatory sequence (and
its native promoter) and the operably linked gene(s) are not
present endogeneously in the cell.
[0287] Typically, a model host cell comprises a bacterial species
or strain which is more easily manipulated under laboratory
conditions than a native strain, e.g. is non-pathogenic or more
susceptible to gene transfer. In general, in the absence of a
n[snare] plasmid or decoy molecule described herein, expression of
the gene(s) is regulated by the cis-regulatory sequence (e.g. in
its promoter). Thus the cell comprises the components necessary for
the cis-regulatory sequence to function in the normal way, e.g.
transcription factor to bind to the sequence and repress or
activate expression of the gene(s).
[0288] A model host cell may comprise a plasmid bearing the
cis-regulatory sequence and gene(s), which has been introduced to
the cell or the cis-regulatory sequence and gene(s) may be
integrated in the host genome. A model host may comprise a genomic
fragment which comprises the cis-regulatory sequence and gene(s),
wherein the fragment has been acquired from a native cell, e.g. by
horizontal transfer.
[0289] A model cell may have acquired genes conferring a given
phenotype of interest e.g. metabolite production, antibiotic
production, antibiotic resistance, solvent tolerance, for example,
a model strain may have acquired a particular phenotype by
horizontal transfer of genes encoding that phenotype, e.g.
antibiotic production and/or antibiotic resistance. Such a model
strain may be prepared without prior knowledge of the coding genes.
This is done by horizontal transfer of DNA from the native strain
(exhibiting the phenotype of interest) to the model strain and by
screening for gain of the phenotype.
[0290] Where the cells are to be host to a custom n[snare] library,
the cells may, for example, comprise the genomic DNA or genomic
fragment from which the library was derived (either endogeneously
or by gene acquisition).
[0291] Suitable host cells are known in the art and are described
herein in the present Examples. For example, Example 6 describes S.
coelicolor strain M600, which is a bacterial model of vancomyin
resistance.
[0292] Reporter Cells
[0293] A host cell may comprise a cis-regulatory sequence of
interest (or a promoter of interest comprising cis-regulatory
sequences) operably linked to a gene(s) the expression of which is
not regulated by that sequence or promoter in its native context (a
non-native gene). Such a gene is referred to as a reporter gene.
Typically, the reporter gene is operably linked to a promoter
sequence which comprises the cis-regulatory sequence of
interest.
[0294] Function of the cis-regulatory sequence can be determined by
determining expression of the reporter gene. Typically the reporter
gene is one whose expression can be easily detected and screened
for. For example, the reporter gene may encode a fluorescent
compound, or antibiotic resistance. Examples of reporter genes are
luciferase, or the neo gene encoding kanamycin resistance. Such
reporter cells are particularly useful where the cis-regulatory
sequences (or promoters) natively control expression of gene(s)
which have no easily scorable phenotype.
[0295] In one embodiment, the reporter gene is negatively regulated
by binding of a transcription factor (transcriptional repressor) to
the cis-regulatory sequence of interest. Such a host cell is
suitable for screening for cis-regulatory sequences which
negatively regulate (repress) expression of their cognate gene(s).
In an alternative embodiment, the reporter gene is positively
regulated by binding of a transcription factor (transcriptional
activator) to the cis-regulatory sequence. Such a host cell is
useful for screening for cis-regulatory sequence which positively
regulate expression of their cognate gene(s).
[0296] Any suitable host strain may be used in the reporter system.
Suitable strains are known in the art and are described herein.
Where appropriate, strains will have been chosen that contain no
genomic copy of the reporter gene. Similarly reporter strains can
be engineered by targeted deletion of genes used as reporters from
the genome prior to the introduction of the true reporter gene.
[0297] The reporter gene and cis-regulatory sequence (e.g. in a
promoter) may be plasmid borne. Alternatively, the reporter gene
and cis-regulatory sequence (or promoter) may be integrated in the
host genome. For example, the reporter system may have been
introduced to the host on an integrative plasmid, e.g. pSET152.
Where the system is plasmid borne, typically the plasmid comprises
a detectable marker gene that allows positive selection for the
plasmid in the host cells. For example, the detectable marker gene
may encode antibiotic resistance, e.g. the aac gene which encodes
resistance to apramycin.
[0298] The inventors have developed a generic reporter cell system
which may be used to screen for the presence of a cis-regulatory
sequence, for example in an n[snare] plasmid library or n[snare]
plasmid described herein. The screening cell and methods allows
screening for the presence of the cis-regulatory sequence on the
basis of cell viability. Expression of the reporter gene is
determinative for cell viability or survival under appropriate
culture conditions. Under the conditions of the assay, host cells
are only viable if a sequence has been introduced into the cell
which is competing with the cis-regulatory sequence of interest
(e.g. in the promoter of interest) for binding of transcription
factor. The system is referred to as a "dead or alive" screening
system. The system can be automated, and has the advantage that it
can be used to screen cells cultured in liquid media.
[0299] When the cis-regulatory sequence of interest functions to
repress expression of an operably linked gene, the reporter gene
typically encodes a product which is necessary for cell survival
under appropriate culture conditions. For example, the reporter
gene may encode antibiotic resistance such that when the host cell
is cultured in the presence of the antibiotic, cell survival is
only possible in the presence of a competing sequence which
inhibits binding of transcription factor to the cis-regulatory
sequence linked to the reporter and allows expression of the
reporter antibiotic gene. Suitable antibiotic resistance genes
include, for example, the kanamycin resistant gene neo. This allows
detection of cis-regulatory sequences that are bound by
transcriptional repressors. Other suitable genes are known in the
art.
[0300] In this case, a host cell typically comprises other
components which are necessary for the cis-regulatory sequence to
function. The cell typically comprises (e.g. expresses) the
transcription factor(s) necessary for binding to the cis-regulatory
sequence. The host cell may already comprise the cis-regulatory
sequence, e.g. a promoter comprising the cis-regulatory sequence.
The host cell may comprise a genomic fragment from which the
cis-regulatory sequence of interest is derived.
[0301] When the cis-regulatory sequence functions to activate
expression of an operably linked gene, the reporter gene encodes a
product which is lethal for the cell under appropriate culture
conditions. Such a gene is often referred to as a suicide reporter
gene. Cells are only viable (under appropriate culture conditions)
in the presence of a competing sequence which inhibits or prevents
transcription factor binding to the cis-regulatory sequence linked
to the reporter. An example of a suicide reporter gene is, for
example, the glucose kinase gene (glkA). This gene converts a
metabolite (2 deoxy glucose (DOG)) to a toxin. Thus, expression of
the glkA gene is lethal to the cell when cultured in the presence
of DOG. This allows detection of cis-regulatory sequences that are
bound by transcriptional activators. Other suitable genes are known
in the art.
[0302] Typically, in this system the host does not otherwise
express the reporter gene but comprises the components necessary
for the cis-regulatory sequence to function. The cell typically
comprises e.g. expresses, a transcription factor(s) necessary for
binding to the cis-regulatory sequence(s). In such a system, any
activity at the cis-regulatory sequence will be due to positive
transcription factors (activators) derived from the host cell
genome. By searching in this background, it is possible to find
upregulators of heterologous expression from the chromosome of the
host. By titrating these activators off the cis-regulatory sequence
linked to the reporter and onto a decoy sequence, e.g. an n[snare]
plasmid, expression of the reporter gene, e.g. glkA, is prevented
and the cells are viable even when grown in the presence of the DOG
metabolite.
[0303] The dead or alive reporter system can be adapted for use
with any suitable cis-regulatory sequence, such as any of those
described and/or identified herein. For example the cin7 promoter,
derived from the cinnamycin biosynthetic cluster in S. cinnamoneous
may be used as described in Example 3. The cin7 promoter may be
operably linked to the neo gene encoding kanamycin resistance as
described in the Examples. This may be comprised in an integrative
plasmid such as the pSET152 backbone used in the examples. The
plasmid also carries a suitable marker gene (the aac gene encoding
apramycin resistance). The plasmid may be introduced into or
integrated into an S. lividans host strain (1326) which
additionally carries the cinnamycin biosynthetic pathway. Such a
reporter cell is suitable for screening, for example, an n[snare]
plasmid library derived from the S. cinnamoneous genome or genomic
fragment comprising the cinnamycin biosynthetic cluster as
described herein.
[0304] Alternatively, the cin7 promoter may be operably linked to
the glkA reporter gene and in the same way integrated into an
integrative plasmid. In this case, a suitable host strain would be
the S. lividans TK24 strain which lacks the cinnamycin biosynthetic
cluster and also lacks the glkA gene.
[0305] In one aspect, the invention relates to the reporter cells
described and prepared according to the present Examples.
[0306] Methods for Identifying and Characterising Cis-Regulatory
Sequences
[0307] The principle of use of the present n[snare] plasmids is
illustrated in FIG. 2. If the snare sequence in the plasmid
comprises a transcription factor binding site that comprises and/or
competes for transcription factor binding with, a cis-regulatory
sequence in the cell into which the plasmid is introduced, this can
be detected as a change in expression of the gene which is
regulated by that cis-regulatory sequence in the cell. Thus
n[snare] plasmids can be used to identify cis-regulatory and decoy
sequences. The plasmid can also be used with a snare comprising a
known decoy sequence, to disrupt gene expression in the cell.
[0308] Thus the n[snare] plasmids of the invention can be used in a
number for a number of different purposes. As above, a library of
n[snare] plasmids can be used to screen for putative cis-regulatory
sequences, e.g. in a fragment of genomic DNA using a custom
n[snare] library. An n[snare] plasmid can be used to test a
particular sequence for decoy function. In any of these methods, a
snare sequence is identified as comprising a cis-regulatory
sequence or a sequence that competes with a cis-regulatory sequence
for transcription factor binding, if the snare in the n[snare]
plasmid is able to titrate transcription factor from the
cis-regulatory sequence present in the host (as determined by an
alteration in gene expression or host cell phenotype). Thus, the
sequences identified using the methods may also act as decoy
sequences, and in one aspect, the methods can be considered as
methods of identifying decoy sequences and molecules. An n[snare]
plasmid comprising a decoy sequence can be used to modulate gene
expression and/or phenotype in a host cell.
[0309] Thus in one aspect the invention provides methods for
identifying and/or characterising cis-regulatory and decoy
sequences. In general such a method comprises: [0310] 1. providing
an n[snare] plasmid or n[snare] plasmid library as described
herein; [0311] 2. introducing the n[snare] plasmid or plasmid
library into a host cell(s) wherein the host cell comprises the
cis-regulatory sequence of interest operably linked to a gene or
genes, the expression of which can be determined directly or
indirectly; and [0312] 3. determining the expression of the gene
(or genes), or alteration of phenotype, in the presence and absence
of the n[snare] plasmid or plasmid library.
[0313] An n[snare] plasmid or n[snare] plasmid library for used in
the method may be as described herein. The snare in each plasmid
comprises the sequence to be tested for competing function.
[0314] In general, a host cell comprises a cis-regulatory element
of interest (e.g. a promoter comprising the cis-regulatory element)
operably linked to a gene or genes, the expression of which can be
directly or indirectly monitored or determined. For example, the
expression may be monitored by determining expression of the
particular gene or genes or by monitoring for a particular
phenotype encoded by the gene or genes.
[0315] Suitable host cells for use in the present methods have been
described herein and include, for example, native strains, clinical
isolates, laboratory models and reporter cells. The "dead or alive"
reporter screening system described herein may be used.
[0316] In Examples 1 and 2 herein it is demonstrated that the
decoy-oligonucleotide approach can be adapted to a plasmid format,
including confirmation that use of a library to identify
cis-regulators of a defined sequence is achievable. A possible
limitation of this iteration of the n[snare] library-based approach
is that it may not be optimally suited to regulators of subtle or
complicated phenotypic changes. In many circumstances the
phenotypic alteration may not be readily detectable and may need a
complicated system of scoring, such as quantifying metabolite
production by chromatographic analysis. There would be obvious
advantage to the creation of a reporter system where the phenotype
scoring was standardized, allowing n[snare] library screening to be
accelerated so that it can be conducted at high-throughput.
[0317] A possible technical limitation of the approach taken in
Example 2, namely of screening all the members of a library for
change of phenotype, is that the need to physically plate or
culture all the members of entire libraries places a practical
limitation on how many colonies can be screened, and hence the
amount of DNA that can be surveyed. It is not feasible to survey
large genomic fragments that would generate libraries with >100
000 members, which equates approximately to 25 kb of sequence. As
described below, both the reporter genes used in a system developed
by the inventors confer viability to cells that have an n[snare]
plasmid containing a candidate cis-regulatory element. Hence,
selection is by viability allowing rapid enrichment of candidate
sequences in liquid culture simply by letting the majority of cells
that do not contain a candidate plasmid perish.
[0318] Hence the inventors have adapted the n[snare] library
approach described herein to address these issues by developing
generic reporter-based assay systems. In this iteration, negative
and positive regulators are detected by their effect on reporter
genes encoding antibiotic resistance (FIG. 7) or metabolite
sensitivity (suicide reporters) as shown in FIG. 8. The target
promoters are introduced into the reporter cassettes to allow
detection of either negative regulation (transcriptional
repressors) or positive regulation (transcriptional activators).
This approach solves the problem of needing a scoreable phenotype
as under normal circumstances each reporter causes cell death, it
is only when an n[snare] plasmid from the library relieves this by
titrating off the transcription factor that the cells can grow.
Hence selection for both reporters relies on cell survival, which
greatly expedites the screening process. Another feature of the
system is that such dead-or-alive screens can be readily automated
as a basis of rapid, comprehensive or high-throughput screens. It
is also possible to identify regulatory sequences controlling
promoters which in their natural context have no easily scoreable
phenotype.
[0319] The plasmid or plasmid library is introduced to host cells
in the present methods by any suitable means. Suitable means are
known in the art. If the n[snare] plasmid is conjugative, the
plasmid may be introduced by conjugation. Other means such as
transfection/transformation may also be used.
[0320] Typically host cells are then monitored for stable
propagation of the plasmid by selecting for expression of a
detectable marker gene on the plasmid as described herein, e.g. an
antibiotic resistance gene. Cells are cultured under conditions
which select for cells expressing the particular marker, e.g. in
the presence of the antibiotic.
[0321] Preferably the method comprises the use of one or more
suitable controls. For example, such controls include host cells
untreated with plasmid, host cells into which has been introduced
an empty plasmid vector, and host cells into which has been
introduced an n[snare] plasmid comprising a scrambled putative
cis-regulatory sequence.
[0322] Once the plasmids have been introduced into the cells, the
cells are screened for expression of the gene or genes operably
linked to the cis-regulatory sequence (or promoter) of interest.
Expression in the presence of the snare sequence (in the n[snare]
plasmid) is compared to expression in the absence of the snare.
Clones which produce an alteration in expression of the test
gene(s) (e.g. an alteration of cell phenotype) are selected as
likely to comprise a cis-regulatory sequence. DNA from these clones
is isolated, and the putative cis-regulatory sequence (or decoy
sequence) isolated.
[0323] Screening for an alteration in gene expression may be
carried out by any suitable method. Screening may comprise
detecting or measuring the expression product of the gene(s), e.g.
by assaying for the function of the gene product, or may comprise
determining a change in a host cell phenotype associated with
expression of the gene(s).
[0324] For example, changes in expression of a gene(s) encoding
production of a metabolite, e.g. an antibiotic, may be monitored by
determining expression of the given metabolite, e.g. the amount (or
presence of absence) of metabolite or function of the
metabolite.
[0325] For example, actinorhodin and undecylprodigiosin are
pigmented antibiotics. Actinorhodin is blue and undecylprodigiosin
is red. Thus expression of these antibiotics can be easily
monitored by colorimetric techniques.
[0326] Production of some metabolites or antibiotics can be
monitored by using indicator bacterial strains and suitable plate
assays. For example, production of cinnamycin can be detected by a
plate assay using the indicator strain Bacillus subtilis, and
suitable media e.g. solid R2YE agar. Cells are typically cultured
on agar plates which have been seeded with the indicator strain.
The expression of the antibiotic is determined by the extent of
reaction, e.g. killing, of the indicator strain, e.g. by the
diameter of a halo on an agar plate.
[0327] Expression of antibiotic resistance gene(s) or phenotype,
e.g. vancomycin resistance, kanamycin resistance, may be determined
by culturing host cells in the presence of the antibiotic and
determining sensitivity to the antibiotic. Typically cells are also
cultured in the absence of the antibiotic as a control.
[0328] Similarly, genes encoding solvent tolerance, e.g. butanol
tolerance, may be monitored by culturing the cells in the presence
of the solvent (again typically a control is carried our in which
cells are cultured in the absence of the solvent).
[0329] In some instances, screening for expression of the relevant
gene(s) comprises determining host cell viability under suitable
culture conditions. For example, when the gene or genes encode
antibiotic resistance, screening may comprise culturing cells in
the presence of the antibiotic and determining whether the cells
are viable. In a screen comprising the "dead or alive" reporter
host cells, under the given culture conditions, cells are only
viable if the n[snare] plasmid introduced into the cells comprises
a sequence which can compete with the cis-regulatory sequence of
interest. Thus screening the cells comprises culturing the cells
under conditions in which expression of the reporter gene(s) (the
gene(s) operably linked to the cis-regulatory sequence of interest)
is determinative for host cell viability, and isolating viable
cells.
[0330] The present methods may comprise culture of the host cells
in liquid media, as described herein, e.g. if the cell phenotype
which is being determined is cell viability. This may have the
advantaged that only a small number of cells remain to be
analysed.
[0331] Screening may comprise use of DNA subtraction techniques as
described herein. This may be particularly useful where expression
of the relevant gene(s) affects cell viability (under suitable
culture conditions).
[0332] Typically a DNA subtraction step comprises subtracting a
population of DNA from cells with a given phenotype and from cells
without the phenotype.
[0333] For example, in some instances, introduction of a sequence
comprising a competing cis-regulatory sequence, will result in host
cells becoming non-viable under suitable culture conditions. This
may be the case if, for example, the cis-regulatory sequence of
interest activates expression of an antibiotic resistance gene(s)
or represses expression of a lethal gene (expression product lethal
under particular culture condition). When a competing
cis-regulatory sequence is introduced into the cells, e.g. in a
snare of an n[snare] plasmid, the competing sequence titrates
transcription factor and results in reduced expression of the
antibiotic resistance gene or expression of the lethality gene.
[0334] Transformed cells are typically cultured (a) under
conditions in which cells with disrupted expression from the
cis-regulatory sequence will be non-viable; and (b) under
conditions in which the cells will be viable. Populations of DNA
are isolated from the two cultures (typically after isolation
and/or amplification of the introduced DNA, e.g. the
n[snare]plasmid or plasmid insert), and subtracted (e.g. by
hybridisation). It is then possible to determine the DNA that is
missing from the cells in culture (a) and which comprises the
likely competing cis-regulatory sequence.
[0335] For example, if the phenotype under investigation is
antibiotic resistance, a cis-regulatory sequence of interest
introduced into a host cell, e.g. in a snare of an n[snare]
plasmid, may restore sensitivity to the antibiotic. Transformed
cells are cultured (a) in the presence of antibiotic; and (b) in
the absence of antibiotic. Cells containing the cis-regulatory
sequence of interest will die in the presence of the antibiotic.
DNA is isolated from both cultures, and nucleic acid comprising the
snare sequences isolated, e.g. by PCR amplification. By subtracting
the populations of snare sequences, it is possible to isolate those
missing from the antibiotic treated samples.
[0336] In one aspect, the enriched population of snare sequences
may then be recloned and the selection process repeated.
[0337] Once cells displaying altered gene expression or phenotype
have been isolated, DNA from these cells is isolated. Typically,
the DNA which was introduced into the cells, e.g. the n[snare]
plasmid DNA and/or the plasmid insert, is isolated. The snare which
caused the altered expression, and which comprises the likely
cis-regulatory (or decoy) sequence can then be determined. This may
be done, for example, by PCR amplification.
[0338] When the present method comprises use of an n[snare] plasmid
library, the method may additionally comprise one or more further
steps.
[0339] For example, the method may comprise a library hybridisation
step. Due to the complexity of libraries used in these sorts of
enrichment procedures it is rare to generate samples that entirely
consist of the desired sequences. Generally there is a background
of false negatives within the enriched sample that need be rejected
and not carried forward for further analysis. The library
hybridization strategy is designed to do so by detecting n[snare]
plasmids that are common to all independent repeats of the
screening process, the logic being that if plasmids carrying the
same sequence are detected in independent repeats at an occurrence
above background then those should be the ones carried forward for
further analysis. An overview of the process is given in FIG.
9.
[0340] Typically when using the library hybridization strategy, x
independent repeats of the above screening method (comprising steps
(1) (2) and (3)) are carried out. For example, x may be 2, 3, 4, 5,
6, 7 or 8.
[0341] In each repeat, clones producing an alteration in gene
expression/phenotype are selected as described. Plasmid DNA is
isolated from the clones and labelled to create a pooled probe
sample (P). For example, the plasmid DNA may be labelled by random
priming PCR, e.g. using a DIG-labelled dUTP molecule as in the
present Examples.
[0342] The clones are also plated onto suitable media at a
concentration which allows individual colonies to be distinguished.
Typically at this stage, a sample of each colony is taken and
further cultured to provide a source of plasmid DNA if necessary at
a later stage.
[0343] Total DNA is extracted from each colony and immobilised on a
suitable matrix, e.g. a nylon membrane, for example by
hybridisation. DNA from each colony is at an addressable position
on the matrix. Each matrix is then separately hybridised with each
of the probe sets.
[0344] Samples which are common to more than one repeat are
typically selected for further analysis.
[0345] Thus for example, if x=4, four probe sets and four matrices,
e.g. 4 nylon membranes are prepared. Each of the 4 probe sets, P1,
P2, P3 and P4 is hybridised to each of the 4 filters, F1, F2, F3
and F4. Hybridisation of P1 with F1 produces a chromatogram where
every sample is detected. F2, F3, or F4 samples which hybridise
with P1 would be potential candidates for further analysis. The
more probe samples that a colony hybridises with, the stronger that
clone is as a candidate.
[0346] Self hybridisation (e.g. P1 with F1) may also be use to
detect false positives in a screen.
[0347] The method of the invention may further comprise repeating
the selection process one or more times, so that selection is
iterative. Thus, clones selected from a first screen may be used to
transform the host cells again and the screen is repeated.
[0348] The n[snare] library approach described herein could be used
to identify cis-regulatory sequences that affect bacterial
phenotypes with important medical consequences, an example being
prevention of induction of antibiotic resistance mechanisms in
pathogenic bacteria infecting humans (FIG. 6). The schematic in
this figure shows how a library of n[snare]plasmids is used to
identify cis-regulatory sequences controlling antibiotic resistance
when nothing is known about the genetic pathway of the mechanism.
In this example the genes conferring antibiotic resistance are
moved into a convenient bacterial host, such as S. coelicolor or E.
coli, by horizontal gene transfer to create a convenient bacterial
model. This can be done without prior knowledge of what the genes
are by screening for the gain of antibiotic resistance. A similar
mechanism will have moved the same resistance genes into the
pathogenic strains detected in the clinic. Libraries of n[snare]
plasmids are created from the genomic fragment carrying the
resistance genes, the entire genome of the naturally resistant or
clinical isolate, or from random oligonucleotides to create
universal libraries conceivably containing every possible
cis-regulatory sequence. Such libraries are introduced into the
bacterial model and the transformant screened for increased
susceptibility to the targeted antibiotic. This is either
accomplished by direct scoring of the phenotype or by using
established DNA-subtraction techniques. Depending on the complexity
of the system, the process is performed singly or iteratively to
identify a single cis-regulatory sequence or cocktail of such
sequences, which is then used to synthesize or manufacture
corresponding TFDs. These are then validated on either the
resistant or clinical isolates, before proceeding to an appropriate
animal model, such as a mouse model, where the efficacy of the
decoys is tested by treating an animal infected with a pathogenic
strain which is resistant to treatment with antibiotic alone.
[0349] As stated, the biology of the genome, the pattern of gene
expression and timing of replication, is primarily controlled by
DNA-protein interactions, and mapping these interactions may be a
prerequisite to the attempts to control the genes. Microarray
analysis can produce a survey of entire genomes to identify which
genes are expressed, but to date no technology exists to identify,
on a similar scale, the proteins (trans-regulators) and their
cognate binding sites (cis-regulatory elements) that determine the
pattern of expression. Example 4 herein and the embodiment of the
invention described therein addresses this deficiency and develops
our current technology to form a generic tool capable of rapidly
identifying the cis-regulatory elements throughout the genome, to
delineate genetic networks and assert control over them. The method
according to this aspect of the invention identifies regulatory
elements throughout the genome which control expression of targeted
genes. As a fast and generic system for delineating regulatory
networks, the tool has the potential to produce gold-standard data
to support the drive towards systems biology, and utility in
defining regions for knowledge-based genetic engineering and
accelerate the mapping of disease-causing genetic variation.
[0350] In some instances there is a priori knowledge of the genetic
machinery underpinning the targeted phenotype, e.g. known promoters
within the cinnamycin cluster. More commonly, little information is
known about the genetic networks and their regulation underpinning
targeted phenotypes. For example many mechanisms of antibiotic
resistance amongst pathogenic infections appearing in clinics are
not understood at the genetic level, and this fact presents a
serious barrier to the development of treatments to solve this
problem. Likewise, in the context of industrial biotechnology, a
technique which could favourably alter complex characteristics of
bacteria without need of prior knowledge of genetic determinants
would be valuable. An example of this would be engineering a
bacterium to be solvent tolerant to improve its efficiency in
fermenting sugars in the process of biofuel production. Examples of
both clinical and industrial applications of the instant technology
are given herein.
[0351] To demonstrate the efficacy of this approach to detect
cis-regulatory elements without prior knowledge of a genetic
pathway, the inventors used n[snare] libraries to increase butanol
tolerance in Eschericihia coli (Example 4). One of the libraries
was derived from the entire E. coli genome using similar techniques
as described in Example 2. The second library was a `universal
n[snare]` library conceivably consisting of direct copies of every
possible 9 nucleotide sequence, or any such length desired. The
universal n[snare] library is made using similar methods to that
described in Example 2 for the creation of a single AfsR
n[snare]plasmid with the exception that instead of having an
oligonucleotide sequenced containing a central section with a
defined cis-regulatory sequence, random sequence is inserted
instead (and at this stage the length of the sequence can be
controlled). The procedure for using such n[snare] libraries to
detect novel cis-regulators of butanol tolerance is similar to that
described in Example 3 in the sense that the targeted phenotype is
scored by measuring viability against increasing concentration of
solvent.
[0352] n[snare] plasmid libraries as described and/or prepared
according to the methods herein, may also be used to identify
cis-regulatory sequences by screening the library directly for
binding by one or more transcription factors.
[0353] The present methods to identify and characterise
cis-regulatory elements may further comprise the use of a mapping
procedure using a combination of footprinting and exonuclease as
described herein.
[0354] Methods for Mapping Boundaries of Cis-Regulatory
Sequences
[0355] In one aspect the invention provides a method for mapping
the boundaries of protein binding sites in DNA. This method may be
used for more precisely mapping the boundaries of such binding
sites.
[0356] The method may be used to map the boundaries of
cis-regulatory sequences. The method may be used in combination
with the method described herein for identifying and characterising
cis-regulatory sequences, e.g. a method using an n[snare] plasmid
or a plasmid library as described herein. By providing more precise
sequence information about cis-regulatory elements and thus the
binding sites for protein regulators such as transcription factors,
the method is useful for designing decoy oligonucleotides which,
when introduced into a suitable cell, will compete with a
cis-regulatory sequence in the cell for binding of the protein
regulator.
[0357] The present method uses a combination of an enzyme or
chemical agent with non-specific DNA nicking capacity, (e.g. DNAse
I) and a 5'-3' exonuclease enzyme (e.g. T7 exonuclease) in a
modified footprinting protocol. Alternatives to DNaseI would
include treatment of the cells with potassium permanganate or free
radicals generated by hydroxides. Another example of a suitable
exonuclease that could be used would be lambda exonuclease. The
protocol is designed to map the 5' boundaries of the protein-DNA
complex on each strand of the DNA and hence define the protected
region of DNA within the complex, comprising the cis-regulatory
element. The DNAse I introduces nicks into the DNA surrounding the
complexes. These serve as substrates for the 5'-3' exonuclease
activity of the 5'-3' exonuclease enzyme. The combined action of
the two enzymes demarcates the 5' boundaries of the protein-DNA
complex on each strand of DNA. Thus the present method has the
advantage that it can detect all of the boundaries of DNA-protein
complexes in a given region, e.g. a promoter region, not just those
closest to restriction sites.
[0358] The principle of the present mapping method is illustrated
in FIG. 13. Typically the method comprises: [0359] 1. providing a
protein-DNA complex; [0360] 2. carrying out a digestion with [0361]
a. an enzyme having non specific DNA nicking ability e.g. DNaseI,
[0362] b. a 5'-3' exonuclease (T7 exonuclease); [0363] c.
optionally, a restriction enzyme that cuts a short distance
upstream of the likely position of the DNA-protein complex; in the
case of the actIIorf4 gene this was determined to be SacI; and
[0364] 3. determining the position of the 5' deletions generated in
each DNA strand in (2) relative to a known fixed point on the DNA
strand.
[0365] The protein-DNA complex may be any complex of interest. In
one example, the method is used to map the boundaries of
protein-DNA complexes in a DNA promoter region, e.g. of protein-DNA
complexes at cis-regulatory sequences. In one example, the
protein-DNA complex may be a transcription factor-DNA complex which
regulates expression of one or more antibiotic resistance genes in
a prokaryote or eukaryote. In another example, the method may be
used to map protein-DNA binding sites in the S. coelicolor
actII-orf4 promoter as in the present Examples.
[0366] Mapping may be carried out during a transcriptional state of
interest e.g. during repression if the cis-regulatory element of
interest is involved in repression i.e. bound by a transcriptional
repressor, or during active expression from a promoter if the
cis-regulatory sequence of interest is bound by a transcriptional
activator.
[0367] Thus, for example, in one aspect, mapping may be carried out
in vivo (e.g. using freshly harvested cells) at a stage when gene
expression in the cells is known to be repressed or activated.
Typically cells are cultured and isolated at the appropriate
transcriptional stage. Cells may be permeabilized with detergents
which allow enzymes to enter the cells. Thus, if the protein-DNA
complexes to be mapped function to repress expression of a given
gene(s) or phenotype, the protein-DNA complexes are tested at a
point when expression of the gene(s) or phenotype is repressed.
Conversely, if the protein-DNA complexes to be mapped function to
activate expression of a given gene(s) or phenotype, the
protein-DNA complexes are tested at a point when expression of the
gene(s) or phenotype is active. Typically, cells can be monitored
for expression of the given gene(s) or phenotype, and isolated at
the appropriate stage.
[0368] For example, if the cis-regulatory sequence of interest (in
the protein-DNA complex) has a role in repression of antibiotic
production and antibiotic production is known to occur late in cell
growth then the producing cells are cultured and harvested prior to
this late stage of growth.
[0369] Cells may be prokaryotic or eukaryotic. In one example, the
cells are prokaryotic, e.g. bacterial cells. For example, an
actinomycete such as a streptomyces species, e.g. S. coelicolor,
for example S. coelicolor A3 (2), (strain M145 or M600), S.
lividans, S. cinnamoneous, or E. coli may be used.
[0370] Typically, the amount of non-specific enzyme (DNaseI) and
exonuclease (e.g. T7) to be used in the method is determined
empirically, as describe herein in the present Examples.
[0371] Use of a restriction enzyme (c) which cuts at a restriction
site upstream of the likely position of the DNA-protein complex
produces a standard 5' end for all of the complexes, to which
oligonucleotides can be annealed in subsequent capture and
amplification steps.
[0372] Once digestion in (2) is complete, the digested nucleic acid
is recovered. Digestion creates 5' deletions on each DNA strand. By
determining the position of these 5' deletions on each strand, e.g.
relative to a known fixed point on the strand, it is possible to
precisely map the protein binding site on the DNA.
[0373] One way of determining the position of the 5' deletions on
each strand is as follows. Once recovered, the digested DNA is
denatured either by heating or treatment with basic solutions, such
as 1M NaOH, and hybridised to a strand of complementary DNA
comprising the binding site(s) of interest. For example, the
complementary DNA may comprise a fragment of a promoter (containing
the cis-regulatory sequence(s) of interest). A PCR fragment of the
promoter can be used. In general the complementary DNA strand
comprises a linker at one end. An amplification reaction can then
be carried out, e.g. PCR, using a labelled primer that binds to the
promoter or linker.
[0374] The sizes of the labelled amplification products are then
determined, e.g. by PAGE, or other methods such as capillary
electrophoresis. In general, although the precise boundaries of the
protein-DNA complex are not known, the approximate position, and
hence the sequence of the DNA region comprising the protein binding
site of interest can be determined from this data. For example, as
the sequence of the promoter region will be known, a comparison of
the size of the labelled fragments, and the position of the primer
binding site with the sequence of the DNA region comprising the
protein binding site(s), will make it is possible to determine the
precise position in the DNA sequence of the 5' boundary of the
protein-DNA binding complex.
[0375] Boundaries on the opposite DNA strand are mapped in the same
way, but using a DNA strand with a linker at the opposite end.
[0376] Typically, the complementary DNA strand is immobilised. For
example, the linker may allow immobilisation to a solid matrix. For
example, the linker may be biotinylated for immobilisation to a
streptavidin matrix. Immobilisation has the advantage that the
digested DNA strands of interest can be easily isolated from the
total digested DNA sample.
[0377] In one aspect the invention relates to a method for
identifying a cis-acting regulator of gene expression of a
prokaryotic or eukaryotic gene, which comprises either or both:
[0378] (a) conducting mapping of protected nucleic acid
sequences;
[0379] (b) providing a library of n[snare] molecules wherein said
library contains sequences representing all possible regulatory
sequences from the genome of said prokaryote or said eukaryote, and
either (i) identifying factors which bind to said library or (ii)
introducing said library of n[snare] molecules into an organism
which can indicate differential activation or suppression of a
target gene as compared to when said n[snare] molecules are not
introduced. The method may be conducted in vivo. Where the method
is in vivo, it may be conducted with a bacterium and may comprise
contacting the bacterium with effective amounts of DNase 1 and T7
exonuclease such that regulatory sequences protected by
transcription factors remain intact while the remainder of the
genome of said bacterium is destroyed.
[0380] Once identified according to the methods herein,
cis-regulatory sequences may be used in screening assays to
identify transcription factors.
[0381] Methods for Modulating Gene Expression and/or Phenotype
[0382] The n[snare] plasmids and methods herein may be used to
identify and characterise sequences which compete with a given
cis-regulatory sequence in a cell for binding to the cognate
transcription factor.
[0383] Such sequences may be used to prepare decoy sequences. A
decoy sequence mimics the native binding site (cis-regulatory
sequence) for a regulatory protein (e.g. transcription factor).
When introduced into suitable host cells comprising the
cis-regulatory sequence (by a method described herein or
otherwise), the decoy sequence competes with the cis-regulatory
sequence in the cell for binding to the cognate transcription
factor.
[0384] When such competition occurs, there is a concomitant
alteration in expression of a gene(s) whose expression is regulated
by the cis-regulatory sequence. This may cause a modulation in cell
phenotype, e.g. antibiotic production, antibiotic resistance,
solvent tolerance, as described herein.
[0385] It will be appreciated that an n[snare] plasmid which causes
an alteration in gene expression or phenotype according to the
methods described herein may be used as a decoy molecule in the
present methods. A snare sequence identified as competing with a
cis-regulatory sequence for transcription factor binding according
to the methods described herein may be used as a decoy
sequence.
[0386] Accordingly, in one aspect the invention relates to methods
for modulating gene expression and or phenotype in a cell,
comprising use of decoy sequences.
[0387] In general, a method for modulating expression of a gene or
genes according to the invention comprises: [0388] (a) providing a
polynucleotide comprising a binding site for a transcription factor
(a decoy sequence); and [0389] (b) introducing the polynucleotide
into a cell, wherein the cell comprises the gene or genes operably
linked to a cis-regulatory sequence which comprises the
transcription factor binding site or which competes with the
transcription factor binding site for binding of transcription
factor.
[0390] Generally, the decoy sequence (transcription factor binding
site) in the polynucleotide is not operably linked to a gene. The
transcription factor binding site may be isolated from any other
elements of a cognate promoter.
[0391] The polynucleotide comprising the decoy sequence may be
referred to as a decoy polynucleotide.
[0392] The decoy polynucleotide may comprise a plasmid vector. For
example the decoy polynucleotide may comprise an n[snare] plasmid
as described herein and/or prepared according to a method described
herein.
[0393] The decoy polynucleotide may comprise more than one copy of
the decoy sequence. The polynucleotide may comprise a multimeric
molecule comprising multiple copies of the decoy sequence. For
example, from 1 to 1000 copies. Typically there are two or more
copies, for example, 2-1000 copies, e.g. at least 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 or 900
copies. For example, there may be 10-200, 10-150, 20-120, 20-100,
30-100, 30-80, 30-50, 30-40 copies. For example, there may be 30
copies of the decoy sequence. Typically there are multiple copies
of the decoy, for example, multiple direct repeats of the decoy
sequence.
[0394] The decoy polynucleotide may comprise additional sequence to
the decoy sequence. Typically the additional sequence results in
increased resistance to degradation of the decoy sequence due to
the action of exo- and/or endonucleases. The decoy polynucleotide
may comprise at least one element of secondary structure. Typically
this secondary structure results in increased resistance to
degradation of the decoy sequence due to the action of exo- and/or
endonucleases. The decoy polynucleotide may comprise modified bases
or sugars to confer greater nuclease resistance. The decoy
polynucleotide may comprise 2' OH nucleotides or amines at the
termini of the polynucleotide to reduce or inhibit exonuclease
activity. In one aspect, the decoy polynucleotide may comprise a
linear oligonucleotide. The decoy polynucleotide may comprise
circular double stranded DNA (a so-called dumbbell structure). In
one aspect, the decoy polynucleotide may comprise a cholesterol
modification at one or at each 5' end of the molecule.
[0395] The decoy polynucleotide may comprise any one or more of the
above features in any suitable combination.
[0396] When introduced into a suitable host cell, a decoy sequence
in a decoy polynucleotide is able to compete with a cis-regulatory
sequence in the cell for binding of the transcription factor which
binds to the endogenous cis-regulatory sequence. Sequences can be
screened for this function by the methods described herein. The cis
regulatory sequence with which the decoy sequence competes may be
any of the cis-regulatory sequences described herein and/or
identified according to the methods herein.
[0397] For example, the cis-regulatory sequence may be one which
regulates expression of a gene or genes which have a role in
metabolite production, e.g. where the metabolite is an antibiotic
(e.g. actinorhodin, undecylprodigiosin or cinnamycin), enzyme or
pharmaceutical. Metabolite production may be increased in the
cell.
[0398] The cis-regulatory sequence may be one which regulates
expression of a gene or genes which have a role in determining
antibiotic resistance. Antibiotic resistance in the cell may be
decreased. The cis-regulatory sequence may be one which regulates
expression of a gene or genes which have a role in determining
solvent (e.g. butanol) tolerance. Solvent tolerance in the cell may
be increased.
[0399] For example, the cis regulatory sequence may comprise
sequence encoding a binding site for AfsR protein (a pleitropic
regulator of antibiotic synthesis in S. coelicolor). An AfsrR
binding site is shown in bold in SEQ ID NO: 1 (Example 1).
[0400] In another example a cis-regulatory sequence may be one in
the S. coelicolor actII-orf4 promoter, for example a repressor
cis-regulatory sequence. For example a cis-regulatory sequence may
comprise sequence A24.1, A24.2, A24.3, A24.4. or A24.5 identified
herein.
[0401] In a further example, a cis-regulatory sequence may comprise
a binding site for the VanR transcription factor, for example, a
VanR binding site located in the vanH promoter of, for example,
Entercoccus faecium or S. coelicolor. An example of a 30 bp VanR
binding site is shown in SEQ ID NO: 21 and a further example is in
SEQ ID NO:26.
[0402] A cis-regulatory sequence may be one which functions to
regulate expression of a gene or genes encoding a particular
function or phenotype of interest, e.g. production of a
metabolite(s) such as an antibiotic (e.g. cinnamycin, actinorhodin,
undecylprodigiosin), tolerance to a particular solvent(s) or
toxin(s), resistance to a particular antibiotic(s), or any other
function or phenotype of interest. Typically the sequence will be
located in the promoter region of such gene(s).
[0403] For example, a cis-regulatory sequence may regulate
expression of cinnamycin biosynthetic genes in S. cinnamoneus, e.g.
a cis-regulatory sequence from the cin7 promoter.
[0404] A cis-regulatory sequence may regulate expression of a
gene(s) encoding solvent tolerance, e.g. in a prokaryote, e.g.
butanol tolerance in E. coli K12.
[0405] A cis-regulatory sequence may be one which regulates
expression of prokaryotic antibiotic resistance gene(s), typically
a cis-regulatory sequence from the promoter of a gene(s) encoding
antibiotic resistance. Any antibiotic resistance of interest may be
targeted. For example, antibiotic resistance to: the class of
antibiotics known as aminoglycosides (such a kanamycin); the
carbapenems (such as meropenem); the cephalosporins (such as
cefepime); the glycopeptides (such as vancomycin and daptomycin);
the penicillins such an ampicillin, carbenicillin and penicillin);
the polypeptide antibiotics (such as polymixcin B); the quinolines
(such a levaquin); the sulfonamides (such a Bactrim); the
tetracyclines (such as tetracycline); and variously,
chloramphenicol, rifampicin and Zyvox.
[0406] In one aspect, a gene or genes encoding resistance to an
antibiotic encode proteins which provide resistance to a specific
antibiotic or, in some cases, a class of antibiotic, such as the
antibiotics and classes listed above. Such antibiotic resistance
genes may encode proteins which target a specific mechanism or
structure of the antibiotic or class of antibiotics. Often such
resistance genes are acquired by a bacterium after exposure to the
antibiotic.
[0407] In one aspect, antibiotic resistance genes may include one
or more of the following genes or types of genes:
[0408] The vanHAX operon genes, regulated by the VanR transcription
factor, which occur in a number of bacteria including Enterococcus
(e.g. E. faecalis, E. faecium), Staphylococcus (e.g. S. aureus)
(Courvalin (2006) Clin. Infect. Dis. 42, S25) and has been reported
as rare occurrences in other pathogenic bacteria (Werner (2008)
Future Microbiol. 3, 547). These genes when expressed provide VanA
type resistance to the antibiotic vancomycin. A decoy sequence
targeting the vanHAX genes comprises a native VanR binding site
(such as that in SEQ ID NO: 21 or SEQ ID NO: 26) or a variant of a
native site, which competes with the native site for VanR binding
in the cell of interest.
[0409] Those genes encoding beta-lactamases, which cause resistance
to the beta-lactam class of antibiotics (in particular the
penicillins, cephalosporins, cephamycins and carabapenems). These
genes are of particular medical importance in Gram-negative
infections which contain a sub-class of beta-lactamases known as
the extended-spectrum-beta-lactamases (ESBLs), which are manifest
in bacteria including C. freundi, P. aeruginosa and increasingly K.
pneumonia, E. coli and Salmonella spp. A survey of the currently
found beta-lactamases can be found in Paterson (2005) Clin.
Microbiol. Rev. 18, 657.
[0410] The main types of beta-lactamases, an example of an organism
and the antibiotics they effect and the gene responsible are as
follows: TEM beta-lactamases, most commonly by the TEM-1 gene
affecting ampicillin resistance in most Gram-negatives, including
E. coli, K. pneumoniae, H. influenzae and N. gonorrhoeae, though in
excess of 140 TEM-type enzymes have been identified (George (2005)
N. Eng. J. Med. 352, 380); SHV beta-lactamases, most commonly SHV-5
and SHV-12 which cause, for example, ampicillin resistance in K.
pneumoniae (Paterson (2003) Antimicrob. Agents Chemother. 47:
3554); CTX-M beta-lactamases, causing, for example, resistance to
cefotaximine and other oxyimino-beta-lactamases (such as
ceftazidime, ceftriaxone or cefepime) in E. coli and S. enterica,
40 CTX-M enzymes have been described (Canton (2008) Clin.
Microbiol. Inf. 14: 134); OXA beta-lactamases, causing, for
example, resistance to oxacillin and cloxacillin in Enterobacteria,
such as E. coli and K. pneumoniae and P. aeruginosa;
inhibitor-resistant beta-lactamases, such as variants of
TEM-beta-lactamases found, for example, in E. coli and K.
pneumoniae, which are resistant to calvulinic acid and other
inhibitors; AmpC-beta-lactamases found in many Gram-negative
bacteria including Enterobacter species that give broad-spectrum
resistance to cephalosporins; carbapenemases that encode resistance
to cephamycins, cephalosporins and carbapenems, this type of
beta-lactamase can be divided into IMP-type (present in
Gram-negative bacteria, particularly Pseudomonas and
Acinetobacter), VIM (an example being the dominant variant being
VIM-2 found predominantly in P. aeruginosa and encoding resistance
to all beta-lactams with the exception of the monobactams) and KPC
(encoding carbapenem resistance in K. pneumoniae).
[0411] Those genes encoding efflux pumps which actively extrude
antibiotics from the bacteria, of which there are five major
superfamilies (Poole (2007) Ann. Med. 39: 162): major facilitator
(MFS), ATP-binding cassette (ABC), small multidrug resistance
(SMR), resistance nodulation (RND) and multidrug and toxic compound
extrusion (MATE). Those antibiotics affected by each of these
efflux system, the genes responsible and the organisms in which
they most commonly occur are given in Table 1 of Poole (2005) J.
Antimicrobial Chemother. 56, 20 and shown in FIG. 27. Medically
important resistance genes include: Macrolide resistance is
commonly encoded for by the Mef(A) gene, for example in
Streptococci (Pozzi (2004) Curr. Drug Targets Infect. Disord. 4,
203) and Gram-Negative bacteria; MsrD encodes ketolides resistance,
for example in S. pneumoniae (Daly (2004) J. Clin. Microbiol. 42:
3570); chloramphenicol resistance is encoded, for example, by the
Cml/CmlAB emux system, as described in P. aeruginosa, K. pneumoniae
and S. enterica (Schwarz (2004) FEMS Microbiol. Rev. 28: 519);
erythromycin resistance is encoded by MexCD-OprJ in Pseudomonas
spp. (Tauch (2003) Mol. Genet. Genomics 268: 570); tetracycline
resistances are encoded by the Tet family--commonly TetA, TetB,
TetC, TetD, TetE, Tet30 and Tet39 in Gram-negative bacteria
(Roberts (1996) FEMS Microbiol. Lett. 19: 1 and Butaye (2003) Int.
J. Antimicrob. Agents 22: 205) and TetK and TetL in Gram-positive
(Butaye (2003) Int. J. Antimicrob. Agents 22: 205); beta-lactam and
aminoglycoside resistance in H. influenzae is encoded for by
AcrAB-TolC (Rosenberg (2000) J. Bac. 182: 1754). Efflux pumps from
the MATE superfamily confers resistance to fluoroquinolines amongst
others, and those genes responsible have been identified in E. coli
(NorE, Morita [1998] Antimicrob. Agents Chemother. 42: 1178), N.
gonorrhoaea (NorM, Roquette-Loughlin [2003] J. Bac 185: 1101), H.
influenzae (HmrM, Xu [2003] Microbiol. Immunol. 47: 937), P.
aeruginosa (PmpM, He J. Bac. 186: 262), C. difficile (CdeA, Kaatz
[2005] Antimicrob. Agents Ther. 49: 1857), S. aureus (MepA, Kaatz
[2005] Antimicrob. Agents Ther. 49: 1857) and E. coli (AcrAB,
Nishino [2001] J. Bac. 183: 5803).
[0412] Those genes encoding resistance to aminoglycosides (such as
streptomycin, kanamycin, tobramycin, amikacin). Resistance is
commonly encoded by genes which produce enzymes that modify the
antibiotics most commonly by N-acctylation (aminoglycoside
acetyltransferases, AAC), adenylylation (aminoglycoside
nucleotidyltransferases, ANT) or O-phosphorylation (Aminoglycoside
phosphotransferases, APH) (Shakil [2008] J. Biomedical Sci. 15: 5).
Examples of such genes are found in both Gram-negative and
Gram-positive bacteria: AAC(6')-Ie APH(2'')-Ia confers broad
spectrum aminoglycoside resistance in enterococci and staphylococci
(Hedge [2001] J. Biol. Chem. 276: 45876); the AAC(3) family is the
largest (Sunada [2003] J. Antibiot. (Tokyo) 52: 809) and, for
example, confers broad resistance to aminoglycosides in enterococci
(Draker [2004] Biochem. 43: 446); ant(2'') and ant(4'') genes
encode resistance to gentamicin and tobramycin in gram-negative
bacteria whilst ant(4) and ant(6) and ant(9) do so in Gram-positive
bacteria (Jana [2006] Appl. Microbiol. Biotechnol. 70: 140); in
Gram-positive organisms the aph(3') gene is widely spread and
confers resistance to a broad range of aminoglycosides, especially
in staphylococci and enterococci (McKay [(1996] Antimicrob. Agents.
Chemother. 40: 2648).
[0413] Additionally the ermB gene is implicated in determining
resistance to erythromycins, such as Zithromax/Azithromycin,
particularly in Streptococcal infections (Richter [2005] Clin.
Infect. Dis. 41: 599).
[0414] In one aspect, the present methods may be used to target
regulation of expression of any one or more of the above resistance
genes or types of genes, e.g. genes encoding beta-lactamases, genes
encoding aminoglycoside modifying enzymes, to thereby alter
resistance to the corresponding antibiotic(s), e.g. in a bacterial
strain listed above. Decoy sequences for targeting regulation of a
gene(s) may be identified and tested as described herein.
[0415] A decoy sequence may comprise a cis-regulatory sequence
itself or, for example, a variant or fragment thereof which retains
the necessary competing function. The cis-regulatory sequence may
comprise any of those described herein and/or identified according
to any of the methods described herein. A decoy sequence may
comprise a snare sequence as described herein and/or as identified
herein, or a variant or fragment thereof which retains the
necessary competing function.
[0416] A decoy sequence for targeting a specific set of genes, e.g.
antibiotic resistance genes, may be prepared based on the native
transcription factor binding site in a cell. For example, sequences
are available in published literature. In one aspect, a test decoy
sequence may comprise an endogeneous binding site or a consensus
sequence or a variant or fragment of a native site.
[0417] A test decoy sequence can then be assessed for decoy
function. Typically, the decoy sequence is prepared as a decoy
polynucleotide as described herein. The decoy polynucleotide is
introduced into a host cell which comprises a binding site for the
given transcription factor, operably linked to a gene or genes, the
expression of which can be detected directly or indirectly: For
example, screening may comprise testing directly for expression of
a regulated gene, or testing for a phenotype which is causally
linked to expression of the gene. A decoy sequence which causes an
alteration in the expression of the gene or genes is said to have
decoy function.
[0418] Suitable methods for testing decoy function, e.g. using the
n[snare] plasmids, reporter systems, are described herein.
[0419] Test decoy sequences can also be determined by the methods
described herein using n[snare]plasmid libraries, and/or methods
for identifying boundaries of protein binding sites. These methods
can be used to examine the genetic basis for phenotype in cells.
For example, to examine the genetic basis for antibiotic resistance
in a clinical isolate.
[0420] A decoy polynucleotide may comprise any of the decoy
sequences described herein and may also comprise additional
sequence as described.
[0421] A decoy polynucleotide may comprise any of the decoy
sequences and/or decoy polynucleotides described in the Examples.
Thus, for example, a decoy polynucleotide comprising the AfsR
binding site may comprise an n[snare] plasmid of Example 1 or the
snare comprised therein, or the oligonucleotide of SEQ ID No. 3
which forms a cyclised decoy dumbbell decoy structure. A decoy
polynucleotide may comprise an n[snare] plasmid identified as
causing an upregulation in cinnamycin production in Example 2 or 3,
or a snare comprised therein. A decoy polynucleotide may comprise
an n[snare] plasmid identified as causing an increase in butanol
tolerance in Example 4, or a snare comprised therein. A decoy
polynucleotide may comprise any of SEQ ID No. 8, 9, 10, 11 or 12 as
described in Example 5. A decoy polynucleotide may comprise the
oligonucleotide of SEQ ID No. 21 which forms a circular dumbbell
decoy, or SEQ ID NO:26, or a species variant of the VanR binding
site.
[0422] Decoy polynucleotides may be prepared by any suitable
method. For example, dumbbell decoys may be prepared by PCR using
appropriate primers, as described in Example 7.2. Each primer
generally contains a portion which will form the stem loop of the
dumbbell structure. Examples of such primers are given in SEQ ID
NOS: 24 and 25. PCR amplification using the primers is typically
followed by restriction digest of the amplification product and
ligation to form the closed circle dumbbell.
[0423] Alternatively, dumbbells can be prepared by restriction
digest of a plasmid as described in Example 7.3. Digestion is
followed by ligation to form the closed circle dumbbell structure.
The present methods may be used to alter gene expression in
prokaryotic or eukaryotic cells. In one aspect, when the method is
applied in eukaryotic cells, the decoy polynucleotide comprises an
n[snare] plasmid as described herein and/or the decoy sequence
competes with a cis-regulatory sequence identified according to the
methods described herein and/or the decoy sequence comprises a
cis-regulatory sequence identified according to the methods
described herein or a variant or fragment thereof.
[0424] A decoy polynucleotide may be introduced to a host cell by
any suitable means. For example, transformation, transfection,
conjugation. For example, where the polynucleotide comprises a
conjugative plasmid, this may be introduced by conjugation. Decoy
polynucleotides, e.g. circular dumbbell structures, may be
introduced to cells by transfection. Cells may be in liquid culture
and the decoy added to the liquid. Alternatively, cells may be
cultured on solid media and decoys transfected from absorbent paper
discs saturated with decoy and overlaid on the media. Typically,
decoy polynucleotide is added to the culture medium and taken up by
cells. Where cells are cultured on solid media, decoy
polynucleotides may be added to a filter disc and taken up by cells
from the disc. A permeability buffer may be used to aid
transfection.
[0425] In one aspect, transfection of decoy polynucleotides may
comprise the use of cholesterol. In particular, the methods may use
linear decoy polynucleotides, bearing a cholesterol modification at
one or both 5' ends. The modification is believed to facilitate
uptake by the cells.
[0426] Decoys may additionally be labelled, e.g. at a 5' end with a
detectable label such as a fluorescence dye, e.g. Cy5. This will
facilitate monitoring of uptake and maintenance in the cell.
[0427] Cholesterol and/or detectably labelled decoys may be
prepared using cholesterol and/or detectable labelled primers, as
described in Example 7.1.
[0428] Transfection of decoy polynucleotide into a cell may
comprise use of R9-cholesterol, which consists of a cholesterol
molecule attached to a linear chain of nine D-arginines (Kim W. J.,
et al., Mol. Ther 2006 14: 343-350).
[0429] In general, the uptake and/or maintenance of the decoy
polynucleotides in the cells is monitored. For example, a plasmid
decoy may comprise a detectable marker e.g. encoding antibiotic
resistance, which allows positive selection for the presence of the
plasmid and monitoring of plasmid propagation. Presence of a decoy
polynucleotide can also be monitored by qrt-PCR.
[0430] In general, the cells into which the decoy is introduced are
those which comprise the cis-regulatory sequence (typically the
promoter containing the sequence) with which the decoy competes for
transcription factor binding, operably linked to the gene(s) whose
expression is to be modulated. Suitable host cells are described
herein. Typically, where the method is used to alter a cell
phenotype, the host cells display the phenotype in the absence of
the decoy. Decoys for alteration of a medically or therapeutically
relevant phenotype, e.g for increasing antibiotic sensitivity, may
be screened in a series of cells. For example, decoys may be tested
first in a bacterial model of the phenotype, e.g. a model of
antibiotic resistance, then further validated in e.g. a pathogen or
clinical isolate. Decoys may be further validated in an animal
model, as described herein.
[0431] Once the decoy polynucleotide has been introduced into the
cells, typically the cells are screened for modulation of gene
expression. Screening may be direct or indirect. Methods of
screening for modulation of gene expression and/or phenotype in
cells are described herein in relation to methods for identifying
cis-regulatory sequences.
[0432] In a further aspect, more than one decoy polynucleotide may
be introduced into a cell to alter phenotype. For example, a
combination of n[snare] plasmids may be used, provided that the
plasmids are compatible.
[0433] In one aspect, the methods described herein are in vitro
methods.
[0434] The methods for modulating phenotype described herein have a
number of applications, for example, in industry and in
therapeutics.
[0435] The methods may be used to alter phenotype in industrially
important cells, for example, to increase the production of useful
metabolites. Accordingly, in one aspect the invention provides a
method of modulating (increasing or decreasing) production of a
metabolite in a cell comprising a method described herein.
Typically, in the methods the cis-regulatory sequence of interest
regulates expression of a gene(s) encoding protein(s) necessary for
production of a metabolite.
[0436] In one aspect the cis-regulatory sequence comprises one of
A24.1, A24.2, A24.3, A24.4. or A24.5 identified herein. The
invention relates to a method of modulating antibiotic production
comprising use of any of A24.1, A24.2, A24.3, A24.4. or A24.5
identified herein, e.g. A24.1. A24.3 A24.5, such as A24.5, or of
decoy molecules which compete with any of the aforementioned for
binding of transcription factor. The invention further relates to a
method of modulating antibiotic production comprising use of any of
SEQ ID NOS 8-12.
[0437] The methods may also be used to render cells, e.g. bacterial
cells, more sensitive to antibiotics. Accordingly, the invention
further provides a method for modulating (increasing or decreasing)
antibiotic sensitivity of cells, e.g. bacterial cells comprising a
method described herein. Typically, in the methods the
cis-regulatory sequence of interest regulates expression of a
gene(s) encoding protein(s) necessary for antibiotic
resistance.
[0438] Thus in one aspect the invention provides the use of a decoy
polynucleotide for modulating antibiotic resistance of a cell. In
general the modulation is caused by an alteration in expression of
antibiotic resistance gene or genes in the cell. The effect may be
specific to a particular antibiotic or in some cases, class of
antibiotics.
[0439] Generally, the cell comprises a gene or genes operably
linked to a cis-regulatory sequence comprising a binding site for a
transcription factor and the decoy polynucleotide comprises a decoy
sequence with the same binding site or a site that competes with
the cellular binding site for transcription factor binding.
Introduction of the polynucleotide into the cell reduces binding of
the transcription factor to the cis-regulatory sequence in the cell
and causes an alteration in expression of the gene or genes in the
cell, thereby modulating antibiotic resistance of the cell.
[0440] Typically the targeted cis-regulatory sequence in the cell
regulates expression of an antibiotic resistance gene or genes as
described herein. The decoy disrupts regulation of expression of
the gene(s) and thereby causes an alteration (e.g. a decrease) in
antibiotic resistance in the cell.
[0441] The cell may be a laboratory model of resistance or a native
resistant strain, e.g. a pathogen or clinical isolate. Suitable
cells are described herein.
[0442] In one aspect, the invention relates to a method for
altering the phenotype of a prokaryote, other than the level of
expression of CO.sub.2-responsive genes in Cyanobacterium, which
comprises providing a decoy comprising an excess of a nucleic acid
encoding a sequence to which a prokaryotic transcription factor
binds, wherein said sequence is identified according to a method
described herein, such that upon contacting said decoy with said
prokaryote, the transcription factor is competitively inhibited
from binding to its cognate binding site in the genome of said
prokaryote.
[0443] The invention may also relate to a method for altering the
phenotype of a prokaryote, other than the level of expression of
CO.sub.2-responsive genes in Cyanobocterium, which comprises
providing a decoy comprising an excess of a nucleic acid encoding a
sequence to which a prokaryotic transcription factor binds, such
that upon contacting said decoy with said prokaryote, the
transcription factor is competitively inhibited from binding to its
cognate binding site in the genome of said prokaryote. The decoy
may comprises a portion of a promoter of a prokaryotic gene. The
decoy may comprises a transcription factor binding site from the
promoter of a prokaryotic gene, e.g. an antibiotic synthetic or
regulatory gene. Binding of the decoy to the transcription factor
may results in susceptibility of the prokaryote to an antibiotic to
which the prokaryote is otherwise resistant. For example, the
prokaryote may be normally resistant to the antibiotic vancomycin
but, in the presence of said decoy, the prokaryote is no longer
resistant. The prokaryote may be a pathogen. Alternatively, binding
of the decoy to the transcription factor results in increased
production by the prokaryotic of an antibiotic.
[0444] In one instance, when the phenotype to be altered according
to the present methods is antibiotic resistance, a putative decoy
sequence is tested first in a laboratory model cell or reporter
cell as described herein. Candidate decoy sequences are then
generally tested in a native resistant cell, e.g. a pathogen cell
or a clinical isolate. In general, the method additionally
comprises testing candidate decoy sequences in a suitable animal
model. For example, a mouse model may be used. In general, the
animal model is infected with a pathogenic strain that is resistant
to treatment with antibiotics alone.
[0445] In some cases, when a decoy sequence (based on a binding
site sequence in a model bacterial species) is tested against a
panel of clinical isolates, it may be found that there are some
isolates which are refractive to the treatment. In such cases those
isolates may be identified and sequence analysis performed to
determine if there are any variations within the regulatory
sequence the TFD mimics. If so the occurrence of those variations
can be estimated and a cocktail of the TFD created that contains
both model and variant sequences in rough proportion to their
incidence in the clinic.
[0446] The present decoy polynucleotides have a number of
applications, including medical and veterinary applications, as
well as in vitro uses.
[0447] For example, decoy polynucleotides which increase cell
susceptibility to one or more antibiotics can be used to treat
bacterial infections in humans or animals, or in ex vivo methods
for killing or inhibiting prokaryotes, e.g in antibacterial
cleaning compositions.
[0448] Typically in use, a decoy will be used in combination with
the antibiotic(s) which it makes the cell more sensitive to. The
antibiotic may be administered simultaneously with, or before or
after the decoy. The antibiotic and decoy may be administered in
the same or in separate compositions.
[0449] Thus, for example, a decoy which targets VanR regulation of
the vanHAX operon to lower resistance to vancoymcin will typically
be used in combination with vancomycin antibiotic.
[0450] The particular infection or associated condition which the
decoy is used to treat is in general dependent upon the pathogenic
cell that the decoy targets.
[0451] For example, vancomycin resistant Enterococci (VRE) such as
E. faecium and E. faecalis are associated with abdominal
infections, skin infections, urinary tract infections, blood
infections. Therefore a decoy targeting VanR regulation of
vancomycin resistance genes in a VRE, may be used for treatment of
such infections, typically in combination with vancoymcin.
[0452] For example, Gram-negative infections (such as E. coli and
K. pneumoniae) carrying genes encoding for Extended-Spectrum
beta-lactamases show broad resistance to penicillins are associated
with serious infections of the urinary tract and gut. In one
aspect, treatment with decoys designed to prevent or downregulate
the expression of those genes encoding the beta-lactamases (such as
the CTM-X family of genes) in combination with a penicillin may be
used for treatment of such an infection.
[0453] In one aspect, Zithromax resistance, caused by expression of
mefA or ermB genes in S. aureus, S. pneumoniae or C. pneumoniae,
and evident in infections of the ear and throat could be treated by
application of decoys targeting mefA and ermB regulation in such
organisms, typically in combination with the antibiotic.
[0454] In one aspect, fluoroguinolone-resistant infections, such as
S. pneumonia, causing pneumonia could be treated by decoys
targeting norA (the gene encoding the efflux pump) regulation and
administered in combination with the antibiotic.
[0455] In one aspect, macrolide-resistance, such as clarithromycins
(for example Biaxin) in C. pneumoniae infections, causing
pneumonia, could be treated by administration of the decoy
targeting the regulation of the resistance gene ermB in combination
with the antibiotic.
[0456] In one aspect, decoys targeting the ampC gene, that encodes
resistance to beta-lactams (such as penicillin, for example,
Augmentin), could be used as a therapy against a wide range of
Gram-positive and Gram-negative nosocomial infections by injection
with the antibiotic in a hospital setting.
[0457] In one aspect the invention relates to a pharmaceutical
composition comprising a decoy polynucleotide and a physiologically
acceptable carrier or excipient. The composition may additionally
comprise one or more antibiotics as described.
[0458] Acceptable carriers or diluents for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington' Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro edit. 1985). The choice of pharmaceutical carrier,
excipient or diluent can be selected with regard to the intended
route of administration and standard pharmaceutical practice. The
pharmaceutical compositions may comprise as, or in addition to, the
carrier, excipient or diluent any suitable binder, lubricant,
suspending agent, coating agent, solubilising agent.
[0459] Decoys may be administered by any suitable means, for
example by intravenous injection, topical application or oral
delivery. As above, administration may be in combination with a
suitable dose of antibiotic, with the antibiotic(s) being
administered at the same time as the decoy, or separately.
[0460] Decoy polynucleotides which increase antibiotic resistance
can also be used in cleaning compositions such as disinfectants,
again typically in combination with the antibiotic(s) to which they
make the cell more sensitive and/or in combination with one or more
antibacterial agents. In one aspect the invention relates to such a
cleaning composition.
[0461] The invention further provides a kit comprising a decoy
polynucleotide as described herein and one or more antibiotics or
antibacterial agents, wherein the decoy and the antibiotic(s) or
antibacterial agent(s) are for combined used in killing or
inhibiting prokaryotes such as bacteria.
[0462] In one aspect therefore the invention further relates to the
use of a decoy polynucleotide as described and/or identified
herein, for the manufacture of a medicament for treating bacterial
infection in a subject. The invention further relates to the decoy
polynucleotide for the treatment of bacterial infection and to a
method for treating a subject for bacterial infection comprising
administering the decoy polynucleotide as described herein.
[0463] The invention further relates to compositions and
medicaments comprising a decoy polynucleotide as described and/or
identified herein. Further aspects include combination therapies in
which a decoy polynucleotide as identified, and/or as described
herein, is administered to a subject in combination with one or
more antibiotics or other antibacterial therapies.
[0464] In further aspects, the invention relates to cis-regulatory
sequences identified according to the methods described herein, and
to their use, e.g. in decoy sequences. In particular, the invention
relates to cis-regulatory sequences identified in the present
Examples, e.g. cis-regulatory sequences comprising A24.1, A24.2,
A24.3, A24.4. or A24.5 identified herein and decoy polynucleotides
comprising them. The invention further relates to decoy
polynucleotides as described herein and/or as described herein,
e.g. to SEQ ID NOS: 3, 8-12 and 21.
[0465] The invention further relates to any of the n[snare]
plasmids, or plasmid libraries described herein and/or prepared
according to the methods described herein, including those prepared
in the present Examples.
[0466] The invention further relates to host cells comprising
n[snare] plasmids and/or decoy polynucleotides as described herein.
In particular the invention relates to such hosts which display
altered gene expression and/or phenotypes. For example, the
invention relates to host cells which display increased production
of a given metabolite, e.g. antibiotic, increased susceptibility to
antibiotic or increased tolerance to a given solvent, including
those prepared in the present Examples.
[0467] Thus in one aspect the invention relates to host cells (such
as those described herein) comprising a decoy polynucleotide as
described herein, including n[snare] plasmids or plasmid libraries
as described herein. Included particularly are those cells having
altered susceptibility to antibiotics (e.g. increased
susceptibility) and prepared according to the methods herein. Thus
in one aspect the invention relates to pathogens or clinical
isolates which have been rendered more susceptible to antibiotics
using decoy polynucleotides as described herein.
Further Description of Specific Embodiments of the Invention
[0468] In a first embodiment according to this invention, we
provide a generic method for identifying cis-regulatory elements.
As with most good science, the Genome Sequencing projects posed
more questions than they answered. Essentially what they delivered
was a list of all the genes within an organism (the genome), but
this raised the question of which of these were being used
(expressed) at any one time, and under what conditions. This is an
important consideration, as patterns of expression provide clues to
the function of the genes and identify those responsible for
important characteristics (phenotypes). Consider human cells: all
contain the same genes, but the pattern of expression will depend
upon (and possibly determine) whether that cell is from the liver
or brain (tissue-specific regulation). Novel technologies have been
developed (microarray technology) capable of describing the pattern
of gene expression, and can, for example, identify sets of genes
responsible for medically-important phenotypes, such as resistance
to disease. One of the promises of the Genome Sequencing projects
was that such knowledge will help speed the design of new drugs and
strategies for treating disease. However new tools will be needed
to realize this potential.
[0469] As we now have the ability to identify genes determining
phenotypes, the next challenge becomes to gain control of these
genes, and by so doing, gain control over phenotypic expression.
This would be of fundamental importance, to our understanding of
biology in general, the identification of novel targets for drugs,
and also to allow us to improve production of medically and
commercially important compounds from industrial bacteria, (e.g.
so-called fermentation products). Fermentation products range from
foods, medicines, and, increasingly, industrial enzymes and fuels.
It is a worthy goal to derive these products from a sustainable
source (bacteria) as opposed to relying on traditional and
environmentally costly (petro)-chemical methods of manufacture.
[0470] Genes are turned on and off by specialist proteins, i.e.,
transcription factors. These have a dual function: they bind to
specific DNA sequences (regulatory elements), usually close to a
target gene, and they alter the likelihood of that gene being
expressed. Earlier attempts focused on changing the properties or
abundance of the transcription factors themselves. Our approach is
to target the regulatory elements to which the transcription factor
binds. One strategy we have developed is to introduce an excess of
these regulatory elements (termed `decoys`), so as to competitively
prevent the transcription factor from binding to the genomic
version and so block any influence on the downstream gene (see the
discussion below pertaining to second and third embodiments
according to this invention). In this embodiment of the invention,
we build libraries of decoys, (containing either all the regulatory
elements from a species, or a universal version with all possible
regulatory elements) that can be rapidly tested to identify which
sets of regulatory elements have the desired effect. A key part of
the approach consists in building a generic `reporter system`, such
that, instead of looking for changes in complex phenomena, such as
increased yield from an industrial bacterium, successful
modification rather is monitored by changes in expression of an
introduced reporter gene which confers resistance to antibiotics
and which is easy to detect. The system is so designed that
screening can be performed at high-throughput. Thus, this aspect of
the invention represents a technology born of the genomic age where
it must be possible to produce high quality data at speed and
relevant to the entire genome. The mixture of a universal decoy
library and a generic reporter system is anticipated to rapidly
improve the delivery of biotechnology products and be an invaluable
research tool.
[0471] More specifically, according to this embodiment of the
invention, we have adapted the `decoy` oligonucleotide approach to
control genetic regulation in a prokaryote and effect control of
antibiotic production in Streptomyces coelicolor. Decoys, however,
are sensitive to degradation, and the approach is not amenable to
high-throughput. We address both of these issues in the method
according to this invention, (see Examples 1-4). Generally, using
Rolling Circle-Mechanism of DNA amplification, an oligonucleotide
was converted into a series of (about thirty) double-stranded
direct repeats. These were cloned into a high-copy shuttle vector
(capable of propagation in both E. coli and Streptomyces) and
introduced into S. coelicolor by conjugation. The plasmid was
maintained by antibiotic selection. We have found that this system
performed better than decoys: there was greater repression of
antibiotic production and it persisted for the length of the
experiment. The method of constructing the library has been adapted
so that generic libraries can be made from synthetic random
oligonucleotides, or species-specific libraries made from fragments
of the entire genome. To create a universal screening system,
reporters for negative and positive selection are created in the
common host for heterologous expression, Streptomyces lividans. The
targeted promoter is cloned upstream of a kanamycin resistance gene
or glkA, which determines sensitivity to the metabolite
2-deoxyglucose (DOG). Following transformation of a library the
transformants are screened in liquid culture for increased
tolerance of kanamycin (detecting negative regulation) or increased
sensitivity to DOG (detecting positive). As selection is in liquid
culture, the vast majority of clones are lost, leaving only a small
and manageable number to take forward for analysis. A validation
system is developed where sets of colonies from independent screens
are tested to see which cross-hybridize with members of other sets.
In this way it is possible to identify clones that recur in the
library and are good candidates for sequence analysis.
[0472] In a second embodiment according to this invention, (see
Example 5), we used decoy oligonucleotides to study the regulation
of the blue-pigmented antibiotic actinorhodin in Streptomyces
coelicolor A3(2). This organism contains (for a prokaryote) a
relatively large genome (8.7 Mb) with a complex and adaptive
pattern of gene regulation, particularly with respect to the
developmental and environmental cues that control antibiotic
production (Bibb (2005) Curr. Opin. Microbiol. 8: 205-215). S.
coelicolor is also the model organism for the actinomycetes, and
increasing the level of understanding of the regulation of
antibiotic production in this strain may inform new strategies for
gaining access to the wide variety of secondary metabolites
produced by these organisms. Many of these compounds have important
applications in medicine, for example as antibiotics, and in
agriculture (Berdy (2005) J. Antibiot. 58: 1-26), and actinomycetes
continue to be a profitable source of new drugs and enzymes (Ward
(2006) Curr. Opin. Microbiol. 9: 279-286).
[0473] Perhaps as a consequence of the complex regulation of
antibiotic production, many pleiotropic mutants identified by
genetic screens are conditional; for example, the antibiotic
non-producing phenotype of a relA null-mutant is highly
medium-dependent (Chakraburtty (1997) J. Bacteriol. 179:5854-5861).
The occurrence of nutritionally conditional phenotypes implies that
genetic screens may underestimate the number of regulatory factors
influencing antibiotic production. Inactivation of a bona fide
transcription factor may be missed if, under the conditions used,
activation of target genes can be mediated by an alternative
transcription factor or regulatory pathway. One of the advantages
of decoys is that they identify and manipulate the sequence
component of DNA-protein interactions controlling gene expression,
potentially blocking the interaction of numerous transcription
factors with a single site. In this embodiment according to the
invention (see Example 5), we demonstrate the relative ease of
targeting regulatory sequences and the ability to rapidly identify
novel regulatory genes in a manner that is complementary to
conventional genetic screens.
[0474] In a third embodiment according to this invention, (see
Example 6), we demonstrate the utility of decoy
oligodeoxynucleotides to confer sensitivity to an antibiotic on a
bacterium that is otherwise resistant to that antibiotic. Those
skilled in the art will appreciate the extreme significance of this
aspect of the invention in an age where so-called "superbugs" are
to be found everywhere, including in our hospitals. Frequently
attributed to the widespread use of antibiotics, the development of
these so-called superbugs has the potential for generating pandemic
infections. By means of this aspect of the invention, however,
those skilled in the art will appreciate that by properly
understanding the mechanisms by which pathogens achieve resistance
to specific antibiotics, decoy oligonucleotides identified
according to the methods of this invention may be used prior to, at
the same time as, or both prior to and at the same time as
administration of the particular antibiotic.
[0475] Many genes are controlled at multiple levels through the
interaction of regulatory factors with cis-regulatory sites. The
nucleoprotein complexes formed may do so in a tissue-specific,
developmental stage-specific, or stimulus-dependent manner. In
short, in vivo DNA-protein interactions are physiologically
determined and represent the interaction between two dynamic
components: the DNA-binding proteins that constitute the
trans-acting environment, and the cis-regulatory sequences. While
our knowledge of the patterns of gene expression has grown
substantially in recent years, this growth has not been paralleled
by a comparable increase in our knowledge of regulatory factors
that control specific genes affecting specific cellular processes.
We anticipate that application of the techniques herein is likely
to give fresh insights into the complexities of prokaryotic genetic
regulation and establish them as a complementary approach to
conventional genetic analysis.
[0476] All documents referred to herein are hereby incorporated by
reference.
EXAMPLES
[0477] Having generally described this invention, including its
preferred embodiments and best mode, the following specific
examples are provided, together with further description, to fully
enable and extend the written description of this invention. Those
skilled in the art will appreciate, however, that this invention
should not be construed to be limited to the specifics of the
Examples. Rather, for purposes of apprehending the scope of this
invention, reference should be made to the claims appended to this
disclosure, including equivalents thereof.
[0478] Although in general many of the techniques mentioned herein
are well known in the art, reference may be made in particular to
Sambrook et al, 1989, Molecular Cloning: a laboratory manual.
Example 1
[0479] Demonstrating the Creation of Plasmid-Borne Versions of
Decoy Oligonucleotides and their Utility in Modifying
Phenotypes
[0480] To achieve this work, it was first necessary to develop a
molecular biology protocol capable of creating an n[snare] plasmid.
A desired feature of such a plasmid vector is that it should
contain a section consisting of direct copies of the same fragment
of DNA. Usually these copies number 30, but may range in number
from one to a thousand, and the length of the repeated sequence is
in the range of 35-54 bp, but can be longer, e.g., up to 1000 bp.
In the example of construction given below, within this repeated
sequence, there is a segment (27 bp) which is common to all
n[snare] plasmids and is a consequence of the method of
manufacture. Also in this example the plasmid vector was chosen:
(i) to allow transformation and propagation of E. coli and S.
coelicolor, (ii) to contain a selectable marker gene encoding
resistance to the antibiotic apramycin; and (iii) to maintain a
high-copy number within the cell, typically approaching 100 copies
per cell.
[0481] Method to Create n[Snare] Plasmid Containing the AfsR
Binding Site
[0482] An overview of the process is given in FIG. 1.
Single-stranded oligonucleotides are synthesized with a chimeric
structure: (A) an annealing site for the T7 primer, and (B) a
region containing the sequence of the cis-regulatory or putative
cis-regulatory sequence. The oligonucleotide is circularized by the
action of Taq ligase, in the presence of a universal joining
oligonucleotide. Following digestion with exonuclease to digest
linear DNA, monomeric circles are recovered from a preparative
acrylamide gel. These are subsequently used as templates in a
Rolling Circle Mechanism amplification reaction using Bst
polymerase and the T7 primer. The same primer is used again in a
PCR reaction and the products separated on an agarose gel. High
molecular weight fragments, typically containing 30-50 repeats, are
isolated and are cloned into a PCR vector before subcloning into a
high-copy shuttle vector, to form an n[snare] plasmid. To determine
function, the n[snare] plasmid is transformed either into the
native strain or into a generic reporter strain. This protocol can
also be used to produce `generic` n[snare] libraries, made from
random oligonucleotides, or species-specific libraries, from
fragmented genomes. An illustration of how n[snare] plasmids affect
expression of targeted genes is given in FIG. 2. In the top left of
this figure, a gene (represented by a horizontal bar "A") is
transcriptionally inactive due to the binding of a repressive
transcription factor (circles "B") to a cis-regulatory sequence
(horizontal bar "C") within the promoter of the gene. The sequence
of the cis-regulatory sequence that constitutes the binding site
for the transcription factor may be determined by various
techniques including bioinformatic analysis, `footprinting` of the
promoter or the various n[snare]methods described herein. This
sequence is incorporated into a decoy oligonucleotide to affect the
expression of the gene, but is shown here cloned into a selectable
plasmid in numerous (typically 30) direct repeats, to create an
n[snare] plasmid. The plasmid is usually a `shuttle` plasmid,
meaning it can be propagated both in E. coli (for ease of genetic
manipulation) and the targeted organism; in addition the plasmid is
usually `high copy`, typically meaning it will produce approaching
100 copies of itself in the host cell. The n[snare] plasmid is
introduced into the targeted prokaryote, by standard means, and its
stable propagation is ensured by selecting for its marker, usually
a gene encoding resistance to an antibiotic. When thus introduced
into a cell the n[snare] plasmid is able to affect expression of
the targeted gene by titrating off the transcription factor "B"
from the genomic promoter to relieve transcriptional repression of
the downstream gene (shown as horizontal box "D", top right).
[0483] A decoy oligonucleotide containing the AfsR binding site was
synthesized. It had the following sequence:
[0484] 5'-Phosphate-aat acg act cac tat agg ggc gtt gag cga acg ttt
ttc gcg gcc gc-3' SEQ. ID. 1
[0485] where the AfsR binding site is shown by the sequence in bold
and a restriction site for NotI is underlined. The oligonucleotide
is synthesized with a phosphate group at the 5' end.
[0486] Also synthesized was a `joining` nucleotide, R-T7, so called
as it contains a partial complement of a commonly used primer,
T7:
TABLE-US-00001 SEQ. ID. 2 5'- ccc tat agt gag tcg tat tgc gg-
3'
[0487] Both primers were resuspended at final concentrations of 250
pmol/.mu.l in a reaction buffer consisting of 1.times.Taq ligase
buffer (as supplied by the manufacturers, New England Biolabs) and
50 U Taq ligase. The mixture was heated to 95.degree. C. for 10
minutes before being allowed to cool to 45.degree. C., upon which
the reaction was supplemented with 4 U/ml Taq ligase and the
reaction incubated overnight at 45.degree. C.
[0488] 1 .mu.l of the reaction was used as a template for rolling
circle amplification (RCA) in an incubation mixture consisting of
1.times. Thermopol reaction buffer (as supplied by the
manufacturers, New England Biolabs), supplemented with 0.2 mM
dNTPs, 70 nM T7 primer (the complement to R-T7) and 120 U/.mu.l of
Bst polymerase (New England Biolabs). The mixture was incubated
overnight at 60.degree. C.
[0489] The amplified DNA was recovered following a clean-up step on
a commercially available column system, (Qiagen PCR Purification
kit), and 1 .mu.l of this DNA was used in a standard PCR reaction
consisting of 1.times.Taq polymerase buffer (supplied by the
manufacturers, Roche Diagnostics) supplemented with 0.2 mM dNTPs,
5% DMSO, 25 pmol T7 primer and 0.5 U Taq polymerase (Roche
Diagnostics). PCR was performed using the following cycling
parameters: 95.degree. C. for 5 min, then 25 repeats of the
following sequence: 93.degree. C. 15s, 55.degree. C. for 15s,
72.degree. C. for 2 min, followed by 72.degree. C. for 2 min.
[0490] The products of the reaction were analyzed by 1% agarose/TBE
gel electrophoresis and visualized by trans-illumination of the
ethidium bromide stained gel.
[0491] A successful preparation appears as a `ladder` of sequences
with a repeat length of approximately 50 bp and with sufficient
amount of the material migrating with an apparent size of 1.5 kb to
allow purification (equivalent to approximately 30 repeats of the
50 bp monomer).
[0492] In general, when carrying out this procedure, if the size of
the products are predominantly small (less than 500 bp in size)
then the amplifications are initially repeated varying the amounts
of templates used, and in the PCR reaction, the number of cycles of
amplification employed. In the event this fails to produce products
of the desired size, an alternate strategy is followed where a NotI
partial digest of product of Bst-amplification is performed, the
fragments are size-fractionated to isolate a 1.5 kb template that
is ligated to NotI compatible linkers in order to allow efficient
amplification in a standard Ligation-Mediated PCR reaction
(LM-PCR).
[0493] The 1.5 kb fragment is subcloned into the commercially
available pGEMT-Easy vector from Promega, which is adapted with 3'T
overhangs for efficient cloning of PCR products. Using standard
molecular biology techniques the fragments are recovered following
EcoRI digestion of DNA isolated from an aliquot of the pGEMT-Easy
library and gel purified. The collection of EcoRI fragments are
then subcloned into a similar restriction site in a shuttle vector,
in our case pIJ86, which is capable of replicating in both E. coli
and S. coelicolor. The pIJ86-borne n[snare] fragment is then
introduced into S. coelicolor by conjugation using standard methods
(Kieser et al. (2000) Practical Streptomyces Genetics. John Innes
Foundation.).
[0494] Use of the afsR n[snare] plasmid to alter the antibiotic
production phenotype in S. coelicolor AfsR is a pleiotropic
regulator of antibiotic production in S. coelicolor and engineered
deletions of this gene have shown that it is essential for the
production of the two pigmented antibiotics, actinorhodin (which is
blue) and undecylprodigisin (which is red) in S. coelicolor (Hong
et al. (1991) J. Bacteriology 173: 2311-2318). A key cis-regulatory
sequence that acts as a binding site for AfsR is found upstream of
the afsS gene (Lee et al., (2002) Molecular Microbiology 43:
1413-1430), and it was this sequence that was used to test the
n[snare]plasmid. It was reasoned that a decoy (made in parallel for
comparison sake) or an n[snare]plasmid containing this site would
be expected to inhibit expression of afsS by titrating off the
activating AfsR protein from the promoter, with a resultant
decrease in production of both of the pigmented antibiotics. Hence,
three growth curves were monitored for untreated S. coelicolor that
would serve as a control, a culture treated with a cyclized TFD
containing the AfsR site (as described above and shown in bold,
using the oligonucleotide: 5'-Phosphate-ata gcg ttg age gaa cgt ttt
tc gcg tttt cgc ga aaa acg ttc get caa cgc tat ag tttt ct-3' SEQ
ID. 3) and a strain transformed with the AfsR n[snare] plasmid
(FIG. 3). In this figure the wild-type promoter of afsS is shown
top left "A", AfsR (oval "B") binds to its cognate site (rectangle
"C") to activate expression of the regulatory gene. The graph "I"
in the top right shows the growth curve of the culture and
production of the two pigmented antibiotics, actinorhodin (Act) and
undecylprodigiosin (Red); (the left ordinate=antibiotic production,
the right ordinate=bacterial growth, the solid line shows bacterial
dry weight measurements, diamonds represent actinorhodin (Act)
production and squares represent undecylprodigiosin (Red)
production). The graph "II" shows introduction of a circular
dumb-bell decoy (+Decoys; middle left) which provide competition
for AfsR binding, repressing expression of the regulator encoded by
afsS, with a concomitant decrease in antibiotic production (middle
right graph II). n[snare] plasmids containing homopolymeric repeats
of the cis-regulatory sequence "C" (bottom left) prove more
effective in suppressing antibiotic production (n[snare]) likely
due to positive selection for the plasmid, and the repressive
effect is observed throught the course of the experiment (graph III
bottom right). n[snare] plasmids are capable of modifying control
of gene expression in a predictable way with a concomitant change
in phenotype. These plasmids are cheaper to produce and easier to
introduce into the cells than decoys, give a more sustained effect
and, crucially, they allow a library approach to discovering key
regulatory elements.
[0495] Hence, what was concluded was that, compared to the control
culture (FIG. 3, top row), antibiotic production is suppressed in
both decoy- and n[snare]-treated cultures. However, with the
decoy-treatment, the effect is largely transient with antibiotic
production recovering after 48 h, whereas with the n[snare]
treatment, the suppressive effect persisted beyond the course of
the experiment. Control experiments using scrambled versions of the
AfsR-binding sequence either in the decoy format or incorporated
into an n[snare] plasmid failed to significantly suppress
antibiotic production. These observations support the conjecture
that n[snare] plasmids can be used to identify and characterize
cis-regulatory sequences, and in some respects (stability and
extent to which the phenotype is modified) they are better than the
extant technique of decoy oligonucleotides.
Example 2
[0496] A Method for Identifying Cis-Regulatory Elements and,
Therefore, Decoys for Modifying Phenotype in Prokaryotes or
Eukaryotes, Using n[Snare] Libraries
[0497] In this example the n[snare] library is made from short DNA
fragments isolated from the 17.083 kb cinnamycin biosynthetic
cluster (Widdick and Bibb (2003) Proc. Natl. Acad. Sci. USA 100:
4316-4321), and the library reintroduced into the producing strain
of Streptomyces cinnamoneus. Detection of the antibiotic is
performed using a plate assay where colonies of the producing
strain are allowed to grow on an agar plate which is overlaid with
a soft nutrient agar seeded with an innoculum of the reporter
strain Bacillus subtilis. A halo of clear nutrient agar appears
around the producer colonies where the antibiotic has killed the
indicator strain where elsewhere the nutrient agar appears turbid
due to the strain's continuing growth. The diameter of the halo is
an indicator of the amount of cinnamycin produced and, following
introduction of the n[snare] library, can be used as a convenient
screen for over-producing exconjugant colonies. In this way it is
possible to determine whether members of the library can upregulate
production of the antibiotic and confirms utility of the approach
as a discovery tool to scan sizeable genomic fragments for
candidate cis-regulatory elements that can be incorporated into
TFDs. The logic of the approach can be expanded to develop
strategies for n[snare] libraries to detect candidate sequences in
much larger surveys (including entire genomes) and demonstrations
of these approaches and their potential utilities are discussed in
later Examples. Those skilled in the art will appreciate, however,
that the method is not limited to the specifics of this
exemplification and other prokaryotic or eukaryotic phenotypes may
be similarly explored using this methodology to achieve phenotypic
alterations of essentially any trait of interest.
[0498] I. Construction of Custom n[Snare] Libraries
[0499] A schematic of the procedure to generate these fragments is
shown in FIG. 4. n[snare]plasmid libraries are created from large
pieces of DNA in such a fashion that every cis-regulatory sequence
within that DNA should be represented in the library. It is shown
in this figure that genomic DNA or fractions of genomes, such as
biosynthetic clusters, are fragmented by sonication and the free
ends repaired by treatment with Taq polymerase and dNTPs, to
generate ends with a single dA overhang at the 3' end of the DNA.
To these biotinylated linkers are ligated, containing restriction
sites for Nt.BbvCI (dark box), MmeI (light box), and a
complementary 5' dT overhang. Unligated adaptor is removed from the
ligation mixture before the modified fragments are digested with
MmeI, an enzyme which cuts 20/18 nucleotides downstream of its
recognition site, allowing for the dT introduced in the overhang;
the resultant molecules consist of the intact biotinylated adaptor
plus a 19/17 nucleotide stretch of genomic DNA, which we refer to
as the `snare`. These molecules are captured onto a
streptavidin-coated matrix and denatured to leave the top strand
(containing the 19 nt stretch) attached to the beads. A further
oligonucleotide is now annealed designed to recreate the Nt.BbvCI
site, and this is subsequently digested with that enzyme to release
a single stranded piece of DNA consisting of a portion of the
adaptor plus the snare sequence. This is used in subsequent steps
to create n[snare] libraries.
[0500] 1.1 Preparation of DNA Fragments
[0501] A DNA fragment containing the approximately 17 kb of the
cinnamycin biosynthetic cluster was isolated from the cosmid it had
been previously cloned into. The fragment was gel purified and
sonicated to an average length of 500 bp as judged by analyzing an
aliquot of the DNA by electrophoresis on a TBE/agarose gel.
Sonication produces a heterogeneous population of DNA terminating
with 3' and 5' overhangs of varying lengths and blunted DNA where a
double stranded break has occurred. In order to convert these to a
homogenous population consisting of ends with a 3' dA overhang, the
DNA was treated with Taq polymerase.
[0502] 1.2 Creation of `Snares` of 18 Nucleotide Length from a
Genomic Fragment
[0503] The term `snare` is used to refer to the short single
stranded portion of DNA derived from the fragments of genomic
sequence that are used to create the n[snare] plasmids.
[0504] Using standard methods a biotinylated adaptor, of the
following sequence (where P stands for Phosphate), is prepared and
ligated to the treated DNA fragments:
TABLE-US-00002 Nt. BbvCI MmeI 5'-Biotin- ggt ccg ggc cac ggt ggt
cta cga gcc tca gcc agg tcc gac t- 3' SEQ ID. 4 3'- agt cgg tcc agg
ctg-P- 5' SEQ ID. 5
[0505] The DNA fragments are then separated from unincorporated
linker using the Qiagen PCR Purification kit and resuspended in 30
.mu.l of Elution buffer (10 mM Tris.HCl pH8). The adapted DNA
fragments are then digested at 37.degree. C. for 16 h in a 200
.mu.l volume reaction buffer (1.times. Buffer 4 [New England
Biolabs] supplemented with 50 .mu.M S-adenosylmethionine)
containing 20 Units of MmeI restriction enzyme (New England
Biolabs). Following digestion the fragments are precipitated and
separated on a 12% non-denaturing acrylamide gel and the bands
analysed. The free adaptor is evident as are a slower migrating
species of adapter plus the 19/16 nt overhang generated by the
asymmetric digestion of the genomic fragment by the type IIS
restriction enzyme MmeI. This band is excised from the gel and the
fragments recovered using standard techniques. The 19 nt on the
upper strand is the portion of genetic material incorporated into
the n[snare] plasmids as candidates for decoy sequences. As these
fragments are biotinylated they are captured onto a paramagnetic
matrix coated with streptavidin, such as M-280 beads from Dynal.
The DNA on these beads is denatured in 0.5 M NaOH followed by
heating to 80.degree. C. and the resultant single stranded DNA
washed in 1.times. Buffer 4 (New England Biolabs). 100 pmol of a
Bbv complementary oligonucleotide (containing the Nt.BbvCI site
which is underlined) of the following sequence:
TABLE-US-00003 SEQ. ID. 6 5'- gga cct ggc tga ggc tcg tag acc acc
gtg gcc cgg acc -3'
[0506] is annealed to the captured single stranded DNA in order to
make a restriction site for Nt.BbvCI. Before digestion the beads
are washed thoroughly to remove any oligonucleotides that fail to
ligate. The mixture is now supplemented with 25 Units of the enzyme
and the reaction incubated for 4 h at 37.degree. C., after which
the mixture is heated to 60.degree. C. for 15 min to denature the
nicked DNA. The `snare` portion is released from the beads whilst
the remainder is retained (FIG. 4). Following capture of the beads
onto a magnetic stand, the DNA from the supernatant is recovered
and used to create n[snare] plasmids using similar methods, as
previously described.
[0507] 2. Use of a Library of n[Snare] Plasmids to Upregulate
Production of the Antibiotic Cinnamycin in its Natural Host
[0508] The n[snare] library created from the cinnamycin
biosynthetic cluster was introduced into the producer strain S.
cinnamoneus by conjugation and plated onto R2YE agar (Kieser et al.
(2000) Practical Streptomyces Genetics. John Innes Foundation) and
allowed to grow for 3 days. Parallel control experiments were
performed where S. cinnamoneus was untreated or conjugated with a
donor strain containing an empty vector (containing none of the
direct repeats). These controls allowed an estimate of the average
size and distribution of `halo` sizes so that measurement of halo
sizes in the n[snare]-treated sample is used to determine the
statistically significant over-producers. An example of an increase
of halo size in a n[snare]exconjugant is shown in FIG. 5. When
plated on solid R2YE medium S. cinnamoneus secretes an antibiotic,
cinnamycin, into the agar that is capable of killing Bacillus
subtilis cells when the producing colony is overlaid with a culture
of that strain (left hand side). The diameter of this halo is used
as a measure of the amount of cinnamycin produced. Production is
increased by introduction of an n[snare] library containing
fragments derived from the 17.083 kb cinnamycin biosynthetic
cluster. Following introduction of members of the library by
conjugation, clones are selected on the basis of increased
production of cinnamycin (right).
[0509] Using this approach it was possible to identify n[snare]
plasmids capable of upregulating antibiotic production, and, by
extension, to find the key cis-regulatory sequences within the
cinnamycin biosynthetic cluster controlling production. In one
application, such sequences in n[snare] plasmids, or incorporated
into TFDs, could be used to manipulate production of antibiotics,
and other industrially valuable biologics. Using this approach, we
have been able to enhance the level of production of previously
identified actinomycete-derived compounds (FIG. 5). This is often a
major stumbling block in the commercial development of a natural
product, and this technology is suitable to exploit the full
commercial potential of actinomycetes by activating or greatly
enhancing the expression of so-called "cryptic" secondary metabolic
gene clusters (Zazopoulos (2003) Nat. Biotech. 21: 187-190)_, or
increase yields in general for industrial biotechnology. This
platform technology is thus anticipated to benefit both human and
animal healthcare, and it exemplifies the collaborative
exploitation of both genome sequencing and developments in
genomics.
Example 3
[0510] A Method for Using n[Snare] Libraries to Identify
Cis-Regulatory Elements and, Therefore, Decoys, for Modifying
Phenotype Using Engineered Reporter Systems.
[0511] 1. Identifying Regulators of Cinnamycin Production with
n[Snare] Libraries and Cin7-Reporter Strains
[0512] The cinnamycin biosynthetic cluster has been previously
described (Widdick and Bibb (2003) Proc. Natl. Acad. Sci. USA 100:
4316-4321) and the role of some of the identified genes confirmed
by bioinformatical and genetic analysis. The cin7 gene is known to
be preceded by the promoter that controls transcription of the
cinMXTH operon that encodes the short protein (cinA) that is
converted enzymatically to give the cinnamycin antibiotic (Sean
O'Rourke and Mervyn Bibb, unpublished data). We created a reporter
system driven by the cin7 promoter and introduced this construct
into a Streptomyces lividans strain carrying the entire cinnamycin
biosynthetic pathway (strain 1326). In order to demonstrate the
approach, the cinnamycin custom n[snare] library (described in
Example 2) was introduced into the strain by conjugation and
exconjugants screened for survival in normally lethal
concentrations of kanamycin.
[0513] 2. Procedure for Detecting Negative Regulation with n[Snare]
Libraries and a Reporter Based System
[0514] The promoter of the targeted gene (cin7) is positioned
upstream of a gene conferring kanamycin resistance, neo, as shown
in FIG. 7. In this figure the targeted promoter of the cin7 gene
from the cinnamycin biosynthetic cluster of the producing strain of
S. cinnamoneus, is used to drive expression of the neo gene
encoding resistance to the antibiotic kanamycin. A cis-regulatory
sequence within the cin7 promoter is shown as a bar "A", to which a
transcriptional repressor is bound (oval "B"), rendering the
downstream neo gene inactive and consequently the strain is
kanamycin sensitive. Typically, this chimeric gene is introduced
into the genome of the producer strain by use of integrative
vectors. A custom n[snare]library, in this instance created from
the fractionated DNA of the S. cinnamoneus genome reporter strain,
is introduced and transformants/exconjugants screened for
resistance to increasing concentrations of kanamycin. Those cells
that are more resistant are carried forward for further
analysis.
[0515] Hence when there is no or little transcription in the
cinnamycin biosynthetic cluster in the heterologous production
strain S. lividans 1326, the cin7 promoter is transcriptionally
inert and the host cell susceptible to kanamycin selection, with
cell death occurring in strains containing a plasmid with a
promoterless neo gene encoding resistance to kanamycin at
concentrations lower than 5 pg/ml (Labes et al. (1997) Microbiology
143: 1503-1512). The cin7-neo chimeric gene is carried on an
integrative plasmid (based on the pSET152 backbone Kieser et al.
(2000) Practical Streptomyces Genetics. John Innes Foundation) that
also carries the aacC gene encoding resistance to the antibiotic
apramycin. We introduced the cassette into S. lividans strain 1326
(containing the cinnamycin biosynthetic cluster) by methods well
known in the art to create a new strain (MM14) with chimeric gene
stably integrated into the genome. The cinnamycin-custom n[snare]
library made as described in Example 2 was introduced into MM14 by
standard methods to generate a spore suspension of the library. A
fixed number of spores were used to inoculate R3 liquid media
(Shirahama et al. (1981) Agric. Biol. Chem. 45: 1271-1273) and
growth in the presence of an increasing concentration of kanamycin
was monitored. Cell viability at each concentration was determined
by withdrawing aliquots of the same volume from the cultures and
spreading these onto agar plates (DNA agar) to determine colony
forming units/ml culture, and from this the percentage of viable
cells. These data show that in this experiment viability at 650
.mu.g/ml kanamycin was less than 0.01%, which is estimated as
equating to 1200 colony forming units in a 50 ml culture. This was
considered to be the optimal concentration of kanamycin as it
greatly enriched for those with increased resistance to the
antibiotic, resulting in a manageable number of cells. Viability at
150 .mu.g/ml was 85%, establishing the baseline activity of the
cin7-neo construct.
[0516] A library hyrbidisation step was carried out. In this
example, in four independent replicates, the MM14 strain was
conjugated with the cinnamycin custom library. The optimal
concentration of kanamycin for each repeat was determined
empirically. Finally 96 clones from each repeat that survived at
the optimal concentration were cultured in R3 media and then split
into two aliquots. From one, plasmids were extracted from the cells
using standard procedures, taking care to prevent contamination
either by RNA or genomic DNA. This plasmid DNA was then labeled by
random priming PCR using a DIG-labeled dUTP molecule according to
standard methods (Roche manual) to create a Probe sample. The
second aliquot was diluted and spread onto DNA agar plates at a
concentration where individual colonies could be clearly seen. The
plates were incubated at 30.degree. C. for a period of 16 h, after
which the colonies still maintained a waxy consistency and had not
yet hardened. 96 such colonies were scrapped with a toothpick,
which was first used to make a streak on a second DNA plate
(supplemented with 50 .mu.g/ml apramycin) and then mixed in 25
.mu.l dimethylsulfoxide (DMSO), heated to 95.degree. C. for 10 min
and then cooled to create a solution of total DNA derived from the
clone. Both the streak and the solution of DNA were numbered so
that if subsequently the DNA sample was found to contain a
candidate n[snare] plasmid a viable exconjugant (MM14 strain plus
n[snare] plasmid) could be easily recovered.
[0517] To identify n[snare] plasmids genuinely able to interfere
with transcription (to distinguish `background` members that form a
considerable part of the total signal from n[snare]plasmids
genuinely able to interfere with transcription), a library
hybridization strategy was developed. In this example, using
standard methods, DNA samples from 96 clones of each repeat were
processed and hybridized to a nylon membrane each with an
addressable position. The strategy pursued is shown in FIG. 9. For
each repeat, four such filters (F1, F2, F3 and F4 derived
respectively from each repeat) were made and then separately
hybridized with different probes, so eventually all of the probe
sets are hybridized to each of the filters. Hybridization of P1 and
F1 detects a chromatogram where every sample is detected. Detection
is not expected to be homogenous as n[snare] sequences that occur
multiple times in the library are proportionally brighter. In this
example, hybridizations of P1 and P2 with F3 and F4 detect samples
common to all libraries. These samples would be considered strong
candidates for continued scrutiny.
[0518] Exemplary data of such a screen is shown in FIG. 10. In this
example two independent repeats of the cinnamycin library were
tested with reporter strains and 96 of the most kanamycin resistant
clones from each used to make a filter containing the n[snare]
plasmids in each replicate (F1 and F2), and two corresponding probe
sets (P1 and P2). All of the clones identified by
cross-hybridization (F1 versus P2 and F2 versus P1) were considered
strong candidates, and priority was given to those common to both
cross-hybridization experiments (circled in red). The sequence of
three of the six clones studied identified a portion of the cin7
promoter and another two surrounding a potential cis-regulator from
another gene within the cinnamycin cluster, cinR, which has been
proposed to have a role in regulation of the cluster. The final
sequence was not identified and was presumed to be derived from the
S. lividans chromosome or be artefactual.
[0519] In some contexts the P1 with F1 hybridization, and other
self hybridization experiments, may be capable of detecting false
positive clones due to enrichment of, for example, repetitive
sequences in eukaryotic libraries. These would generate far
stronger signals due to their abundance in the library, i.e.
greater than true positives that may be expected to occur more than
once in the library but not as many times as repetitive DNA (types
of which can account for 10% of the human genome by mass).
[0520] 3. Adapting Reporters to Detect Positive Regulation
[0521] A simple adaptation is used to make the reporter system
capable of detecting positive regulation, by creating a cassette
consisting of a chimeric reporter gene incorporating the coding
sequence for the glucose kinase gene (glkA) driven by the targeted
promoter. This enzyme converts the metabolite 2-deoxyglucose (DOG)
to the toxic, phosphorylated version of the compound. Hence by
making a chimeric reporter gene consisting of the cin7 promoter
linked to the glkA gene and stably integrating this into a S.
lividans strain containing a glkA' deletion such a reporter system
is provided (FIG. 8).
[0522] In the context of this experiment, the TK24 strain of S.
lividans is used. This does not contain the cinnamycin biosynthetic
cluster (as did the strain used in detecting negative
cis-regulatory elements controlling production), and as such any
activity of the cin7 promoter shall be due to positive regulators
derived from the S. lividans genome. Hence, by searching for
positive cis-regulatory sequences in this background it is expected
to find up-regulators of heterologous expression from the
chromosome of the S. lividans host. Titrating these positive
regulators off the cin7 promoter and onto an n[snare] plasmid,
prevents expression of glkA the cells grow in the presence of DOG.
To date considerable work has been done, using conventional genetic
screens, to detect such generic regulators of heterologous
production, which may be caused by overexpression of certain
components of the transcriptional machinery, such as sigma factors
and components of the translational machinery. In addition a class
of transcription factors known as SARPs (Butler et al. (2003) Appl.
Microbiol. Biotechnol. 61: 512-516) have been identified and the
cis-regulatory elements to which these bind are potential targets
for this screen. The screen is conducted in the same way as
described previously with similar advantages; compatibility with
high-throughput processing, standardized methods and easily
scoreable phenotypes.
Example 4
[0523] A Method for Using n[Snare] Libraries to Identify
Cis-Regulatory Elements and, Therefore, Decoys for Modifying
Phenotype in Undefined Genetic Systems
[0524] 1. Creation of Genomic n[Snare] Libraries
[0525] Purified E. coli K12 strain genomic DNA was sonicated to
give an average size of 500 bp.
[0526] The fragments were then treated as described in Example 2:
adapted with a biotinylated linker containing convenient
restriction sites, digested to produce adaptors plus a 19/17
nucleotide overhang, captured on a streptavidin matrix, before
restriction and denaturation to release a single stranded DNA
fragment containing a portion of the adaptor plus the 19 nucleotide
overhang. These types of molecules are then used to create
libraries of n[snare] plasmids using techniques as described in
Example 2.
[0527] One version of the E. coli K12 genomic n[snare] library
(hereafter referred to as `K12 library`) was estimated to have 1,
560,000 members, which, considering the size of both the genome and
the fragments used to construct the n[snare] library, corresponds
to up to 99.8% coverage of the entire genome using standard
analysis. Hence if there is no sequence bias in the creation of the
libraries, then three independent versions of the K12 library
should allow complete coverage of the genome. In order to assess
whether there was any bias in the library, plasmids were isolated
from an aliquot of said grown-in-liquid culture. This mixture of
plasmids was digested with EcoRI to regenerate the fragments used
to create the n[snare]libraries, and these were subjected to MmeI
digestion to confirm the molecular biology of construction had
worked: as expected the inserts were of large size and theses
collapsed to a 30 bp monomer on digestion to completion with MmeI
(Figure ii), while partial digestion gave rise to intermediate
products with sizes which were multimers of the 30 bp repeat.
Sequencing of the collection of plasmids clearly confirmed the
sequence of the `spacer` element derived from the adaptor and that
spacing was well preserved over at least ten copies. As expected
the `snare` portion of the plasmid collection was essentially
random sequence with little nucleotide bias at any of the 19
positions.
[0528] 2. Creation of Universal n[Snare] Libraries
[0529] Oligonucleotides were synthesized with the following
sequence: [0530] 5'-Phosphate-aat acg act cac tat agg gnn nnn nnn
ngc ggc cgc-3' SEQ. ID. 7
[0531] where a restriction site for NotI is shown underlined, and a
randomized nucleotide is represented by `n`. In this example the
number of randomized nucleotides is 9, but this number can be
varied. The type of sequences encoded by the variable region can
also be modified, for example by inclusion of constant regions in
the sequence (i.e. a defined core sequence flanked by variable
sequences), or by introducing nucleotide bias to the variable
regions (i.e. by stipulating that `n` should be a dGTP or dCTP 60%
of the time to create randomized sequences with a higher
GC-content). The protocol for creating n[snare] plasmids with this
oligonucleotide containing randomized sequence, and hence, a
universal n[snare]library is the same as that given in Example 2.
We refer herein to these n[snare] libraries derived from randomized
oligonucleotides as Universal libraries.
[0532] The titre of one version of the library was found to exceed
2 million. Using standard analysis, the confidence that a library
of such titre would contain all possible combinations of 9 bp
sequences would be 7.5.times.10.sup.5. However, it is felt unlikely
that such a library shall ever be truly universal as certain
sequences will be depleted from the library for many reasons such
as PCR-bias against GC-rich sequences or sequences with extensive
secondary structure and those sequences in the collection that
contain either/or MmeI or NotI sites. However it will be
appreciated that what has been created has the potential to be a
substantial and comprehensive collection of potential
cis-regulatory sequences to use in the n[snare] library
approach.
[0533] 3. Procedure for Using n[Snare] Libraries to Detect
Cis-Regulators of Phenotypes with Undefined Genetic Network
[0534] The E. coli strain K12 was transformed with either the
genomic n[snare] library or a version of the universal library and
grown in liquid culture. When growth had reached mid log-phase as
estimated by reading the absorbance of the culture (an A.sub.600
reading of 0.4) and aliquots used to seed media supplemented with
either no solvent or concentrations of butanol varying from 0.5% to
5%. The cultures were incubated for a further 1 h and then
colony-forming units were measured in order to calculate cell
viability. Cells were isolated from cultures in which viability had
decreased 10,000 fold (to 0.01% or less) and washed in media free
from solvent and used to reinoculate fresh medium without solvent.
The experimental cycle was repeated; this culture grown to a
density of 0.4 and used to inoculate media supplemented with
increasing concentrations of butanol. This process was repeated
four times and the cell viability measured each time (FIG. 12).
Iteration of the selection process (meaning the library from
existing members following one passage through butanol, were
extracted and used to transform E. coli K12) increased the
viability count as a function of the number of repeats of the
selection procedure. E. coli that were not treated with the K12
n[snare] library were designated `0` (represented by the filled
diamonds), those with 1 treatment were represented by filled
squares, those with 2 by filled triangles, those with 3 by hollow
circles and those with 4 by asterisks. What became evident was that
bacteria transformed with either n[snare] library compared to
control transformations, consisting of plasmid backbone with no
n[snare] insert or untransformed, gave substantially higher cell
viability accounts when cultured in the presence of high
concentrations of butanol. In addition, it became evident that
iteration of the selection process increased the viability count as
a function of the number of repeats of the selection procedure.
[0535] The E. coli K12 custom library performed better than the
universal n[snare] library, consistent with this library being more
representative of the genome of the strain.
Example 5
[0536] Manipulating and Understanding Antibiotic Production in
Streptomyces coelicolor A3(2) with Decoy Oligonucleotides
[0537] What has been described to this point is a suite of
innovative approaches to identify key cis-regulatory sequences
implicated in the control of phenotypes. The technologies are
universally applicable to all organisms. It was next demonstrated
that knowledge of these sequences could be used to create tools
capable of modifying gene expression in vivo. These tools are again
functional in all organisms, but the focus of the following
examples is use in prokaryotes. The transcription factor decoy
approach is adapted for use in prokaryotes and shown to be a
powerful way to mobilize the information generated by the n[snare]
protocols to develop a system capable of modifying gene expression.
The combination of the two approaches is referred to herein as
"regulatory engineering".
[0538] In the example below the cis-regulatory sequences
influencing antibiotic production in Streptomyces coelicolor are
determined by a novel mapping procedure and these sequences used to
design decoy oligonucleotides. The decoys are tested by
introduction into S. coelicolor and their uptake, stability and
effect of antibiotic production monitored. A complete description
of the methods used is given in section 7 of this example.
[0539] 1. Mapping Regulatory Elements within the actII-Orf4
Promoter
[0540] The DNA-protein interactions controlling expression of
actII-orf4 were studied by in vivo DNaseI/T7 exonuclease mapping.
This method was developed to identify the boundaries of cis-acting
regulatory elements so that the deduced sequences could be used to
design decoy oligonucleotides for functional studies. A schematic
summary of the approach is given in FIG. 13. To map the in vivo
boundaries of DNA-protein complexes at specific locations within
the S. coelicolor genome, high concentrations of DNaseI and T7
exonuclease were added to freshly harvested cells. The DNaseI
introduced `nicks` into the DNA surrounding the complexes which
then served as substrates for the 5' to 3' exonuclease activity of
T7 exonuclease. Hence, the joint actions of the enzymes demarcate
the 5' boundaries of the complexes. DNA is recovered from the
treated cells and the fragments from the targeted promoter
(actII-orf4) captured by hybridization to an immobilized strand of
a PCR fragment of the promoter, which incorporates a biotinylated
linker. The positions of boundaries within the population of
captured fragments are mapped by performing a PCR reaction using a
second DIG-non-radioactively labeled primer (incorporating
dioxygenin, DIG [Roche]) that hybridizes to the biotinylated
linker. The sizes of the labeled products are determined by PAGE
and chemo-luminescent detection. Boundaries on the opposite strand
are mapped in a similar manner, using a PCR product with the
biotinylated linker at the opposite end. The labeled PCR fragments
were size fractionated following 12% polyacrylamide gel
electrophoresis and the presence of the fragments and their size
determined following chemo-luminescent detection. The novel
combination of T7 exonuclease and DNaseI in a footprinting protocol
allows detection of all the boundaries of DNA-protein complexes
within a promoter region, and not just those closest to a chosen
restriction site.
[0541] Since our aim was to discover regulatory elements involved
in the repression of actII-orf4 expression, we mapped the
transcriptionally down-regulated promoter in a rich liquid medium
(R5). Under these conditions repressors may occupy the promoter and
prevent expression. Growth curves were derived by measuring cell
density (A.sub.430 of the culture), and production of the two
pigmented antibiotics (the blue actinorhodin, and the red
undecylprodigiosin [Kieser et al. (2000) Practical Streptomyces
Genetics. John Innes Foundation), and the transcriptional activity
of actII-orf4 determined. The latter is known to be induced during
later stages of growth and thus samples for mapping were harvested
prior to the visually detectable onset of actinorhodin production
(indicated by the arrow in FIG. 14A). Cells of S. coelicolor M145
were harvested from a culture grown in rich (R5) media at a time
point (indicated by arrow) preceding visible actinorhodin
production. Cell growth (diamond) and production of actinorhodin
(triangle) and undecylprodigiosin (circle) were monitored
throughout. The amounts of the enzymes required for digestion were
determined empirically, with the concentration of DNaseI needing
careful optimization. For example, an excess of DNaseI resulted in
loss of signal clarity due to cutting within the DNA-protein
complexes, whereas too little enzyme was ineffectual for mapping.
250 U of T7 exonuclease per reaction worked well in most cases; it
was necessary to add an excess of the enzyme as it is not highly
processive, nor was the digestion performed in an optimal buffer.
Boundaries were mapped on both strands, as described in FIG. 13,
and their positions determined following size analysis by 12%
non-denaturing PAGE, followed by chemo-luminescent detection of
DIG-labeled products. (FIG. 14B), and by comparison with a standard
size ladder it was possible to define the boundaries of the
DNA-protein complexes in the transcriptionally silent actII-orf4
promoter region (FIG. 14C). The sequence of the actII-orf4 promoter
showing the positions of the putative cis-regulatory elements
(relative to the primers used in the mapping protocol). Boxed areas
indicate the coding sequences of the upstream gene (actII-orf3) and
actII-orf4 itself. Capitalized sequence marks the candidate
regulatory elements with their names shown above. The underlined
sequences indicate the -35 and -10 boxes for the actII-orf4
promoter, the asterisk shows the position of the transcriptional
start site, and the convergent arrows indicate the inverted repeat
present in A24.4.
[0542] We referred to the sequences defined by these regions as
regulatory elements, and in total five were seen. The regulatory
elements were labeled A24.1 to A24.5 (going towards the
transcriptional start site) and these sequences were used to design
the decoy oligonucleotides used in the subsequent functional
studies.
[0543] 2. Rapid Screening for Decoy Function on Agar Plates
[0544] We developed a rapid agar plate-based assay to test whether
the decoys had any effect on antibiotic production. R2YE agar was
used since it promotes the production of both of the pigmented
antibiotics at levels that are readily detectable by eye. Plates
were inoculated with a dilute spore suspension and incubated for 24
h at 30.degree. C. so that confluent lawns of S. coelicolor formed.
At this stage undecylprodigiosin (which is red) production had
begun but not actinorhodin (which is blue). The plates were covered
with a thin layer of SNA in 0.5% agarose and before this set, small
disks of Whatman paper (Antibiotic Assay disks) were laid onto the
medium and saturated with 15 .mu.l of a 10 pmol/.mu.l solution of
decoy oligonucleotide, or control solution, as indicated in FIG. 15
Filter discs were saturated with solutions of decoys or, as
control, buffer (as shown) and applied to a lawn of S. coelicolor
M145 overlaid with SNA medium (Soft Nutrient Agar. 0.5% Difco Agar
w/v). As the bacteria continued to grow, incidences of enhanced
antibiotic production could be recognized by early accumulation of
the pigmented antibiotics within and around the disks (evident 48
hours after addition of the decoys). Negative controls (buffer
alone or a `scrambled` version of decoy A24.5) did not show
precocious production. All samples were prepared in a buffer
containing 0.5% (v/v) of the two non-ionic detergents NP-40 and
Triton X-100, which were found to improve the efficiency of
transfection. The controls consisted of the buffer alone or a
scrambled decoy oligonucleotide, where the sequence of the A24.5
decoy was randomized. The plate was incubated further at 30.degree.
C. and inspected regularly to determine whether there were any
effects on the amount or the timing of antibiotic production. A
purple/red halo was seen around the disk soaked with A24.5, and to
a lesser extent A24.3 and the cocktail of decoys A24.1 to A24.4
(FIG. 15, 48h; at earlier time points actinorhodin appears red as
the molecule acts as a pH indicator, though the halos were clearly
visible above the background of secreted undecylprodigiosin). To
confirm that actinorhodin synthesis had begun, the disks at 48
hours were recovered, pigments extracted and quantified
spectrophotometrically; decoy A24.5 strongly activated early
actinorhodin production, while A24.3 and the cocktail of decoys did
so to a lesser extent (data not shown). No early actinorhodin
production was seen surrounding the control disks. By 96 hours the
entire lawn of S. coelicolor M145 had produced large amounts of
both antibiotics (not shown), and no zones of growth inhibition
were apparent around any of the disks. The ability of some of the
decoys to enhance production of actinorhodin at early time points,
potentially by interfering with the binding of repressors to their
identified sites within the actII-orf4 promoter region,
demonstrated the efficacy of the approach and stimulated us to
perform more extensive functional studies by introducing the decoys
into cells grown in liquid culture.
[0545] 3. Uptake and Stability of Decoys in Liquid Cultures
[0546] The next issues to address were whether or not a decoy could
be efficiently introduced into mycelium in liquid culture, and if
so, for how long would it persist? Six liquid cultures of S.
coelicolor M145 were set up in SMM media (Kieser et al. (2000)
Practical Streptomyces Genetics. John Innes Foundation) and grown
to mid-exponential phase. Cells were collected by gentle
centrifugation and resuspended in one tenth of the original volume
of a permeabilizing buffer (containing the same concentrations of
detergents as used in the plate assays) before transfection with
varying amounts of decoy A24.1 (0 mM, 5 mM, 10 mM, 20 mM, 50 mM and
100 mM). Following brief incubation, the cells were resuspended in
the retained media and incubation continued. Uptake of the decoy
oligonucleotide was measured by qrt-PCR, using primers designed to
amplify a small fragment (40 bp) spanning the join introduced in
the formation of the dumbbell decoy. Reference to a standard curve
was used to calculate the absolute number of copies of decoy
present within the cells (following centrifugation and two wash
steps), and this was corrected by reference to a genomic control.
To assess stability, aliquots were withdrawn at various time points
(0 to 72 hours). The rate of uptake saturated above concentrations
of 20 mM. Optimal uptake was achieved with a 20 mM transfection, 2
hours following which 45% of the decoy had entered the cells (FIG.
16). Actively growing cells were transfected with a solution
containing decoy and the uptake of the oligonucleotide and its
stability estimated by quantitative real-time PCR (qrt-PCR).
Following transfection with 20 mM A24.1, cells were harvested and
washed before qrt-PCR was used to estimate copies of decoy
remaining in the cell as a function of time. The data represents
three independent determinations. The decoy could be detected
intracellularly 72 hours after addition, with 22% (corresponding to
half that had entered the cells) persisting after 36 hours. Since
the cultures were transfected when nearing stationary phase, it was
assumed that the decoys were being slowly degraded by endogenous
nucleases as opposed to being lost due to dilution. Thus the decoy
was able to enter the mycelium and persist for a prolonged period,
suggesting that this approach could be used to alter gene
expression in liquid cultures.
[0547] 4. Use of Decoy Oligonucleotides to Control Antibiotic
Production
[0548] The five decoys, corresponding to the identified regulatory
elements, were used to transfect liquid cultures of S. coelicolor
M145 grown in R5 or SMM liquid media. Transfection was timed to
precede the expression of actII-orf4, providing the decoys with the
opportunity to interact with their cognate transcription factors
and potentially influence actinorhodin production. Control
experiments were similar to those used in the plate assays and
consisted of either a mock transfection procedure or the
introduction of three scrambled decoys (based on the sequences of
decoys A24.1, A24.3 and A24.5). In each experiment, the growth of
the culture was measured, as well as the production of the
pigmented antibiotics and the expression of actII-orf4, at fixed
intervals following transfection. None of the decoys altered the
growth of the cells, but several did have an effect on the
production of actinorhodin, and in some instances
undecylprodigiosin. In R5 medium, where antibiotic production is
relatively high, decoy A24.5 up-regulated actinorhodin production,
increasing yield by 95% at the 96 hour time point (FIGS. 17A and
B). S. coelicolor M145 was grown for 20 hours before transfection
(indicated by arrows) with (A) a no-decoy control or with (B) the
A24.5 decoy. Cell growth (diamonds) and the amount of
undecylprodigiosin produced (circles) was similar in the two
cultures, and the only variation seen was in the accumulation of
actinorhodin (triangles), which was stimulated following treatment
with decoy A24.5. The data represent the average of three
independent determinations and the bars show the standard error.
Decoys A24.1 and A24.3 had milder effects, causing up-regulation of
both pigmented antibiotics. In all of the treatments where an
increase in actinorhodin production occurred, there was a
corresponding increase in the absolute level of actII-orf4
expression (determined by qrt-PCR; data not shown). Hence the
results were largely consistent with those seen on the plates,
suggesting that the effect of the decoys is specific and
predictable. The decoys were also tested in SMM medium, a minimal
medium that supports less antibiotic production than R5.
Transfection with decoy A24.5 led to a doubling in actinorhodin
production (FIGS. 18 A and B). Comparison of the data obtained with
(A) a mock-transfected control and (B) a decoy A24.5-treated
culture revealed that the decoy oligonucleotide caused a pronounced
increase in actinorhodin production. The data are presented as in
FIG. 17 and are is the average of three independent determinations;
bars show the standard error. In this medium, increases in
actinorhodin production were also seen for decoys A24.1 and A24.3
(data not shown). Hence, the decoys acted as convenient tools to
validate the identification of cis-acting regulatory elements
within the actII-orf4 promoter, allowing us to influence the onset
of antibiotic production. As indicated above, several of the decoys
also upregulated undecylprodigiosin production and possible reasons
for this co-regulation are addressed below.
[0549] 5. Discovery of a New Modifier of Actinorhodin
Production
[0550] The combination of a novel mapping technique and decoy
oligonucleotides has been used to validate three regulatory
elements controlling expression of the actinorhodin regulatory gene
actII-orf4. We next asked whether these regulatory motifs occurred
in the promoter regions of any other genes; such genes might also
be involved in the regulation of antibiotic production. Performing
a BLAST search with all five decoy sequences identified one such
gene. SCO5812 contained strong matches to A24.1 (a run of 10 out of
14) and A24.3 (a run of 11 out of 18). The gene itself is a
potential homologue of ribonuclease HII, and as such may have a
similar function to a previously identified pleiotropic mutant in
antibiotic production, absB (SCO5572; Adamidis & Champness
(1992) J. Bacteriol. 174:4622-4628), which is a homologue of
RNaseIII and thought to be involved in transcript processing (Chang
et al. (2005) J. Biol. Chem. 280:33213-33219). To establish the
role, if any, of SCO5812 in antibiotic production, we deleted the
gene from M145 and compared production with the parental strain on
R5 and SMMS media (FIG. 19). In this figure M145 (left side of
plates) and M145 .DELTA.SCO5812 (right side of plates) were
streaked on (A) R5 agar medium or (B) SMMS agar medium and
incubated for 72 hr and 96 hr, respectively. While deletion of
SCO5812 dramatically reduced actinorhodin production on R5 agar, it
enhanced undecylprodigiosin production on SMMS. It is of interest
to note that cells transfected with either A24.1 or A24.3 showed a
slight up-regulation of undecylprodigiosin production, suggesting
that the transcription factors that bind to these sites may
influence the expression of both of the antibiotic biosynthetic
gene clusters. Also one of the decoys tested, A24.4, which contains
a 6 bp inverted repeat (FIG. 14), was found to have a match (8 out
of 12) in the promoter region of redD, the pathway-specific
activator gene of the undecylprodigiosin biosynthetic cluster
(Takano et al. (1992) Mol. Microbiol. 6:2797-2804). This motif is a
predicted binding site for the nucleoid protein IHF (on the basis
of sequence homology to previously identified sites), raising the
possibility that the site has an architectural role in the
nucleoprotein complex instead of directly affecting
transcription.
[0551] 6. Concluding Remarks
[0552] We have used novel techniques to identify and validate a
binding site for a repressor of antibiotic production in S.
coelicolor. Validation was performed with decoy oligonucleotides:
transfection of copies of these regulatory elements led to
derepression of the targeted gene and increased production of
actinorhodin. The intention of our work was to demonstrate that
decoy oligonucleotides can be a valuable tool in prokaryotes to
rapidly delineate genetic networks. Has it been successful? In two
senses it has been. Three of the five decoys tested showed the
expected activity. The decoy with the strongest effect, A24.5, has
been shown recently to be bound by a TetR-like transcriptional
regulator, AtrA, when the promoter is active (Uguru et al. (2005)
Mol. Microbiol. 58:131-150). Our own work, using affinity
purification to identify repressors of actII-orf4, has similarly
identified TetR-like transcription factors bound to this site; it
is our assumption that as decoy A24.5 is added prior to
transcription of actII-orf4, it allows expression by diminishing
the binding of these putative repressors.
[0553] 7. Materials and Methods
[0554] 7.1 Strains and Growth Conditions
[0555] A standard reference for general techniques concerning the
handling of streptomycetes is Kieser et al. (2000) Practical
Streptomyces Genetics. John Innes Foundation. Spores of S.
coelicolor A3(2) strain M145 were germinated synchronously by heat
treatment and grown in SMM or R5 medium at 30.degree. C. with
continual shaking. Growth of the culture and actinorhodin
production were measured as described previously. R2YE and SNA were
used for agar plate assays of decoy-induced antibiotic production.
Deletion of SCO5812 was accomplished by PCR-targeting (Gust et al.
(2003) Proc. Natl. Acad. Sci. USA 100: 1541-1546.).
[0556] 7.2 In Vivo T7 Exonuclease-Mapping
[0557] Prior to treatment with the footprinting reagent, cultures
of S. coelicolor mycelial fragments were supplemented with 0.5 mM
CaCl.sub.2 and 50 units DNaseI (to remove extracellular DNA) and
incubated for a further 15 min at 30.degree. C. The mycelium was
harvested by low speed centrifugation and washed extensively in TES
buffer (13) supplemented with 5 mM EDTA. For DNaseI footprinting,
the cells were washed in TES supplemented with 0.5% (v/v) NP-40 and
0.5% (v/v) Triton X-100, and incubated at 30*C for 15 min. The
cells were then washed in DNaseI Digestion Buffer (DDB) preheated
to 30.degree. C. and digested with varying amounts of DNaseI for 5
minutes. Reactions were stopped by addition of an equal volume of
STOP buffer (50 mM Tris.HCl, 5 mM EDTA, 0.2% (w/v) SDS, 10 mg/ml
Proteinase K, pH8) and incubated overnight at 55.degree. C. Nucleic
acids were ethanol precipitated following two rounds of
phenol-chloroform extraction. The samples were digested with RNaseA
before reprecipitation and quantification.
[0558] 7.3 Decoy Oligonucleotide Studies
[0559] The sequences of all oligonucleotides used are listed in
Table 1. Decoy oligonucleotides were synthesized (Invitrogen) and
ligated with CircLigase (Epicentre) to create dumbbells (Mann &
Dzau (2000) J. Clin. Investigation 106: 1071-1075.). 100 pmol of
each decoy oligonucleotide were mixed in a 20 .mu.l reaction volume
of 1.times. CircLigase buffer supplemented with 50 .mu.M ATP, 2.5
mM MnCl.sub.2, 500 U CircLigase, and incubated for 1 hour at
60.degree. C. The mixture was treated with an excess of exonuclease
1 to remove linear DNA, and the remaining covalent circles
precipitated. Each preparation was analyzed by 12% non-denaturing
gel electrophoresis to check that the majority of products were
monovalent covalent circles. The sample was resuspended at 200
pmol/V .mu.l in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) buffer. 10
.mu.l of each decoy were spotted onto 3 mm antibiotic assay disks
which were then pressed gently into a thin layer of SNA agar that
had been poured over a 24 hour old confluent lawn of S. coelicolor
M145 grown on an R2YE agar plate. Induction of antibiotic
production would result in a localized increase in pigmentation
surrounding the disk. Alternatively the dumbbell decoy
oligonucleotides were used to transfect mycelium from exponentially
growing cultures of S. coelicolor M145. Transfection involved
washing the cells in equal volumes of TES buffer supplemented with
0.5% (v/v) NP-40 and 0.5% (v/v) Triton X-100, then resuspending
them into 1/10.sup.th of their original volume in TES plus
detergents and supplemented with decoy oligonucleotide. The cells
were incubated at 30.degree. C. for 15 min with gentle mixing,
diluted in the retained culture medium and incubation continued.
Growth rate slowed temporarily following this treatment but soon
recovered. Samples were taken thereafter to assess the levels of
production of the two pigmented antibiotics, actinorhodin and
undecylprodigiosin (13). Samples were simultaneously withdrawn for
RNA analysis.
[0560] 7.4 Gene Expression and Decoy Copy Number Analysis
[0561] Expression analysis was performed by quantitative real-time
PCR (qrt-PCR). Preparation of cDNA was performed as previously
described (Ryding et al. (2002) J. Bacteriol. 184: 794-805.).
Briefly, 1 .mu.g of RNA was thermally denatured (incubated at
70.degree. C. for 10 min and then placed on ice) and mixed with 0.5
U AVM Reverse Transcriptase (Amersham), 1 mM dNTPs, 25 pmol custom
primer in 20 .mu.l of the supplier's recommended buffer. The
reaction was incubated at three successive temperatures, 45.degree.
C., 50.degree. C. and 55.degree. C., each for 30 min. 4 .mu.l of
this reaction was used as a template in a 20 .mu.l qrt-PCR reaction
prepared in Invitrogen's SYBR Greener reaction mix supplemented
with 10% (v/v) DMSO and 25 pmol of the custom forward and reverse
primers for the target gene (actII-orf4) and an internal reference
(SCO4742, a conserved hypothetical protein that was found to show
little variation in expression following microarray analysis
[http://www.biomedcentral.com/1471-2164/8/261/abstract]) (Table 1).
Amplification and analysis were performed on a BioRad Chromo4
machine. Determination of the copy number of decoys was similarly
performed using SYBR-green detection. Primers were designed (Table
1) to detect circularized decoy oligonucleotides and their copy
number determined by sonication of mycelium and comparison to known
amounts of exonuclease-treated decoy; these values were corrected
for the number of genomes in the sample by reference to the number
of copies of SCO4742 present.
TABLE-US-00004 TABLE 1 Oligonucleotide primers Name No. Sequence
SEQ. ID. Decoy oligonucleotides A24.1 5'-
P-atatcactctcgatgtcggcgttttcgccgacatcgagagtgatatagttttct- 3' 8
A24,2 5'- P-atatcaggaatgccagatgcgttttcgcatctggcattcctgatatagttttct-
3' 9 A24.3 5'-
P-atactgcctctcggtaagcgttttcgcttaccgagaggcagtatagttttct- 3' 10 A24.4
5'- P-atatgcagctcgctgcacgcgttttcgcgtgcagcgagctgcatatagttttct- 3' 11
A24.5 5'- P-ataaatctgttgagtagggcgttttcgccctactcaacagatttatagttttct-
3' 12 A24.5 scrambled 5'-
P-atagcaatttagatggtggscgttttcgccaccatctaaattgctatagttttct- 3' 13
Quantitative PCR for decoy copy number A24.1 copy f 5'-
cgacatcgagagtgatatagtttt- 3' 14 A24.1 copy r 5'-
gccgacatcgagagtgatat- 3' 15 Quantitative PCR for expression data
A24 custom 5'- tgtcgcccccaggagacggag- 3' 16 qA24f 5'-
cttaaatcctcgaaggcgacccag- 3' 17 qA24r 5'- tcctcgagccggttctcctcg- 3'
18 q4742f 5'- gctggtggacatcggtct- 3' 19 q4742r 5'-
gcccgtacttgtcgctctc- 3' 20 The cis-regulatory sequences in each of
the decoy oligonucleotides are underlined.
Example 6
[0562] Demonstration that Decoy Oligodeoxynucleotides May be Used
in the Therapeutic Context to Overcome Antibiotic Resistance in
Pathogens
[0563] The overall approach taken to identify a decoy capable of
altering susceptibility to vancomycin, or indeed other antibiotic
resistance mechanisms that have been characterized at the genetic
level, is presented in FIG. 20. This schematic demonstrates how
TFDs are used to counter known resistance mechanisms in pathogenic
bacteria. Resistance to the prescribed antibiotic vancomycin is
used as an example. This occurs in strains of Entercoccus faecium,
a human pathogen, and Streptomyces coelicolor. In both bacteria
resistance is encoded by the vanHAX operon which is induced by the
VanR protein. By designing decoys to interfere with the binding of
VanR to its cognate site in the genome it is possible to prevent
induction of vanHAX and so render the bacteria susceptible to
vancomycin. In this example decoys are functionally validated in
bacterial (non-pathogenic) models containing similar resistance
mechanisms to the pathogens or the actual mechanisms moved into the
model by horizontal gene transfer. Subsequently the decoy is tested
on pathogenic models, such as clinical isolates and finally moved
to animal models, such as mice infected with vancomycin resistant
Entercoccus (VRE) or vancomycin resistant S. aureus. It should be
appreciated that the approach could be applied to a broad range of
bacterial phenotypes which are controlled by such genetic switches.
These include, but would not be limited to the discovery of other
decoys or combinations of decoys that could render bacteria
susceptible to other antibiotics such as penicillin,
chlormaphenicol, tetracycline, daptomycin, etc.
[0564] The structure of the vancomycin resistance operon in
Streptomyces coelicolor is shown in FIG. 21 (From Hong et al. 2004
Molecular Microbiology 52: 1107-1121.). The vancomycin operons
consist of four operons, as indicated by the arrows. The vanRS
operon encodes a two component regulatory system that acts to sense
the presence of vancomycin (directly or indirectly) and induce the
expression of the vanHAX operon which encodes the vancomycin
resistance mechanism. The vanHAX operon is induced by the
phosphorylated version of VanR binding within its promoter, VanS in
turn is the kinase that phosphorylates VanR on detection of
vancomycin. We expect our decoy to disrupt the binding of
phosphorylated VanR to the site in vanH promoter.
[0565] It should be noted that the evolutionary source of the
resistance genes in Entercoccus faecalis, which is the cause of
some of the vancomycin-resistant infections seen in the clinic is
thought to be a vancomycin-producing actinomycete.
[0566] The sequence of the vanH5 decoy used is shown below, with
the binding site for phosphorylated VanR capitalized:
TABLE-US-00005 SEQ ID. NO. 21 5'-P- ata tctatatgaa gcgacgtggt
cgatgagccg cagcg tttt cgctg CGG CTC ATC GAC CAC GTC GCT TCA TAT AGA
tatag tttt ct- 3'
[0567] The oligonucleotide was prepared as a circular dumbbell
decoy as shown in FIG. 22. The oligonucleotide was resuspended at a
final concentration of 100 pmol/ul in a T4 DNA ligase buffer (as
supplied by the manufacturer of the enzyme, New England Biolabs)
and 400 U of T4 DNA ligase and incubated at 16 degrees Centigrade
for various times. During incubation the oligonucleotide formed a
stem-loop structure which, due to the activity of the ligase, can
become converted into a covalently joined single-stranded
`circular` molecule of DNA. The extent of this cyclization is
dependent upon the incubation time with ligase; the longest
incubation (16 h) led to near complete conversion. Hence, following
incubation of the oligonucleotide with T4 DNA ligase for 0, 1, 2,
4, 6 and 16 h (a-f respectively), aliquots were taken from each
reaction, heat treated to inactivate the enzyme and the DNA was
recovered by ethanol precipitation before being analyzed by 6%
polyacrylamide gel electrophoresis and vizualization with ethidium
bromide staining. Lanes a-f show conversion of slower migrating
linear single-stranded DNA molecule into a circular dumbbell closed
circular duplex which migrates at higher rate, (lane f).
[0568] The decoy is in a `circular dumbbell` configuration (Ahn J
D, Kim C D, Magae J, Kim Y H, Kim H J, Park K K, Hong S, Park K G,
Lee I K, Chang Y C (2003) Biochemical Biophysical Res Comm
310:1048-1053), although this is not required. Other forms of
oligodeoxynucleotide may be used, including but not limited to, for
example (a) longer double stranded oligodeoxynucleotides comprising
this sequence, such that exonucleases would need to substantially
reduce the length of the oligonucleotide before loss of decoy
function; (b) oligodeoxynucleotides comprising modified bases or
sugars to confer greater nuclease resistance; (c) 2'-OH nucleotides
or amines attached to the termini of the oligonucleotides, which
would block exonuclease activity (which is all the dumbbells are
doing); (d) small circular double-stranded DNA molecules; or (e)
multimeric molecules comprising multiple copies of the active decoy
sequence.
[0569] Essentially, what is required is that the ODN used has a
double stranded region incorporating the targeted sequence within
the vanH promoter bracketed by small stem loop structures, or other
sequences. For the circular dumbbell structure, the molecule is
synthesized as a linear oligonucleotide and cyclized by incubation
with T4 DNA ligase.
[0570] A diagram showing the introduction of a decoy called vanH5
into a liquid culture of Streptomyces coelicolor strain M600 is
given in FIG. 23. S. coelicolor strain M600 was grown in liquid
MMCGT medium (Molecular Microbiology 52: 1107-1121), growth was
measured by recording the absorbance of the culture at 430 nm (Cell
Density) and plotted as a function of time of incubation. Growth of
four cultures was monitored: (1) with nothing added to the media
(diamonds `M600`); (2) the media supplemented with a sub-lethal
concentration of 20 .mu.g/ml of the antibiotic vancomycin at 0 h
(squares `plus 20 .mu.g/ml VAN); as in (2) but with the decoy
oligonucleotide H5 added to a final concentration of 64 pM
following 20 h of incubation (triangles `VAN plus 64 pM H5`); as in
(2) but with the decoy oligonucleotide H5 added to a final
concentration of 256 pM following 20 h of incubation (circles `VAN
plus 256 pM H5`).
[0571] From this data, it is evident that M600 grows comparably
well in the presence or absence of 20 ug/ml vancomycin (added or
not added at the start of the culture process), confirming that the
strain has resistance to vancomycin. In the presence of 20 ug/ml
vancomycin, the addition of the cyclized decoy vanH5 to a final
concentration of 64 pM has little detectable effect, while addition
to 256 pM causes the cells to cease growing over a period of
approximately 18 h. This may colloquially be referred to as a
"therapeutic window", where the decoys have entered the cells, have
interfered with the binding of the vanR regulator to the vanH
promoter, and thereby prevented the induction of the resistance
mechanism. After 18 h, our evidence from other systems suggests,
degradation of the decoys (by endonucleases within the cell)
occurs, which may affect their efficacy.
[0572] As for why the vanH decoy seemingly has a bacteriostatic
effect, we believe this is due to the mechanism of resistance being
a modification to the bacterial cell wall which prevents the action
of vancomycin. When vancomycin is added to the culture, this is at
the beginning of the experiment. The decoy is added at 20 hours. At
this point, the bacteria will have been growing with a modified
cell wall. Upon addition of the decoy, from that point on until the
decoy is dissipated, the ability to modify the bacterial cell wall
is blocked. As a result, any new bacterial growth is rendered
sensitive to vancomycin, although older cells are able to persist.
Upon depletion of the decoy, the persistent cells with residual
vancomycin resistance are able to once again proliferate. Adding
the decoy prior to the vancomycin would have little effect, as the
resistance operon is not on. We anticipate, however, that
administration of the decoy ODNs at the same time induces a
bacteriocidal effect.
[0573] Thus, this example demonstrates sensitivity of S. coelicolor
to vancomycin in the presence of the TFD's and not in their
absence. This would be accepted by those skilled in the art as an
acceptable surrogate for demonstrating this effect in a pathogen,
such as E. faecalis. The E. faecium van operon is induced by the
same mechanism (vanR binding to vanH), even though homology on a
sequence level between the two systems is not very high. Those
skilled in the art would be able to easily modify the decoy
sequence for use in the pathogen.
[0574] The vanH5 decoy is one example of oligodeoxynucleotides
which can bind to a prokaryotic transcription factor such that
binding of the transcription factor to its cognate target in the
genome of the prokaryote is diminished or obliterated.
[0575] 2. Designing TFDs to Combat Antibiotic Resistance in
Pathogens
[0576] The principle of how TFDs can be used to restore vancomycin
sensitivity has been discussed above. For situations where the
genes responsible are unknown, the universal n[snare]libraries or
custom libraries created from the genomic DNA of the pathogen
provided by this invention are used. As shown in FIG. 6 the library
can either be introduced into the pathogenic bacteria under
laboratory conditions or the genetic elements determining
resistance can be cloned and introduced into a non-pathogenic
laboratory strain to act as a model system, which can then be
transformed. The cells will then be cultured in the absence of the
chosen antibiotic and the library introduced into the cells by
transformation whilst in liquid culture, the sample is now split
and the antibiotic added to half of the sample and incubation
continued. The populations of cells are recovered and the
concatamerized TFDs amplified from the plasmids by PCR. These two
populations are subtracted to isolate the TFDs missing from the
antibiotic-treated sample, and hence those that conferred
sensitivity. This enriched population of TFDs is recloned and the
selection process repeated until it is sufficiently enriched in
TFDs capable of rendering the cell antibiotic sensitive.
[0577] Potential targets for such an approach would include
investigation of the mechanisms of resistance to many of the
clinically prescribed antibiotics, and future ones for which
antibiotic resistance begins to limit their efficacy. Examples of
the current antibiotics that would be considered for investigation
using the n[snare] methodologies and subsequent treatment with TFDs
to defeat antibiotic resistance would include: those from the class
of antibiotics known as aminoglycosides (such as kanamycin); from
the carbapenems (such as meropenem); the cephalosporins (such as
cefepime); the glycopeptides (such as vancomycin and daptomycin);
the penicillins (such as ampicillin, carbenicillin and penicillin);
the polypeptide antibiotics (such as polymixcin B); the quinolines
(such as levaquin); the sulfonamides (such as Bactrim); the
tetracyclines (such as tetracycline); and variously,
chloramphenicol, rifampicin, Zyvox.
Example 7
[0578] 7.1 Cholesterol Labeled TFDs in the Presence of
Streptolysin-O
[0579] Using oligonucleotide primers, one of which has a 5'
cholesterol modification and the other a similar modification at
its 5' end or some other (such as a fluorescent dye, such as Cy5,
so that the uptake of the TFD can be easily measured) a TFD is
prepared by PCR. If the TFD has been previously cloned into a
vector (pGEMT-Easy) the primers are designed to anneal to the
vector sequences immediately flanking the insert, for example:
TABLE-US-00006 SEQ ID NO: 22 Chol_TEf: 5' Cholesterol-TEG-ggc cgc
cat ggc ggc cgc ggg aat tc SEQ ID NO: 23 Cy5_TEr: 5' Cy5- AGG CG
CCG CGA ATT CAC TAG TG.
[0580] If the sequence to be used for a TFD has not been cloned it
may either be directly synthesized (if short enough) and annealed
to form the TFD, or amplified directly from genomic DNA using
primers designed to anneal within the TFD.
[0581] The PCR product is ethanol precipitated and resuspended in
TE buffer at a concentration of 500-1000 ng/.mu.l. Typically
antibiotic sensitivity assays are performed using 96 well plates,
each well containing 200 ml of broth. For example, in the case of
Enterococcus faecium this broth is BHI media (from Becton
Dickinson) supplemented with 0.2 U/ml Streptolysin-O (Sigma) and 5
.mu.g/ml of vancomycin antibiotic and inoculated with a
vancomycin-resistant strain of E. faecium.
[0582] 7.2 Preparation of Dumbbells by PCR
[0583] Dumbbell decoys are covalently closed single stranded DNA
characterised by a double-stranded centre, containing the binding
site for the targeted factor, flanked by stem-loop structures. The
stem-loops stabilize the decoy by preventing action of exonucleases
which would otherwise degrade the decoy polynucleotide. Hence
Dumbbell decoys (DB) are so called because of their characteristic
shape.
[0584] DBs are prepared by PCR using as a template a
pGEMTEasy-derived plasmid containing the targeted binding site, as
described in 7.1. The primers used in amplification are:
TABLE-US-00007 (SEQ ID NO: 24) DBTEF: 5' P- CTTGG TTTTT CCAAG
AGAAGAGC ccg cca tgg cgg ccg cgg gaa ttc (SEQ ID NO: 25) DBTEr: P-
CCG TCT TTT TGA CGG CGA AGA GCA GGC GGC CGC GAA TTC ACT AGT GA.
[0585] The portion of the primers which will form the stem-loops
are underlined. Amplification with the appropriate vector gives the
DNA product shown in FIG. 24, where the portion of the DB which
will bind to the transcription factor is given by `NNN NNN`. The
sequences in bold represent a binding site for the nicking
restriction enzyme Nt.BspQ1. In the second part of FIG. 24 the
consequence of digesting the PCR product with Nt.BspQ1 is shown;
this exposes the stem-loop structures as single stranded regions
which will form a stem-loop and can subsequently be ligated by
treatment with T4 DNA ligase to form a covalently closed circle and
DB.
[0586] 7.3 Preparation of Dumbbell Oligonucleotides by Restriction
Digest of Plasmid
[0587] Alternatively DBs can be made by cloning the blunted PCR
product shown in FIG. 24 into a suitable PCR-cloning vector, such
as pGEMTEasy, confirming its identity and preparation of the
plasmid. The plasmid can then be digested to release the insert
which is additionally digested with Nt.BspQ1 to release the
fragment shown in the second portion of FIG. 24. This can be
similarly treated with T4 DNA ligase in order to covalently close
the DNA molecule and form a DB.
[0588] The advantage of this approach is that is more amenable,
both practically and economically, to scaling up should the DB be
required in large quantities.
[0589] 7.4 Transfection with R9-Cholesterol Agent.
[0590] R9-cholesterol has been described for its properties of
aiding transfection of siRNA (or other nucleic-acid based therapy)
molecules and the like in eukaryotic cells (US Patent:
20070207966). Here we describe its utility in transfecting various
bacteria with TFDs.
[0591] R9-cholesterol, which consists of a cholesterol molecule
attached to a linear chain of nine D-arginines, was synthesized as
previously described (Kim W. J., et al., Mol. Ther 2006 14:
343-350). TFDs were mixed with increasing amounts of R9-cholesterol
in a TE based buffer supplemented with 5% glucose. The mixture was
incubated at room temperature for 1 hour and then either used
directly in transfections or analysed by agarose gel
electrophoresis. Typically the minimum-amount of R9-cholesterol was
used that caused the complex with DNA not to run in the gel; i.e.
the charge of the nucleic acid backbone had been neutralized by
binding of poly-arginine. The cholesterol molecule helps the TFD
associate with the bacterial membrane and so enter the cell.
[0592] TFD/R9-cholesterol conjugates were mixed at various
concentrations into 200 .mu.l of culture in a 96 well plate. For
example, in the case of Enterococcus faecium this broth is BHI
media (from Becton Dickinson) supplemented with 0.2 U/ml
Streptolysin-O (Sigma) and 5 .mu.g/ml of vancomycin antibiotic and
inoculated with a vancomycin-resistant strain of E. faecium.
Example 8
[0593] Use of a Van Decoy Sequence to Sensitize Enterococcus
faecium to Vancomycin
[0594] 8(a) Using Cholesterol Labeled TFDs in the Presence of
Streptolysin-O
[0595] Cholesterol/Cy5-labeled decoy polynucleotides were prepared
as in Example 7.1. The assays were performed using 96 well plates,
each well containing 200 ml of broth consisting of BHI media (from
Becton Dickinson) supplemented with 0.2 U/ml Streptolysin-O (Sigma)
and 5 .mu.g/ml of vancomycin antibiotic and inoculated with a
vancomycin-resistant strain of E. faecium.
[0596] 1 .mu.l of various concentrations of a
Cholesterol/Cy5-labeled decoy were added to each well and their
effect on bacterial growth of E. faecium monitored by measuring
absorbance of the broth at intervals during incubation. The plates
were incubated at 37.degree. C. with shaking and absorbance
readings (at 450 nM) were taken using a plate reader. Two decoys
were tested: VAN contains the regulatory element controlling
induction of VanA-type antibiotic resistance; CON is a decoy
sequence that does not occur in the E. faecium genome and was used
as a negative control. The sequence of the VAN decoy sequence (that
part cloned into pGEMT-Easy vector to create a plasmid used in the
PCR amplification step with the Chol_TEf and Cy5_TEr primers)
was:
TABLE-US-00008 (SEQ ID NO: 26) VAN TFD 5' AAA AAA GAA TCA TCA TCT
TAA GAA ATT CTT AGT CAT TTA 3'
[0597] The resultant growth curves are shown in FIG. 25. All data
points were performed in triplicate. It is evident that treatment
with the VAN TFD at concentrations as low as 40 nM resensitized the
E. faecium strain to vancomycin, whereas the CON negative control
had no effect. Uptake of TFDs was confirmed with qPCR and
fluorescence microscopy.
[0598] 8(b) Using Transfection with R9-Cholesterol Agent
[0599] The array used a 96 well plate, each well containing 200 ml
of culture, consisting of BHI media (from Becton Dickinson)
supplemented with 0.2 U/ml Streptolysin-O (Sigma) and 5 .mu.g/ml of
vancomycin antibiotic and inoculated with a vancomycin-resistant
strain of E. faecium.
[0600] 1 .mu.l of various concentrations of the TFD/R9-cholesterol
conjugates were added to each well and their effect on bacterial
growth monitored by measuring absorbance of the broth at intervals
during incubation. The plates were incubated at 37.degree. C. with
shaking and absorbance readings (at 595 nM) were taken using a
plate reader. Two Dumbbell TFDs were tested: VAN contains the
regulatory element controlling induction of VanA-type antibiotic
resistance (containing the same sequence as Seq. 26); CON is a
decoy sequence that does not occur in the E. faecium genome and was
used as a negative control (containing the same sequence as CON in
Example 8(a)).
[0601] The resultant growth curves are shown in FIG. 26. All data
points were performed in triplicate. It is evident that treatment
with the VAN TFD at concentrations as low as 40 nM resensitized the
E. faecium strain to vancomycin, whereas the negative control had
no negative impact on cell growth.
Sequence CWU 1
1
31147DNAArtificial SequenceSynthetic sequence Decoy oligonucleotide
containing the AfsR binding site 1aatacgactc actatagggg cgttgagcga
acgtttttcg cggccgc 47223DNAArtificial SequenceSynthetic sequence
Joining polynucleotide R-T7, which contains a partial complement of
a commonly used primer, T7 2ccctatagtg agtcgtattg cgg
23364DNAArtificial SequenceSynthetic sequence Oligonucleotide
containing the AfsR binding site and used to create a cyclised
decoy sequence 3atagcgttga gcgaacgttt ttcgcgtttt cgcgaaaaac
gttcgctcaa cgctatagtt 60ttct 64443DNAArtificial SequenceSynthetic
sequence Strand of a double stranded adaptor molecule 4ggtccgggcc
acggtggtct acgagcctca gccaggtccg act 43515DNAArtificial
SequenceSynthetic sequence Strand of a double stranded adaptor
molecule 5gtcggacctg gctga 15639DNAArtificial SequenceSynthetic
sequence Bbv complementary oligonucleotide 6ggacctggct gaggctcgta
gaccaccgtg gcccggacc 39736DNAArtificial SequenceSynthetic sequence
An oligonucleotide comprising a NotI site and a randomised
nucleotide sequencemisc_feature(20)..(28)n is a, c, g, or t
7aatacgactc actatagggn nnnnnnnngc ggccgc 36854DNAArtificial
SequenceSynthetic sequence Decoy oligonucleotide designed based on
cis-regulatory sequence A24.1 8atatcactct cgatgtcggc gttttcgccg
acatcgagag tgatatagtt ttct 54954DNAArtificial SequenceSynthetic
sequence Decoy oligonucleotide designed based on cis-regulatory
sequence A24.2 9atatcaggaa tgccagatgc gttttcgcat ctggcattcc
tgatatagtt ttct 541052DNAArtificial SequenceSynthetic sequence
Decoy oligonucleotide designed based on cis-regulatory sequence
A24.3 10atactgcctc tcggtaagcg ttttcgctta ccgagaggca gtatagtttt ct
521154DNAArtificial SequenceSynthetic sequence Decoy
oligonucleotide designed based on cis-regulatory sequence A24.4
11atatgcagct cgctgcacgc gttttcgcgt gcagcgagct gcatatagtt ttct
541254DNAArtificial SequenceSynthetic sequence Decoy
oligonucleotide designed based on cis-regulatory sequence A24.5
12ataaatctgt tgagtagggc gttttcgccc tactcaacag atttatagtt ttct
541354DNAArtificial SequenceSynthetic sequence Decoy
oligonucleotide designed based on a scrambled A24.5 sequence
13atagcaattt agatggtggc gttttcgcca ccatctaaat tgctatagtt ttct
541424DNAArtificial SequenceSynthetic sequence PCR primer used in
quantitative PCR 14cgacatcgag agtgatatag tttt 241520DNAArtificial
SequenceSynthetic sequence PCR primer used in quantitative PCR
15gccgacatcg agagtgatat 201621DNAArtificial SequenceSynthetic
sequence PCR primer used in quantitative PCR 16tgtcgccccc
aggagacgga g 211724DNAArtificial SequenceSynthetic sequence PCR
primer used in quantitative PCR 17cttaaatcct cgaaggcgac ccag
241821DNAArtificial SequenceSynthetic sequence PCR primer used in
quantitative PCR 18tcctcgagcc ggttctcctc g 211918DNAArtificial
SequenceSynthetic sequence PCR primer used in quantitative PCR
19gctggtggac atcggtct 182019DNAArtificial SequenceSynthetic
sequence PCR primer used in quantitative PCR 20gccggtactt gtcgctctc
192188DNAArtificial SequenceSynthetic sequence A vanH5 decoy
oligonucleotide containing a binding site for phosphorylated VanR
21atatctatat gaagcgacgt ggtcgatgag ccgcagcgtt ttcgctgcgg ctcatcgacc
60acgtcgcttc atatagatat agttttct 882226DNAArtificial
SequenceSynthetic sequence Oligonucleotide primer used for
amplification of a target sequence from the pGEMT-Easy vector
22ggccgccatg gcggccgcgg gaattc 262323DNAArtificial
SequenceSynthetic sequence Oligonucleotide primer used for
amplification of a target sequence from the pGEMT-Easy vector
23aggcggccgc gaattcacta gtg 232447DNAArtificial SequenceSynthetic
sequence Oligonucleotide primer used for amplification of a target
sequence from the pGEMT-Easy vector in the production of dumbbell
decoys 24cttggttttt ccaagagaag agcccgccat ggcggccgcg ggaattc
472547DNAArtificial SequenceSynthetic sequence Oligonucleotide
primer used for amplification of a target sequence from the
pGEMT-Easy vector in the production of dumbbell decoys 25ccgtcttttt
gacggcgaag agcaggcggc cgcgaattca ctagtga 472639DNAArtificial
SequenceSynthetic sequence VAN decoy sequence containing the
regulatory element controlling induction of VanA type resistance in
E. faecium. 26aaaaaagaat catcatctta agaaattctt agtcattta
3927300DNAStreptomyces coelicolor 27tcactctcga tgtcggccgg
tggatgtggt ggccgcaccc gctcgcccgg cgcgaggacc 60cttccgagga cccagccgta
tcaggaatgc cagattctat tgattcggaa gcctcgacca 120ctgcctctcg
gtaaaatcca gcaaaaatta atcagtgcag ctcgctgcac tgattaattt
180ttgatcaata ggagatcgct tgtgacggca agcacattga aatctgttga
gtaggcctgt 240tattgtcgcc cccaggagac ggagaatctc gacgggggcg
cagatgagat caacttattg 30028101DNAArtificial SequenceSynthetic
sequence Amplification product obtained using the primers in SEQ ID
NOs 24 & 25 and an appropriate vector
substratemisc_feature(48)..(53)n is a, c, g, or t 28cttggttttt
ccaagagaag agcccgccat ggcggccgcg ggaattcnnn nnntcactag 60tgaattcgcg
gccgcctgct cttcgccgtc aaaaagacgg a 10129100DNAArtificial
SequenceSynthetic sequence Amplification product obtained using the
primers in SEQ ID NOs 24 & 25 and an appropriate vector
substratemisc_feature(48)..(53)n is a, c, g, or t 29ccgtcttttt
gacggcgaag tgcaggcggc cgcgaattca ctagtgannn nnngaattcc 60cgcggccgcc
atggcgggct cttctcttgg aaaaaccaag 1003085DNAArtificial
SequenceSynthetic sequence Consequence of digesting PCR product
with Nt.BspQ1misc_feature(48)..(53)n is a, c, g, or t 30cttggttttt
ccaagagaag agcccgccat ggcggccgcg ggaattcnnn nnntcactag 60tgaattcgcg
gccgcctgct cttcg 853185DNAArtificial SequenceSynthetic sequence
Consequence of digesting PCR product with
Nt.BspQ1misc_feature(48)..(53)n is a, c, g, or t 31ccgtcttttt
gacggcgaag tgcaggcggc cgcgaattca ctagtgannn nnngaattcc 60cgcggccgcc
atggcgggct cttct 85
* * * * *
References