U.S. patent application number 10/087187 was filed with the patent office on 2003-03-27 for unmarked deletion mutants of mycobacteria and methods of using same.
Invention is credited to Jacobs, William R. JR., Pavelka, Martin S. JR..
Application Number | 20030059441 10/087187 |
Document ID | / |
Family ID | 23375012 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030059441 |
Kind Code |
A1 |
Pavelka, Martin S. JR. ; et
al. |
March 27, 2003 |
Unmarked deletion mutants of mycobacteria and methods of using
same
Abstract
Disclosed is a recombinant slow-growing mycobacterium comprising
at least one mycobacterial gene containing an unmarked mutation,
where an "unmarked mutation" is a mutated nucleotide sequence
introduced into a mycobacterium where the introduced mutated
nucleotide sequence does not contain a selectable marker, such as a
gene conferring antibiotic resistance to the recombinant
mycobacterium incorporating the mutated nucleotide sequence. Also
disclosed is a method for preparing a recombinant slow-growing
mycobacterium comprising at least one mycobacterial gene containing
an unmarked mutation, as well as a vaccine comprising a recombinant
slow-growing mycobacterium having at least one mycobacterial gene
containing an unmarked mutation dispersed in a physiologically
acceptable carrier. Further disclosed is a method of treating or
preventing tuberculosis in a subject comprising administering the
vaccine of the present invention in an amount effective to treat or
prevent tuberculosis in the subject.
Inventors: |
Pavelka, Martin S. JR.;
(Rochester, NY) ; Jacobs, William R. JR.; (City
Island, NY) |
Correspondence
Address: |
Craig J. Arnold
Amster, Rothstein & Ebenstein
90 Park Avenue
New York
NY
10016
US
|
Family ID: |
23375012 |
Appl. No.: |
10/087187 |
Filed: |
February 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10087187 |
Feb 28, 2002 |
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09350047 |
Jul 8, 1999 |
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6423545 |
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Current U.S.
Class: |
424/200.1 ;
435/252.3; 435/253.1; 435/476 |
Current CPC
Class: |
C12R 2001/32 20210501;
A61P 31/04 20180101; C12N 9/0004 20130101; C12N 1/205 20210501;
C12N 15/74 20130101; A61K 39/00 20130101 |
Class at
Publication: |
424/200.1 ;
435/252.3; 435/253.1; 435/476 |
International
Class: |
A61K 039/02; C12N
001/21; C12N 015/74 |
Goverment Interests
[0001] This invention is supported by NIH Grant Nos. AI26170 and
AI33696. As such, the U.S. Government has certain rights in this
invention.
Claims
What is claimed is:
1. A recombinant slow-growing mycobacterium comprising at least one
mycobacterial gene containing an unmarked mutation.
2. The recombinant slow-growing mycobacterium of claim 1 wherein
the mutant mycobacterial gene comprises a deletion, addition,
substitution or point mutation.
3. The recombinant slow-growing mycobacterium of claim 2 wherein
the mycobacterial gene is a gene that encodes an enzyme essential
in the biosynthetic pathway of a nutrient, structural component or
an amino acid.
4. The recombinant slow-growing mycobacterium of claim 3 wherein
the mycobacterium is auxotrophic for lysine.
5. The recombinant slow-growing mycobacterium of claim 4 wherein
the mycobacterial gene is lysA.
6. The recombinant slow-growing mycobacterium of claim 5 which is
selected from the group of M. bovis BCG, M. tuberculosis, and M.
leprae.
7. A method for preparing the recombinant slow-growing
mycobacterium of claim 1, comprising: (a) introducing a vector into
a slow-growing mycobacterium, said vector comprising a selectable
marker, a counterselectable marker, and an unmarked mutant
mycobacterial gene; (b) selecting for primary recombinants
incorporating the selectable marker; (c) culturing the primary
recombinants incorporating the selectable marker; (d) selecting for
secondary recombinants that have lost the counterselectable marker;
and (f) isolating the secondary recombinants comprising the desired
unmarked mutant mycobacterial gene.
8. The method of claim 7, wherein the vector is a suicide
plasmid.
9. The method of claim 8, wherein the selectable marker confers
antibiotic resistance and the counterselectable marker is one of
rpsL, pyrF, and sacB.
10. The method of claim 9, wherein the counterselectable marker is
sacB.
11. The method of claim 7, wherein the recombinant slow-growing
mycobacterium is auxotrophic for lysine.
12. The method of claim 11, wherein the mycobacterial gene is
lysA.
13. A vaccine that comprises (i) a recombinant slow-growing
mycobacterium comprising at least one mycobacterial gene containing
an unmarked mutation and (ii) a physiologically acceptable
carrier.
14. The vaccine of claim 13, wherein the unmarked mutation is a
deletion, addition, substitution or point mutation.
15. The vaccine of claim 14, wherein the slow-growing mycobacterium
is auxotrophic for lysine.
16. The vaccine of claim 15, wherein the gene is lysA.
17. The vaccine of claim 16, wherein the slow-growing mycobacterium
is selected from M. bovis BCG, M. tuberculosis, and M. leprae.
18. A method of treating or preventing tuberculosis in a subject
comprising administering the vaccine of claim 17 in an amount
effective to treat or prevent tuberculosis in the subject.
Description
BACKGROUND OF THE INVENTION
[0002] Mycobacterium tuberculosis, the agent of tuberculosis, is
the leading cause of death in adults worldwide (14). The emergence
of drug resistant strains (48) and the problems associated with
tuberculosis in HIV-infected populations (18) have brought
tuberculosis research to the forefront. The development of genetic
techniques to study the biology of the organism is an important
goal of mycobacterial research.
[0003] Considerable effort has gone into the development of allelic
exchange methods to selectively disrupt genes of various
mycobacterial species. Several groups have used either small linear
DNA fragments (4, 25, 43), long linear DNA fragments (5), or
suicidal plasmids, (37, 44) (9, 27, 39, 41, 42) to achieve allelic
exchange in both fast and slow-growing mycobacteria. Slow-growing
mycobacteria such as M. tuberculosis and M. bovis BCG can integrate
exogenous DNA into their chromosome by both illegitimate and
homologous recombination (2, 25). Allelic exchange in fast-growing
mycobacteria such as M. smegmatis is easier than in the
slow-growing species; this has led to the idea that the homologous
recombination machinery of slow-growing mycobacteria is rather
inefficient (32).
[0004] Thus far, the only mutants constructed in the slow-growing
mycobacterial species are those with genes disrupted with an
antibiotic marker. However, in many cases an antibiotic marker may
not be desirable. It may not be known whether or not a gene is
essential and targeted disruption does not let one ascertain
essentiality. The failure to obtain a mutant might be due to the
failure of the methodology and not to the essentiality of the gene.
Furthermore, the possibility of polar effects from an inserted
antibiotic marker can prevent the disruption of a non-essential
gene if that gene is located in an operon upstream of an essential
gene. Also, there are a limited number of antibiotic resistance
genes available for use in mycobacteria and making a marked
mutation excludes one antibiotic from further consideration. In
addition, mutants that are potential vaccine candidates should not
contain antibiotic resistance determinants.
[0005] An ideal allelic exchange system is one that can be used for
the exchange of unmarked deletion alleles as well as alleles with
point mutations. Constructing knockout mutants by in-frame
deletions would negate the concerns with using a targeted
disruption method. Such mutants are antibiotic sensitive, cannot
revert, and the mutations should not be polar on the expression of
downstream genes. By extension, the same technique could be used
for allelic exchange of point mutations, allowing for a finer
dissection of gene function. This allelic exchange methodology,
utilizing a plasmid unable to replicate in the organism of interest
and selectable and counter-selectable markers (15), has been
successfully used in M. smegmatis (27, 41). The inventors sought to
determine if such an allelic exchange methodology would
reproducibly work for the slow-growing mycobacteria, such as M.
bovis BCG and M. tuberculosis.
[0006] The inventors describe herein a new mycobacterial suicide
plasmid for allelic exchange of unmarked mutations utilizing sacB
sucrose counter selection. This counter selectable marker was
previously reported to work in mycobacteria, including M.
tuberculosis and M. bovis BCG (40) (42) (9). However, the
previously described mycobacterial sacb vector systems were used
for allelic exchange of genes disrupted with an antibiotic
resistance marker. The present invention demonstrates the
reproducibility of this system for allelic exchange of unmarked
deletions in the chromosome of M. smegmatis, M. bovis BCG and M.
tuberculosis. The inventors have also constructed lysine auxotrophs
of these three organisms by allelic exchange of lysA, the gene
encoding meso-diaminopimelate decarboxylase, the last enzyme in the
lysine biosynthetic pathway (52). To the best of the inventors'
knowledge, this is the first report of the construction of unmarked
deletion mutations in the genome of slow-growing mycobacteria.
SUMMARY OF THE INVENTION
[0007] The present invention discloses a slow-growing recombinant
mutant mycobacterium comprising at least one mycobacterial gene
containing an unmarked mutation. The invention further provides a
method for preparing the recombinant mutant mycobacterium of the
present invention comprising introducing a vector into a
slow-growing mycobacterium, where said vector comprises a
selectable marker, a counter selectable marker, and an unmarked
mutant mycobacterial gene, culturing the slow-growing mycobacterium
and selecting for primary recombinants incorporating the selectable
marker. The primary recombinants are then cultured, and secondary
recombinants that have lost the counter selectable marker are
selected for, followed by isolation of the secondary recombinants
incorporating the desired unmarked mutant mycobacterial gene.
[0008] Also provided is a vaccine comprising the slow-growing
recombinant mutant mycobacterium of the present invention contained
in a physiologically acceptable carrier, as well as a method of
treating or preventing tuberculosis in a subject comprising
administering the vaccine of the present invention in an amount
effective to treat or prevent tuberculosis in the subject.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 depicts a map of the suicide vector pYUB657. The
vector pYUB657 cannot replicate in mycobacteria, but has the ColE1
origin of replication for E. coli. The P.sub.groEL-sacB cassette is
indicated along with the sacR regulatory region (50). The vector
has the bla gene, conferring resistance to ampicillin in E. coli
and the hyg gene, conferring resistance to hygromycin in
mycobacteria. This vector is also a double cos, PacI-excisable
cosmid cloning vector (5). Useful cloning sites are indicated.
[0010] FIG. 2 illustrates Southern blots of genomic DNA from four
mycobacterial lysa deletion mutants. Panel A depicts genomic DNA
from wild-type M. smegmatis mc.sup.2155 (Lane 1) and the M.
smegmatis auxotroph mc.sup.21493 (Lane 2), digested with EcoRI and
probed with a 3.3-kb EcoRI fragment from plasmid pYUB617,
encompassing the .DELTA.lysA4 allele. The wild-type fragment is the
expected 4.4-kb, while the mutant has the expected 3.2-kb fragment.
Panel B depicts genomic DNA from wild-type BCG substrain Pasteur
(Lane 1), BCG substrain Pasteur auxotroph mc.sup.21604 (Lane 2),
wild-type BCG substrain Connaught (Lane 3), BCG substrain Connaught
auxotroph mc.sup.22519 (Lane 4), wild-type M. tuberculosis H37Rv
(Lane 5), and M. tuberculosis H37Rv auxotroph mc.sup.23026 (Lane
6), digested with BssHII and probed with a lysA PCR product
obtained from BCG Pasteur wild-type genomic DNA. Digestion of
wild-type genomic DNA with BssHII splits the lysA gene over two
fragments, one which is 1.1-kb in size, the other which is 1.2-kb.
Digestion of genomic DNA from the deletion mutants yields the same
1.2-kb fragment seen in wild-type with a 0.9-kb fragment,
corresponding to the deletion site, replacing the 1.1-kb fragment.
The blots in Panel B show the expected shift in size of the 1.1-kb
fragment down to 0.9-kb in all three mutants (Lanes 2, 4, and 8).
The invariant 1.2-kb fragment shows a lower intensity in the blot
due to a lower percentage of homology to the probe, relative to the
1.1 and 0.9-kb fragments.
[0011] FIG. 3 illustrates the effect of AEC on the growth of
wild-type M. bovis BCG, and M. tuberculosis H37Rv. Growth curve
data were obtained as described in the Materials and Methods. Panel
A illustrates growth of M. bovis BCG substrain Pasteur; Panel B
illustrates growth of M. tuberculosis H37Rv. (Key: Basal (7H9
medium), AEC (Basal with 3 mM AEC), Thr (Basal with 3 mM
threonine), AEC/Thr (Basal with AEC and threonine at 3 mM
each.)
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention provides a method for yielding
recombinant unmarked mutants of mycobacteria, wherein the
recombinant mutant mycobacteria comprises at least one
mycobacterial gene containing an unmarked mutation. As used herein,
an "unmarked mutation" is a mutated nucleotide sequence (i.e., a
mutant DNA substrate) that is homologous to and replaces a wildtype
nucleic acid sequence of the mycobacteria via homologous
recombination, where said mutant DNA substrate does not contain a
selectable marker, such as a gene conferring antibiotic resistance
to the recombinant mycobacterium incorporating the mutated
nucleotide sequence. The term "recombinant unmarked mutant
mycobacteria" as used herein means that the recombinant unmarked
mutant mycobacterium comprises at least one unmarked mutation, such
that the expression or function of the mutant DNA substrate
incorporated into the recombinant mycobacterium is varied with
respect to the non-mutated nucleotide sequence in the parent
strain. The method of the present invention is particularly suited
for generating mutants via allelic exchange in Mycobacterium
tuberculosis complex organisms, preferably strains of M.
tuberculosis, M. bovis and Bacille-Calmette-Geurin (BCG), which are
slow-growing mycobacteria, as well as in other slow growing
mycobacteria, although the method may be used with nontuberculosis
fast-growing mycobacteria commonly encountered in biological
samples isolated from human subjects, e.g., M.
avium-intracellulare, M. kansasii, M. xenopi, M. scrofulaceum, M.
simiae, M. szulgai, M. gordonae, M. gastri, M. smegmatis, and M.
chelonae.
[0013] The method for preparing a recombinant unmarked mutant of
the present invention comprises introducing a vector into a
slow-growing mycobacterium, where said vector comprises a
selectable marker, a counter selectable marker, and a mutant DNA
substrate for allelic exchange, then growing the mycobacterium and
selecting for primary recombinants incorporating the selectable
marker, then culturing the primary recombinants incorporating the
selectable marker and selecting for secondary recombinants that
have lost the counter selectable marker, and isolating the
secondary recombinants comprising the desired unmarked mutation.
The method of the invention may also be used to produce recombinant
unmarked mutant mycobacteria that are fast-growing mycobacteria,
including recombinant mutant strains of M. smegmatis or M. avium,
but is preferably used to produce recombinant unmarked mutant
strains of slow-growing mycobacteria, and more preferably,
recombinant unmarked mutants of M. tuberculosis or M. bovis BCG
strains.
[0014] The vector of the present invention is a plasmid which is
unable to replicate in mycobacteria (i.e., a suicide plasmid),
having a selectable marker and counter selectable marker on the
plasmid backbone. Selectable marker genes which may be included on
the plasmid are well known in the art and include, but are not
limited to, genes encoding resistance to antibiotics, including
carbenicillin, viomycin, thiostrepton, ampicillin, tetracyline,
hygromycin, kanamycin or bleomycin. In a preferred embodiment of
the invention, the selectable marker genes included on the vector
are genes encoding for ampicillin and hygromycin resistance. The
counter selectable marker which is included on the vector confers
susceptibility to a specific agent, and preferably is one of the
rpsL, pyrF, or sacB genes, and more preferably is the sacB gene
encoding for levansucrase and conferring susceptibility to
sucrose.
[0015] The mutant DNA substrate for allelic exchange may be of any
origin, but is preferably from a mycobacterium. In a preferred
embodiment of the invention, the mutated DNA substrate for allelic
exchange is from a mycobacterium and is homologous to a wildtype
nucleic acid sequence of the mycobacterium in which it is desired
to introduce the mutated DNA substrate in lieu of the wildtype
nucleic acid sequence.
[0016] The DNA substrate for allelic exchange contains the mutation
of interest, which through allelic exchange, is introduced into and
replaces the homologous region of the mycobacterium nucleic acid.
As used herein, "mutated DNA substrate" refers to the nucleotide
sequence for at least one allele that has been modified by
addition, substitution or deletion of at least one nucleotide, and
lacks any selectable marker. In a preferred embodiment of the
invention, the mutated DNA substrate comprises a deletion or point
mutation of the wildtype nucleic acid sequence. Mutations,
including but not limited to deletion, point, substitution, or
insertion mutations, may be generated by any number of methods
known in the art, including but not limited to treatment with
restriction endonucleases, inverse PCR, subcloning techniques and
other methods of in vitro mutagenesis. The wildtype nucleic acid
sequence may encode a protein or polypeptide, and in a preferred
embodiment of the invention encodes an enzyme essential in the
biosynthetic pathway of a nutrient, structural or cell wall
component of the mycobacterium, or an amino acid, such as lysine,
leucine, methionine, etc. It is also within the confines of the
present invention that the wildtype nucleic acid of the
mycobacterium may comprise an operon or cluster of alleles encoding
a number of proteins or polypeptides, or one or more promoters,
enhancers or regulators that are involved in the expression and
translation of mycobacterial proteins and polypeptides. In a
preferred embodiment of the invention, the wildtype nucleic acid
comprises the lysA gene.
[0017] The suicide vector, comprising a selectable marker, a
counter selectable marker, and the mutant DNA substrate for allelic
exchange, is introduced to the mycobacteria using any suitable
method known in the art, including by electroporation. Primary
recombinants incorporating the selectable marker are directly
selected for using the appropriate agent, for instance, by exposing
the mycobacterium to hygromycin and obtaining Hyg.sup.r clones
where the selectable marker confers resistance to hygromycin.
Secondary recombinants that have lost the counter selectable marker
are directly selected for by using the appropriate agent, for
instance, by exposing the mycobacterium to sucrose and obtaining
suc'clones where the counter selectable marker is sacB. Once
suspected secondary homologous recombinants comprising the desired
unmarked mutation are isolated, the unmarked mutation genotype may
be confirmed by methods known in the art, such as PCR screening or
Southern blot analysis.
[0018] The method of the present invention may be used to generate
numerous strains of auxotrophic recombinant unmarked mutant
mycobacteria that are auxotrophic for a particular nutrient or
nutrients by reason of the substitution via allelic exchange of a
wildtype nucleic acid sequence of a mycobacterium with a mutated
DNA substrate. As used herein, the term "auxotrophic recombinant
unmarked mutant mycobacterium" is defined as a mycobacterium having
an unmarked mutation resulting in the nutritional requirements of
the mycobacterium being altered. For example, some auxotrophic
mutants are unable to synthesize amino acids, or may require
specific amino acids that are not needed by the parental or
prototrophic strain. Specific auxotrophic recombinant unmarked
mutant mycobacteria of the present invention include slow-growing
mycobacteria which are auxotrophic for lysine, although other
auxotrophic recombinant unmarked mutants of slow growing
mycobacteria are provided for, including recombinant unmarked
mutants that are auxotrophic for leucine, threonine, methionine,
etc. Preferably, the auxotrophic recombinant mutant mycobacteria
are strains of M. bovis BCG or M. tuberculosis, but the invention
is not limited to these species of mycobacteria. In a specific
embodiment of the invention, the auxotrophic recombinant unmarked
mutant mycobacteria that is auxotrophic for lysine comprises an
unmarked mutation of the lysA gene.
[0019] The present invention provides a vaccine comprising an
auxotrophic recombinant unmarked mutant mycobacterium. The
invention also provides a method of treating or preventing
tuberculosis in a subject comprising administering the vaccine of
the present invention in an amount effective to treat or prevent
tuberculosis in the subject. In this regard, the vaccine containing
the recombinant unmarked mutant slow-growing mycobacteria of the
present invention may be administered in conjunction with a
suitable physiologically acceptable carrier. Mineral oil, alum,
synthetic polymers, etc., are representative examples of suitable
carriers. Vehicles for vaccines and therapeutic agents are well
within the skill of one skilled in the art. The selection of a
suitable vaccine is also dependent upon the manner in which the
vaccine or therapeutic agent is to be administered. The vaccine or
therapeutic agent may be in the form of an injectable dose and may
be administered intramuscularly, intravenously, orally,
intradermally, or by subcutaneous administration.
[0020] Further, mycobacteria have well known adjuvant properties
and so are able to stimulate a subject's immune response to respond
to their antigens with great effectiveness. Their adjuvant
properties are especially useful in providing immunity against
pathogens in cases where cell mediated immunity is critical for
resistance. In addition, the mycobacterium stimulates long-term
memory or immunity and thus a single inoculum may be used to
produce long term sensitization to protein antigens. The vaccine of
the present invention may be used to prime long-lasting T-cell
memory, which stimulates secondary antibody responses which will
neutralize infectious agents or toxins, e.g., tetanus and diptheria
toxins, pertussis, malaria, influenza, herpes virus and snake
venom.
[0021] In addition, the recombinant unmarked mutant mycobacterium
of the present invention that is auxotrophic for lysine may be used
in the construction of DAP auxotrophs (peptidoglycan mutants).
[0022] The present invention is described in the following
Experimental Details Section which is set forth to aid in the
understanding of the invention, and should not be construed to
limit in any way the scope of the invention as defined in the
claims which follow thereafter.
EXPERIMENTAL DETAILS SECTION
A) Materials and Methods
[0023] Bacterial strains and culture methods: The bacterial strains
used are listed in Table 1. The genetic nomenclature for strains
bearing an integrated suicide plasmid (DUP) is as previously
described (37). E. coli cultures were grown in LB (Luria-Bertani)
broth or on LB agar (DIFCO). Mycobacterial cultures were grown in
Middlebrook 7H9 broth (DIFCO) with 0.05% Tween-80, or on 7H9 medium
solidified with 1.5% agar or on Middlebrook 7H10 or 7H11 media
(DIFCO). All cultures were incubated at 37.degree. C. All
Middlebrook media were supplemented with 0.2% (v/v) glycerol and
with 1X ADS (0.5% bovine serum albumin, fraction V (Boehringer
Mannheim), 0.2% dextrose, and 0.85% NaCl) for M. bovis BCG and M.
tuberculosis cultures. Basal media were 7H9 and 7H10 supplemented
as described above. Sucrose was used in the medium at a
concentration of 2% (w/v), added after the medium was autoclaved
and cooled to 55.degree. C. Casamino acids (acid-hydrolyzed casein,
DIFCO) was used at a concentration of 0.2 % (w/v). Individual amino
acids were obtained from Sigma Chemical (St. Louis, Mo.) and used
at a concentration of 40 .mu.g/ml, unless indicated otherwise. The
lysine analog S-(.beta.-aminoethyl)-L-cysteine (AEC) was obtained
from Sigma Chemical, dissolved in water and used at a concentration
of 3 mM. When required, the following antibiotics were used at the
specified concentrations; carbenicillin (50 .mu.g/ml; E. coli),
kanamycin A monosulfate (25 .mu.g/ml; E. coli, M. smegmatis, M.
bovis BCG), hygromycin B (50 .mu.g/ml; E. coli, M. bovis BCG, and
M. tuberculosis, 150 .mu.g/ml; M. smegmatis,). Hygromycin B was
purchased from Boehringer Mannheim (50 mg/ml in phosphate buffered
saline), all other antibiotics were purchased from Sigma Chemical.
It was often found that pYUB412 and pYUB405-based plasmids were
only stable in E. coli using both carbenicillin and hygromycin at
50 .mu.g/ml in solid and liquid media. M. smegmatis plates were
incubated for 3-5 days, while M. bovis BCG and M. tuberculosis
plates were incubated for 3-4 weeks. M. smegmatis liquid starter
cultures were inoculated from plates into 10 ml of medium in 30 ml
plastic media bottles, grown for 1-2 days on a shaker platform at
100 rpm and then subcultured 1:100 in fresh media within 250 ml
glass baffle flasks. M. bovis BCG and M. tuberculosis starter
cultures were inoculated using 1 ml frozen stocks in 10 ml of media
in 30 ml plastic media bottles and incubated for 5-7 days on a
shaker platform at 100 rpm. Larger cultures were inoculated from
the starter cultures at a 1:50 dilution in 50 ml or 100 ml of
medium within 490 cm.sup.2 roller bottles (Corning) and incubated
on a roller apparatus at 8 rpm for 5-7 days. For growth curves, mid
to late exponential phase cultures were centrifuged, washed with
fresh media lacking supplements, and the cells resuspended
appropriately and inoculated into test media. Samples of M.
tuberculosis and BCG cultures were mixed 1:1 with 10%
phosphate-buffered formalin and fixed for at least 1 hour prior to
spectrophotometric measurement at O.D..sub.600.
[0024] DNA methodologies: DNA manipulations were done essentially
as previously described (29). The plasmids used in this study are
listed in Table 2. Plasmids were constructed in E. coli HB101 or
DH5.alpha. cells and prepared by an alkaline lysis protocol (22).
Plasmids used for recombination were purified using Qiagen columns
as recommended by the manufacturer (Qiagen, Inc., Chatsworth,
Calif.). DNA fragments used for plasmid construction were purified
by agarose gel electrophoresis and recovered by absorption to glass
fines (GeneClean, Bio 101, Vista, Calif.).
[0025] Genomic DNA was prepared either as previously described (23)
or by a modified guandinium thiocyanate protocol (34). Briefly, the
cells from a 10 ml culture are lysed with 1.3 ml of a 3:1 mixture
of chloroform: methanol. The lysate is mixed with 1.3 ml of
Tris-equilibrated phenol and a 2 ml of GTC solution (4 M guandinium
thiocyanate, 0.1 M Tris pH 7.5, 0.5% sarcosyl, with
.beta.-mercaptoethanol added to a final concentration of 1% prior
to use). The upper phase is collected after centrifugation and the
genomic DNA precipitated with isopropanol. Southern blotting and
hybridization were done as previously described (37).
Oligonucleotides for sequencing and PCR were synthesized by the
Albert Einstein College of Medicine oligonucleotide synthesis
facility.
[0026] Cloning and sequencing of the M. smegmatis lysA operon: The
inventors used a library of genomic DNA from wild-type M. smegmatis
mc.sup.2155 constructed in the cosmid vector pYUB412 to clone the
lysA gene. The vector pYUB412 is an integration-proficient,
PacI-excisable cosmid vector (6). This cosmid vector has the
mycobacteriophage L5 attachment site (attP), the L5 integrase gene
(int), and the hyg gene, conferring resistance to hygromycin. This
vector efficiently integrates into the mycobacteriophage L5
attachment site (attB) of the mycobacterial chromosome and is
stable (28). The pYUB412::mc.sup.2155 library was electroporated
into the strain MCK3037, a lysine auxotrophic mutant of mc.sup.2155
generated by EMS mutagenesis (33). Transformants were selected on
7H10 media lacking lysine and Lys' clones screened for the
hygromycin resistance marker carried on the cosmid vector backbone.
One Lys.sup.+ Hygr clone was chosen for study and the genomic DNA
insert within the integrated cosmid recovered by .lambda. in vitro
packaging (GigaPak III, Strategene). The recovery procedure is as
follows: the library insert DNA is flanked by PacI restriction
endonuclease sites present in the cosmid vector, and since PacI
sites do not exist in mycobacterial genomic DNA (26), PacI
digestion of the genomic DNA will release the cosmid insert DNA.
This DNA fragment is re-packaged into PacI-digested arms of the
cosmid vector pYUB412 by .lambda. in vitro packaging, and a new
cosmid (pYUB601) with the insert recovered in E. coli. The cosmid
pYUB601 insert DNA was subcloned to a 4.4-kb EcoRI fragment bearing
the lysA gene in plasmid pYUB604. The plasmid pYUB604, and two
subclones, pYUB605 and pYUB607, were templates for DNA sequencing
using the Applied Biosystems Prism Dye Terminator Cycle Sequencing
Core kit with AmpliTaq DNA polymerase (Perkin Elmer) and an Applied
Biosystems 377 automated DNA sequencer. Sequence data for both
strands of the lysA operon of M. smegmatis were obtained from these
subclones and by primer walking.
[0027] Construction of sacB suicide vector pYUB657: A 2.5-kb PstI
fragment from the E. coli sacB vector pVCD442 bearing sacB and its
upstream regulatory region sacR, were subcloned into the PstI site
of the shuttle vector pMV261 downstream of the mycobacterial groEL
(Hsp60) promoter, yielding the plasmid pYUB631. A 3.5-kb NotI-NheI
fragment from pYUB631, bearing P.sub.groEL-sacB was cloned into the
cosmid vector pYUB405, resulting in the final construct, pYUB657
(see FIG. 1). The vector pYUB405 is a PacI-excisable cosmid vector
unable to replicate in mycobacteria and encodes resistance to
ampicillin and hygromycin (6).
[0028] Construction of the M. smegmatis .DELTA.lysA4 suicide
plasmid pYUB618: The plasmid pYUB604 was used as the template in an
inverse PCR reaction to produce a deletion within the lysA gene.
Oligonucleotide primers Pv44 (5'-CCCGTCGTACGTACGAACCAGGTTGCGC-3')
and Pv45 (5'-CGAGTCGATACGTACTGCTGTGCCGCCC-3') were used at 50 pmol
each in an inverse XL-PCR reaction in a Perkin Elmer 9600
temperature cycler with the following program: 95.degree. C./5 min,
1 cycle; 93.degree. C./1 min-68.degree. C./5 min, 16 cycles;
93.degree. C./1 min-68.degree. C./5 min with .DELTA.Th=15 sec, 12
cycles; 72.degree. C./30 min. The reaction produced a 7.7-kb
fragment with a 1.2-kb deletion within the lysA ORF (spanning nt
positions 2051 . . . 3251 of GenBank accession AF126720) marked
with a unique SnaBI site. The PCR product was gel purified,
digested with SnaBI and self-ligated to yield the plasmid pYUB617.
A 3.2-kb EcoRI fragment from pYUB617 bearing the .DELTA.lysA4
allele was cloned into the PacI sites of the mycobacterial sacB
suicide vector pYUB657, resulting in the M. smegmatis .DELTA.lysA4
suicide plasmid pYUB618.
[0029] Construction of the M. bovis BCG/M. tuberculosis
.DELTA.lysA5:: res suicide plasmid pYUB668: The lysA gene of M.
tuberculosis was originally cloned and sequenced by Anderson et al.
(3). The plasmid pET3d.lysA contains the lysA gene of M.
tuberculosis strain Erdman cloned by PCR using primers designed
from the previously published sequence (3)(16). A 1.3-kb XbaI-BamHI
fragment bearing the lysA gene was cloned from pET3d.lysA into the
same sites in pKSI.sup.+ to produce pYUB635. This plasmid was used
as the template in an inverse PCR reaction with the oligonucleotide
primers Pv7: (5'-GATAGCGGTCACGCGTCTCGTGCGCGGTGGA-3') and Pv8
(5-TCCGTACGATACGCGTCAGCCACATCGGTTCG-3') to generate a 95-bp
deletion within the lysA gene marked with a unique MluI restriction
endonuclease site. The inverse XL-PCR reaction was done using a
Perkin Elmer 9600 temperature cycler and the program described
above for plasmid pYUB617. The resulting 4.1-kb PCR product was
gel-purified, digested with MluI and self-ligated to yield the
plasmid pYUB636. The lysA deletion was marked with the aph gene,
conferring kanamycin resistance, by insertion of a specialized aph
cassette via the unique MluI site to yield pYUB638. This
specialized cassette has an aph gene flanked by two .gamma..delta.
resolvase sites from the E. coli transposon .gamma..delta. (Tn1000)
(20). The presence of the resolvase sites makes it possible to
excise the antibiotic marker by expressing the .gamma..delta.
resolvase in mycobacteria after the cassette has been inserted into
the mycobacterial chromosome (8). In the present case, however, the
res-aph-res marker was removed from pYUB638 by resolvase excision
in E. coli DH5.alpha. prior to introduction into mycobacteria (see
below).
[0030] To include more DNA on both sides of the M. tuberculosis
.DELTA.lysA construct, cosmid cosY373 from the Sanger Centre M.
tuberculosis H37Rv genome sequencing project (12) was used. An
11-kb SnaBI fragment from cosY373, containing lysA situated in the
middle, was subcloned into the EcoRV site of pKSI.sup.+ to yield
plasmid pYUB659. To replace the wild-type lysA allele in pYUB659
with the .DELTA.lysA::res-aph-res allele constructed above in
pYUB638, the inventors exchanged an internal NheI-BglII fragment of
lysA encompassing the deletion region between these two plasmids.
Because there is an additional Nhel site at the 5' end of the
res-aph-res cassette, this exchange resulted in an additional
deletion of 236-bp within the lysA gene. The resulting plasmid,
pYUB665, contains a deletion within lysA totaling 331-bp and the
res-aph-res cassette. Plasmid pYUB665 was passaged in E. coli
DH5.alpha. (which has a .gamma..delta. element capable of excising
the aph gene from the _.DELTA.lysA::res-aph-res allele) and
isolated a Kn.sup.s derivative, plasmid pYUB667. DNA sequence
analysis of pYUB667 showed that the aph cassette was absent and a
single res site remained that was in-frame with respect to the lysA
open reading frame. The mutant lysA allele in pYUB667 is designated
.DELTA.lysAS::res and has a total deletion of 331-bp of an internal
portion of the lysA gene, but with the addition of the 136-bp res
site, the net change in size of .DELTA.lysA5::res compared to
wild-type is a decrease of 195-bp. To produce the final suicidal
plasmid for allelic exchange in M. bovis BCG and M. tuberculosis, a
8.4-kb HpaI fragment from pYUB667 was cloned into the PacI sites of
the sacB suicidal vector pYUB657, resulting in plasmid pYUB668.
This plasmid has approximately 4-kb of DNA flanking each side of
the .DELTA.lysA5::res allele.
[0031] Electroporation of mycobacteria: M. sniegmatis was
electroporated as previously described (37). M. bovis BCG and M.
tuberculosis were electroporated as per M. smegmatis, except that
all manipulations were done at room temperature instead of on ice
and the expression step proceeded overnight for approximately 12
hours prior to plating.
[0032] Nucleotide sequence accession number: The DNA sequence of
the 4462 bp EcoRI fragment encoding the M. smegmatis lysA gene was
submitted to GenBank and assigned the accession number
AF126720.
B) Results
[0033] Allelic exchange methodology: The basic procedure for making
mutants with the sacB suicidal vector pYUB657 (FIG. 1) is a
two-step allelic exchange (15) (38). A suicidal recombination
plasmid is electroporated into cells and primary recombinants
selected upon hygromycin medium. Since the plasmid cannot
replicate, any hygromycin resistant clones must have integrated the
plasmid into the chromosome by a single crossover event. Because of
the presence of the sacB gene on the pYUB657 vector backbone, the
Hyg.sup.r clones are also sensitive to sucrose (Suc.sup.s). Plasmid
integration at the desired locus results in a tandem duplication
(given the designation DUP) of the cloned region with the vector
DNA in the middle. One such DUP clone is grown to saturation in
supplemented medium, during which time individuals within the
population undergo a second homologous recombination event between
the duplicated regions. In this event, the plasmid vector loops out
and is lost along with the hyg and sacB genes, leaving behind
either the wild-type or mutant allele, depending upon which side of
the mutation the second recombination event occurred. This second
recombination event occurs at a low frequency, thus there must be a
selection for the desired secondary recombinants. To select these
clones one takes advantage of the loss of the sacB gene; any clone
losing the plasmid is now sucrose resistant (Suc.sup.r). The
culture is plated on supplemented media containing sucrose to kill
any clones that did not undergo a second recombination event. The
sucrose resistant clones are then screened for hygromycin
sensitivity and the mutant phenotype.
[0034] Cloning of the mycobacterial lysA genes: The present system
by constructing lysine auxotrophs via deletion of the lysA gene,
encoding meso-diaminopimelate decarboxylase, in M. smegmatis, M.
bovis BCG, and M. tuberculosis. The lysA gene of M. tuberculosis
was already available and could also be used for allelic exchange
in M. bovis BCG due to the conservation of DNA sequences between
the two species, however, the lysA gene of M. smegmatis was not
available. The M. smegmatis lysA gene and resident operon was
cloned as described in the Materials and Methods. The lysA gene of
M. smegmatis is 1424-bp in length and shares 77% homology with the
lysA gene of M. tuberculosis, while the two LysA proteins share a
80% identity (17). The structure of the lysA operon is conserved
between several mycobacteria and the related organism
Corynebacterium glutamicum. In M. tuberculosis, the gene order is:
argS (arginyl-tRNA synthetase), lysA(meso-DAP decarboxylase), hdh
(homoserine dehydrogenase), thrC (threonine synthase), PGRS-17
(poly GC-rich repeat 17), and thrB (threonine kinase) (http). The
sequence from M. smegmatis spans upstream of argS through the hdh
gene. A similar argS-lysA operon arrangement is seen for M. leprae
(37) and Brevibacterium glutamicum (renamed Corynebacterium
glutamicum) (35). The hdh gene product supplies homoserine, the
precursor for Met and Thr biosynthesis (30); while the thrC and
thrB genes are responsible for threonine synthesis (36).
[0035] Construction of an unmarked lysA deletion mutant of M.
smegmatis: M. smegmatis mc.sup.2155 was electroporated with the
.DELTA.lysA4 suicidal plasmid pYUB618 (see Materials and Methods
for plasmid construction) and obtained an average of 15 Hyg.sup.r
clones per transformation, with primary recombination efficiencies
of 10.sup.-5 (see Table 3). Two cultures of one strain,
mc.sup.21492, were grown to saturation in 7H9/lysine media and
dilutions plated onto 7H10/lysine medium supplemented with sucrose.
Sucrose resistant clones were obtained at a frequency of 10.sup.-4,
and 100 clones from each set were screened for Suc.sup.r,
Hyg.sup.s, and auxotrophy. Three basic phenotypes were found:
Suc.sup.r/Hyg.sup.r/prototrophic, Suc.sup.r/Hy.sup.s/prototrophic,
and Suc.sup.r/Hyg.sup.s/auxotrophic (see Table 4, exps. 1 and 2).
The largest group was the Suc.sup.r/Hyg.sup.r/prototrophic class
and likely resulted from inactivation of the sacB gene, since the
clones were still resistant to hygromycin and did not appear to
have arisen from a secondary recombination event. The other two
Suc.sup.r classes were Hyg.sup.s and appeared to result from
secondary recombination events; the first class retained the
wild-type allele, while the second class retained the mutant allele
and were auxotrophic for lysine. One mutant was given the
designation mc.sup.21493 and allelic exchange of lysA confirmed by
Southern blot (see FIG. 2, panel A). The mutant grows equally well
on defined 7H9 medium supplemented with lysine or on complex media
(7H9 supplemented with casamino acids or LB medium).
[0036] Construction of an unmarked lysA deletion mutant of M. bovis
BCG substrain Pasteur: The suicide plasmid pYUB668 (see Materials
and Methods) was used to construct an unmarked, in-frame deletion
of lysA (.DELTA.lysA5::res) in the genome of M. bovis BCG substrain
Pasteur. After electroporation of BCG substrain Pasteur with the
suicide plasmid, an average of 5 Hyg.sup.r clones were obtained per
transformation with a primary recombination efficiency of 10.sup.-4
(see Table 3). Several Hyg.sup.r, Suc.sup.s clones were screened by
PCR to determine which of the primary clones were homologous
recombinants. Three out of four clones examined had incorporated
the suicide plasmid pYUB668 at the lysA locus, while the fourth
appeared to be the result of an illegitimate recombination event
(data not shown). Two clones were chosen for further study,
mc.sup.21601 (DUP3) and mc.sup.21602 (DUP4) both of which had
integrated pYUB668 at lysA but had differed in the orientation of
the duplication (see Table 1). The two strains were grown to
saturation in 7H9 media supplemented with lysine, methionine, and
threonine and then plated upon the same type of media containing
sucrose. This combination of amino acids was used to ensure that
any unforeseen polar effect of the .DELTA.lysA5::res allele on the
downstream Met and Thr biosynthetic genes would not prevent the
isolation of mutants. The results of the sucrose selection are
shown in Table 4, exp 3 and 4. Suc.sup.r clones were obtained at a
frequency of 10.sup.-4 and observed the same three classes of
secondary recombinants that we saw in the M. smeginatis
experiments. Allelic exchange was confirmed in strain mc.sup.21604,
a mutant derived from DUP3 strain mc.sup.21601 (see Southern blot,
FIG. 2, panel B). The auxotroph mc.sup.21604 does not revert, and
no suppression was observed in two independent cultures of
5.times.10.sup.9 CFU each.
[0037] The kinetics of allelic exchange of lysA in M. bovis BCG
substrain Pasteur was surprisingly similar to that of M. smegmatis
prompting examination of the reproducibility of this system.
Sucrose selection was repeated with M. bovis BCG substrain Pasteur
DUP3 strain mc.sup.21601 using cultures grown in Basal medium or
media supplemented with Lys, Met+Thr+Lys, or casamino acids
(acid-hydrolyzed casein). Suc.sup.r clones were obtained from each
of the respective cultures at a frequency similar to those seen in
the previous experiment with mc.sup.21601 (See Table 4, exps. 5
through 8, compare to exp. 3). The distribution of the three
phenotypic classes in the Suc.sup.r population was also similar
except that no lysine auxotrophs were obtained from cultures grown
in Basal medium lacking lysine (as expected) or, surprisingly,
casamino acids medium (Table 4, exps. 5 and 8).
[0038] Using allelic exchange to distinguish homologous from
illegitimate primary recombinants: When using the two-step allelic
exchange methodology with the slow-growing mycobacteria, it is
important to identify primary recombinants that resulted from
illegitimate recombination and those which resulted from homologous
recombination. This can be done by PCR screening (as we did for the
above experiment) or Southern blot, although these screening
methods are difficult when using large recombination substrates.
The inventors reasoned it should be possible to distinguish between
the two types of recombinants by observing the phenotypic
frequencies in the pool of Suc.sup.r secondary clones. Presumably,
any primary recombinant resulting from a homologous integration of
the plasmid at lysA would be able to undergo a second recombination
event and loop out the plasmid, while a recombinant that had
integrated the plasmid via illegitimate recombination would be
unable to do the same. Any Suc.sup.r clones arising from an
illegitimate recombinant would result from inactivation of the sacB
gene as seen above and all these clones should also be
Hyg.sup.r.
[0039] This idea was tested in a series of lysA allelic exchange
experiments with BCG substrain Connaught. Electroporation of BCG
Connaught with the suicide plasmid pYUB668, yielded an average of 2
Hyg.sup.r clones per clecti-oporation for a primary recombination
efficiency of 10.sup.-3 (see Table 4). 7 Hyg.sup.r, Suc.sup.s BCG
Connaught::pYUB668 primary recombinants were chosen, grown in media
supplemented with lysine and plated for sucrose resistant clones.
The Suc.sup.r clones were screened for hygromycin sensitivity and
auxotrophy (see Table 4, exps 9 through 15). Three of the seven
primary recombinants (clones 3, 9, and 10) gave rise to similar
phenotypic populations as that seen for M. bovis BCG substrain
Pasteur DUP strains mc.sup.21601 and mc.sup.21602 (compare results
in Table 4). Therefore, these three primary clones (3, 9, and 10)
were homologous primary recombinants. Two clones (2 and 11) yielded
only Suc.sup.r, Hyg.sup.r, prototrophs, while the remaining clones
(4 and 8) yielded a majority of Suc.sup.r, Hyg.sup.r, prototrophs
and a small number of Suc.sup.r,Hyg.sup.s, prototrophs (Table 4).
These four primary clones (2, 4, 8, and 11) were classified as
illegitimate recombinants. One BCG substrain Connaught lysine
auxotroph, derived from clone 3 was designated strain mc.sup.22519,
and allelic exchange confirmed by Southern blot (see FIG. 2, panel
B).
[0040] Construction of an unmarked, in-frame lysA deletion mutant
of M. tuberculosis strain H37Rv: The same methodology and suicide
plasmid, pYUB668, described above was used to construct a lysA
deletion mutant of M. tuberculosis H37Rv. Primary recombination
efficiencies were observed that were similar to those observed in
experiments with BCG substrain Pasteur (see Table 3). Six
Hyg.sup.r, Suc.sup.s primary recombinants were chosen, grown in
lysine supplemented medium and plated for sucrose resistant
recombinants. All 6 primary recombinants gave rise to phenotypic
populations similar to the results seen with the BCG mc.sup.21601
DUP3 strain grown in Basal medium in Table 4, exp. 5 (data not
shown). It was concluded that these primary clones were all likely
homologous recombinants, but that something was wrong with the
system since we did not isolate any auxotrophs. The sucrose
selection was repeated with two of these primary recombinant
strains, mc.sup.22998 and mc.sup.22999, grown in several types of
media: Basal, Lys, Met+Thr+Lys, and casamino acid (see Table 4,
exps. 16 through 23). The frequency of sucrose resistance was in
the range of 10.sup.5 to 10.sup.4 (see Table 4). Again, auxotrophs
were not obtained and it was confirmed that the phenotypic
frequencies within the Suc.sup.r population were similar to the
failure to isolate Lys- BCG mutants on Basal medium (compare Table
4, exps. 17 and 18 with exp. 5). Furthermore, the results from the
M. tuberculosis primary recombinants were unlike the results
obtained with the BCG Connaught illegitimate primary recombinants.
Thus, these results suggested that the primary recombinants were
indeed homologous, but for some reason any auxotrophs resulting
from a secondary recombination event were nonviable. Apparently,
the media could not support the growth of a M. tuberculosis lysine
auxotroph. It was decided to determine if the inability to isolate
a lysine auxotroph of M. tuberculosis was due to the inability of
the organism to transport lysine.
[0041] Transport of lysine in mycobacteria: To investigate lysine
transport in M. tuberculosis, the toxic lysine analog
S-(.beta.-aminoethyl)-L-cysteine (AEC) was used. AEC is transported
via lysine importers; the lysine permeases of E. coli (LysP), and
Corynebacterium glutamicum (LysI) were identified using
AEC-resistant mutants (46, 49). AEC inhibits aspartokinase, the
enzyme catalyzing the first step of the aspartate amino acid family
pathway responsible for the synthesis of Met, Thr, lIe, Lys, and
DAP (meso-diaminopimelate), the latter begin a component of the
cell envelope peptidoglycan and the precursor to lysine (45) (24).
AEC alone is capable of inhibiting the growth of E. coli, but
requires the addition of threonine to inhibit the growth of C.
glutamicum (45). Presumably, full AEC sensitivity in corynebacteria
requires repression of the threonine branch of the pathway by
threonine.
[0042] The growth curves of M. tuberculosis strain H37Rv and BCG
substrain Pasteur in media with or without AEC and Thr are shown in
FIG. 3. A molar concentration of 3 mM was used for AEC and Thr, a
concentration that is close to the 40 .mu.g/ml used for amino acid
supplementation in the inventors' studies. As seen in FIG. 3,
panels A and B, neither AEC or Thr alone have an inhibitory effect
upon the growth of the two species, however the combination of the
two does inhibit growth, with BCG experiencing the greatest
inhibition compared to M. tuberculosis. One interpretation of the
results of this experiment is that lysine uptake is not as
efficient in M. tuberculosis compared to BCG. The BCG lysine
auxotrophic mutant mc.sup.21604 does not grow well in media
supplemented with lysine at concentrations below the standard
concentration of 40 .mu.g/ml (data not shown). This suggests that a
decrease in transport efficiency of M. tuberculosis compared to
that of BCG might preclude isolation of a M. tuberculosis lysine
auxotroph. Since the inability to isolate a lysine auxotroph of M.
tuberculosis might be due to inefficient lysine transport by the
organism, another attempt was made using media with increased
amounts of lysine.
[0043] Identification of media that support the growth of a M.
tuberculosis H37Rv lysine auxotroph: Allelic exchange with the M.
tuberculosis primary pYUB668 homologous primary recombinant strain
mc.sup.22999 was repeated using modified media with increased
amounts of lysine. Experiments utilizing media containing lysine at
200 .mu.g/ml, or 200 .mu.g/ml with 0.05% Tween-80, or lysine at 1
mg/ml did not yield any auxotrophs (Table 4, exps. 24-26). However,
auxotrophic mutants were isolated when media containing lysine at 1
mg/ml with 0.05% Tween-80 was used (Table 4, exp. 27). The mutants
produce colonies that are much smaller than wild-type and were
easily identified on the sucrose selection plates (see Table 4, exp
27).
[0044] One mutant was designated mc.sup.23026 and allelic exchange
of lysA was confirmed by Southern blot (see FIG. 2, Panel B). No
reversion or suppression was seen in 3.times.10.sup.9 CFU. The
mutant grows slowly, requiring approximately 4-5 weeks to form a
large colony on solid media and has an approximate doubling time of
48 hours in liquid medium (data not shown). Surprisingly, the
mutant can grow on 7H10 solid media supplemented with casamino
acids and also grows on 7H11 (supplemented with casitone, a
pancreatic digest of casein), but requires high concentrations of
lysine if lysine is the sole supplementation. It has an absolute
dependency upon Tween-80 regardless of the type of solid media.
C) Discussion
[0045] Several groups have demonstrated the use of suicide plasmids
for allelic exchange in fast and slow-growing mycobacteria. The
most efficient are those systems using a counter selectable marker;
for mycobacteria, workers have successfully used rpsL (37, 44),
pyrF (27), and sacB (42). The most promising counter selectable
system for the slow-growing mycobacteria is sacB, which confers
sensitivity to sucrose. Methodologies using sacB were used for the
targeted disruptions of ureC in BCG (42) and M. tuberculosis (39);
and the erp gene of BCG and M. tuberculosis (9).
[0046] It was decided to construct a new sacB suicidal vector,
pYUB657, and test it for the construction of unmarked, in-frame
deletion mutants in the slow-growing mycobacteria. These studies
provided an opportunity to examine homologous recombination in the
mycobacteria from a practical standpoint. The bane of allelic
exchange in slow-growing mycobacteria has been the propensity with
which these organisms incorporate exogenous DNA into their genome
via illegitimate recombination (25) (2, 32). Allelic exchange in M.
smegmatis is relatively easy, and this species does not appear
integrate DNA via illegitimate recombination. Several workers have
suggested that the homologous recombination machinery is rather
inefficient in the slow-growing mycobacteria. It is generally
believed that illegitimate recombination occurs at a higher
frequency than homologous recombination in the slow-growing
mycobacteria, but this does not necessarily mean that homologous
recombination is defective in these organisms (32).
[0047] In any allelic exchange technique with the slow-growing
mycobacteria, it is important to distinguish homologous primary
recombinants from illegitimate recombinants; in a method of the
present invention, this was done by observing the frequencies of
the phenotypes in the Suc.sup.r populations. The inventors'
experiments with BCG substrain Connaught and M. tuberculosis
pYUB668 recombinants showed that one using the present method could
reproducibly determine if they had a primary homologous
recombinant, obtain the mutant or discover that the mutation was
not permitted, all at once. The illegitimate pYUB668 recombinants
of BCG substrain Connaught were apparently unable to undergo a
second recombination event, since virtually all of the Suc.sup.r
clones were sacB inactivated clones. A small number of Suc.sup.r
Hyg.sup.s clones from Connaught::pYUB668 clones 4 and 8 may have
arisen from deletions within the integrated plasmid.
[0048] The results of this work suggest that homologous
recombination in M. bovis BCG and M. tuberculosis is as efficient
as that in M. smegmatis. First, the frequency of integration of
suicidal plasmids into the chromosomes of the fast and slow-growers
is similar, within the 10.sup.-4 to 10.sup.-5 range (except for
BCG-Connaught which was 10.sup.-3; this might be an inflated value
however, due to an unusually low electroporation efficiency with
the control vector pYUB412). While the number of primary
recombinants obtained in BCG and M. tuberculosis is often less than
that obtained in M. smegmatis, the differences in the number of
primary recombinants and recombination frequencies are small, and
the electroporation frequencies are at best, only an approximation.
It is suspected that any significant differences in primary
recombination frequencies between slow-growers and M. smegmatis
likely reflect a difference in DNA entry into the cells, since it
is generally agreed that higher electroporation efficiencies are
possible with M. smegmatis than with the slow-growers.
[0049] The recombination frequencies for the slow-growing
mycobacteria includes both homologous and illegitimate
recombinants, thus a direct comparison between the frequencies of
primary recombination in fast and slow-growing mycobacteria may not
be valid. However, more illegitimate recombination may occur with
linear DNA than that which occurs with plasmid DNA. Electroporation
of digested, linear insert DNA from the recombination plasmids of
the present invention into BCG yielded 10 fold more clones than
electroporation with the plasmids, but all clones were illegitimate
recombinants (data not shown). In addition, we rarely obtained
hygromycin resistant clones were rarely obtained when the sacB
suicide vector pYUB657 lacking a DNA insert for recombination was
electroporated into BCG or M. tuberculosis (data not shown).
[0050] Comparing homologous recombination frequencies among these
three species is more straightforward when one examines the
frequencies of secondary recombination events. When cultures were
subjected to sucrose selection, sucrose resistant clones were
obtained in the range of 10.sup.-4 to 10.sup.5 for all three
species; the same as the frequency seen for the primary
recombination of the plasmid into the chromosome. In the sucrose
resistant population, three phenotypic classes were observed, two
of which resulted from a recombination event and one that the
inventors believe did not. The latter class, the Suc.sup.r,
Hyg.sup.r prototrophs were designated "sacB inactivated" clones,
since they were still hygromycin resistant. Inactivation of sacB at
a similar frequency to that observed in this study has been noted
previously (42). Counter-screenable markers can be inactivated at
an approximate frequency of 10.sup.-5 in M. smegmatis by the action
of mobile insertion elements (11). A similar phenomenon, at a lower
frequency, has been seen using the rpsL system for allelic exchange
in M. smegmatis (37).
[0051] In this study, mutants were constructed with a deletion in
lysA, conferring a lysine auxotrophic phenotype. Unexpectedly, the
lysine auxotrophs described herein have different lysine
requirements. The M. smegmatis mutant is the most flexible in its
requirements, growing on chemically defined media supplemented with
lysine as well as medium supplemented with casamino acids. In
contrast, auxotrophs of BCG Pasteur could not be isolated using
casamino acids-containing media, even though the compositional
analysis of the casamino acids used in this study showed that the
media should have a lysine concentration that is three-fold greater
than the amount required for the BCG lysine auxotrophs (13).
Neither the BCG Pasteur or Connaught lysine auxotrophs are able to
grow on solid media if casamino acids or casitone (a pancreatic
digest of casein) is used as the source of lysine. Previously
studied Met, Ile-Val, and Leu auxotrophic mutants of BCG can grow
on all of these media, unlike the BCG lysine auxotrophs described
in this study (31) (25). In more recent work with transposon
mutagenesis of BCG; there were attempts to assay the efficiency of
mutagenesis by screening for amino acid auxotrophy (7). The only
mutants that were obtained were Leu auxotrophs, as isolated
previously. This led to some concern that the transposition
mechanism might not be random which would be detrimental to a
mutagenesis system (6). However, all of these attempts utilized
media containing casein preparations. Under such conditions, lysine
auxotrophs would not be isolated. It is possible that the casein
phenomenon described here is more widespread and could explain the
dearth of auxottophs in the above experiments. The inventors are
currently investigating why the BCG lysine auxotrophs fail to grow
on media containing casein.
[0052] Lysine auxotrophs of M. tuberculosis H37Rv were not isolated
until media with a high concentration of lysine and 0.05% Tween-80
was used. As in the case for BCG, M. tuberculosis mutants could not
be isloated using casamino acids, however, once a mutant was
obtained, the inventors found that it could grow on casamino acids
media or casitone, as long as there was Tween-80 in the media.
Since the M. tuberculosis mutant is dependent upon the presence of
Tween-80, the inventors assume that the failure to obtain a mutant
using casamino acids media was due to the absence of Tween in the
selection media. It is important to note that Tween-80 does not
allow the BCG auxotrophs to form colonies of casamino acids media.
Based upon the AEC toxicity data, it can be concluded that M.
tuberculosis H37Rv does not transport lysine as effectively as BCG.
Alternatively, since AEC toxicity requires transport of threonine
as well, the AEC results could be explained by inefficient
threonine transport. However, the high lysine requirement of the
mutant and the dependency upon Tween-80 would support the former
conclusion, since Tween-80 is believed to increase the permeability
of the mycobacteria cell envelope (21). The primary phenotypic
difference between the BCG and the M. tuberculosis mutants is that
the BCG mutants require lysine supplementation alone, while the M.
tuberculosis mutant requires Tween-80 along with either lysine at
high concentration or casamino acids.
[0053] The auxotrophic mutants obtained herein will be useful in a
variety of applications. The BCG and M. tuberculosis lysine mutants
may be usable for the construction of DAP auxotrophs (peptidoglycan
mutants), as the inventors have done for M. smegmatis (37). A
series of vectors bearing the lysA gene are also being developed
that could be used for the expression of foreign antigens in the
BCG auxotrophs; the presence of the lysA gene would maintain the
plasmids in vivo in the absence of antibiotic selection. The
behavior of the BCG mutants in animals is being tested in the hope
that they could be used in HIV infected populations as a safer
alternative to live, wild-type BCG vaccine. One major goal of
mycobacterial research is the development of attenuated strains of
M. tuberculosis that could be used as potential vaccine strains.
Such mutant strains would be unable to grow in a host, or grow only
for a short time, lasting long enough to prime the immune system.
To this end, the inventors are currently examining the growth
kinetics of the M. tuberculosis auxotroph in animal models.
1TABLE 1 Strains used in this study Strain Description Reference E.
coliK-12 HB101 F-.DELTA.(gpt-proA)62 leuB1 glnV44 ara-14 lacY1
hsdS20 rpsL20 xyl-5 mtl-1 recA13 (10) DH5.alpha.
F-[.phi.80d.DELTA.lacZM15].DELTA.(lacZYA-ar- gF)U169 deoR recA1
endA1 hsdR17 glnV44 thi-1 gyrA96 relA1 (19) M smegmatis mc.sup.2155
ept-1 (47) mc.sup.21492 ept-1 DUP2 [(argS .DELTA.lysA4
hdh')*pYUB657*(argS lysA hdh)] This work mc.sup.21493 ept-1
.DELTA.lysA4 This work M. bovis BCG Pasteur Vaccine strain Statens
Seruminstitut mc.sup.21601 Pasteur DUP3 [(argS lysA hdh
thrC')*pYUB657*(argS .DELTA.lysA5::res hdh thrC')] This work
mc.sup.21602 Pasteur DUP4 [(argS .DELTA.lysA5::res hdh
thrC')*pYUB657*(argS lysA hdh thrC')] This work mc.sup.21604
Pasteur .DELTA.lysA5::res This work Connaught Vaccine strain AECOM
mc.sup.21618 Connaught::pYUB668 homologous primary recombinant,
clone 3 This work mc.sup.22519 Connaught .DELTA.lysA5::res This
work M. tuberculosis H37Rv Virulent AECOM mc.sup.22998
H37Rv::pYUB668 homologous primary recombinant, clone 1 This work
mc.sup.22999 H37Rv::pYUB668 homologous primary recombinant, clone 2
This work mc.sup.23026 .DELTA.lysA5::res This work
[0054]
2TABLE 2 Plasmids Used in this study Name Description Reference
pKSI.sup.+ Ap.sup.r, high copy number cloning vector Stratagene
pMV261 Km.sup.r, E. coli-mycobacterial shuttle vector (51)
pET3d.lysA M. tuberculosis Erdman lysA gene cloned into pET3d (16)
pCVD442 Ap.sup.r, sacB (15) pYUB328 Ap.sup.r, PacI-excisable cosmid
vector, ColE1 (5) pYUB405 Ap.sup.R, Hyg.sup.r, PacI-excisable
cosmid vector, ColE1, does not replicate in mycobacteria (6)
pYUB412 Ap.sup.r, Hyg.sup.r, E.coli-mycobacteria shuttle
PacI-excisable cosmid vector, ColE1 origin, int attP, (6)
nonreplicative but integration proficient in mycobacteria pYUB601
in vitro repackaged pYUB412::lysA.sup.+ cosmid from mc.sup.2155
library This work pYUB604 4.4-kb EcoRI fragment from pYUB601 cloned
in the EcoRI site of pMV261 This work pYUB605 5.5-kb NotI
self-ligated subclone of pYUB604 This work pYUB607 3.4-kb NotI
fragment from pYUB604 cloned into NotI site of pKSI.sup.+ This work
pYUB617 7.7-kb inverse XL-PCR product from pYUB604, containing a
1.2-kb deletion of lysA (.DELTA.lysA4) marked with This work unique
SnaBI site. pYUB618 3.2-kb EcoRI fragment from pYUB617, bearing
.DELTA.lysA4, blunt cloned into PacI sites of pYUB657 This work
pYUB631 2.5-kb PstI fragment from pCVD442, bearing sacB, cloned
into same of pMV261 This work pYUB635 1.3-kb XbaI-BamHI lysA gene
from pET3d.lysA, cloned into same sites of pKSI.sup.+ This work
pYUB636 3-kb inverse XL-PCR product from pYUB635, containing 95-bp
deletion of lysA marked with unique MluI site This work pYUB638
1.4-kb MluI res-aph-res cassette cloned into MluI site in pYUB636
This work pYUB651 pYUB412 containing lysA.sup.+ of M. tuberculosis
Erdman, under control of the BCG groEL (Hsp60) promoter pYUB657
3.5-kb NotI-NheI fragment from pYUB631, bearing groEL (Hsp60)
promoter and sacB, cloned into the EcoRV This work site of pYUB405
pYUB659 11-kb SnaBI fragment from cosY373 cloned into the EcoRV
site of pKSI.sup.+ This work pYUB665 1.7-kb NheI-Bg/II fragment
from pYUB638 (.DELTA.lysA::res-aph-res) replacing 300 bp NheI-Bg/II
This work (lysA.sup.+) fragment in pYUB659 pYUB667 pYUB665 with the
aph gene resolved by passage in E. coli DH5.alpha., Km.sup.s This
work pYUB668 8.4-kb HpaI fragment from pYUB667 cloned into the PacI
sites of pYUB657 This work cosY373 pYUB382::M. tuberculosis H37Rv
cosmid bearing the lysA operon (1)
[0055]
3TABLE 3 Electroporation efficiencies and primary recombination
frequencies for lysA allelic exchange Suicide Ave. # Hyg.sup.r
Electroporation Recombination Species/strain plasmid (N).sup.a
clones.sup.b efficiency.sup.c frequency.sup.d M. smegmatis pYUB618
2 15 .+-. 3 3 .times. 10.sup.5 5 .times. 10.sup.-5 mc.sup.2155 M.
bovis pYUB668 10 5 .+-. 3 1 .times. 10.sup.4 5 .times. 10.sup.-4
BCG-Pasteur M. bovis pYUB668 5 2 .+-. 1 1 .times. 10.sup.3 2
.times. 10.sup.-3 BCG-Connaught M. tuberculosis pYUB668 10 3 .+-. 3
3 .times. 10.sup.5 1 .times. 10.sup.-5 H37Rv .sup.a(N) = number of
electroporations for each species/plasmid combination. Each set was
done with the same stock of electrocompetent cells. .sup.bAverage
number of Hygromycin resistant clones (.+-. standard deviation)
from each set of electroporations done with the suicide plasmids.
.sup.cElectroporation efficiency is the number of Hyg.sup.r clones
obtained from electroporations done with pYUB412, an attP/int
Hyg.sup.r vector that integrates into the attB site of the
mycobacterial genome. The number of Hyg.sup.r clones from pYUB412
electroporations is an indicator of the electroporation efficiency
of the cells; the number of transformants obtained with an attP/int
vector is equivalent to the number obtained with a # replicating
vector. We have never observed spontaneous resistance to hygromycin
in the species studied in this paper. .sup.dRecombination frequency
is calculated by dividing the average number of Hyg.sup.r clones
obtained per electroporation with suicide plasmids, divided by the
electroporation efficiency obtained with the vector pYUB412.
[0056]
4TABLE 4 Recombination products from segregation of lysA DUP in
different mycobacterial species Frequency of phenotypes in
Suc.sup.r population.sup.e (sacB inactivated) (secondary
recombinants) Relevant Suc.sup.r Hyg.sup.r Hyg.sup.s Hyg.sup.s
Species Exp Strain genotype.sup.a Media.sup.b freq..sup.c (N).sup.d
prototrophs prototrophs auxotrophs M. smegmatis 1 mc.sup.21492 DUP2
K 4 100 67 24 9 2 mc.sup.21492 K 3 100 60 31 9 M. bovis BCG Pasteur
3 mc.sup.21601 DUP3 K,M,T 4 48 2 63 35 4 mc.sup.21602 DUP4 K,M,T 9
46 26 33 41 5 mc.sup.21601 DUP3 Basal 0.2 92 9 91 0 6 mc.sup.21601
K 0.9 86 15 73 12 7 mc.sup.21601 K,M,T 3 90 11 61 28 8 mc.sup.21601
CAA 6 78 8 92 0 Connaught 9 clone 3 Hom. pYUB688 K N.D. 47 15 51 34
10 clone 9 " K N.D. 48 6 54 40 11 clone 10 " K N.D. 47 10 77 13 12
clone 2 Illeg. pYUB668 K N.D. 48 100 0 0 13 clone 4 " K N.D. 48 96
4 0 14 clone 8 " K N.D. 47 98 2 0 15 clone 11 " K N.D. 95 100 0 0
M. tuberculosis 17 mc.sup.22998 K 0.3 41 10 90 0 18 mc.sup.22998
K,M,T 1 45 16 84 0 19 mc.sup.22998 CAA 0.6 40 23 77 0 20
mc.sup.22999 Hom. pYUB688 Basal 0.5 42 26 74 0 21 mc.sup.22999 K
0.9 38 13 87 0 22 mc.sup.22999 K,M,T 2 44 36 64 0 23 mc.sup.22999
CAA (a) 0.7 34 6 94 0 24 mc.sup.22998 Hom. pYUB688 K200 2 39 44 56
0 25 mc.sup.22998 K200/TW 10 287 20 80 0 26 mc.sup.22998 K1 0.3 96
20 80 0 27.sup.f mc.sup.22998 K1/TW 1 L 96 L 17 L 83 L 0 L 0.8 S 63
S 0 S 0 S 100 S .sup.aDUP designation is used for strains with
pYUB688 integrated at lysA with known orientation (see TABLE 1).
"Illeg. pYUB688" refers to primary Hyg.sup.r Suc.sup.s clones in
which pYUB688 integrated into the chromosome via illegitimate
recombination. "Hom. pYUB688" refers to primary Hyg.sup.r Suc.sup.s
clones in which pYUB688 integrated at lysA but the orientation of
the duplication is unknown. .sup.bType of media used for outgrowth
(Middlebrook 7H9) and sucrose selection (Middlebrook 7H10): Basal
(no supplementation), K (lysine @ 40 .mu.g/ml), K,M,T (lysine,
methionine, and threonine each @ 40 .mu.g/ml), CAA (0.2% casamino
acids, acid-hydrolyzed), K200 (lysine @ 200 .mu.g/ml), K200/TW
(lysine @ 200 .mu.g/ml plus 0.05% Tween-80), K1 (lysine @ 1 mg/ml),
K1/TW (lysine @ 1 mg/ml plus 0.05% Tween-80) .sup.cNumber of
Suc.sup.r CFU/ml divided by the viable CFU/ml, (expressed as N
.times. 10.sup.-4). .sup.d(N) = number of Suc.sup.r clones
screened. .sup.eFrequency of phenotypes expressed as a percentage
of the number of sucrose resistant clones screened. Hyg.sup.r
prototrophs (not secondary recombinants-"sacB inactivated"),
Hyg.sup.s prototrophs (secondary recombinants, wild-type lysA),
Hyg.sup.s auxotrophs (secondary recombinants, .DELTA.lysA).
.sup.fFor exp. number 27, "L" refers to large colonies, while "S"
refers to small colonies seen on the sucrose selection medium. N.D.
(not determined)
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[0109] All publications mentioned herein above are hereby
incorporated by reference in their entirety. While the foregoing
invention has been described in some detail for purposes of clarity
and understanding, it will be appreciated by one skilled in the art
from a reading of the disclosure that various changes in form and
detail can be made without departing from the true scope of the
invention in the appended claims.
* * * * *