U.S. patent application number 16/795726 was filed with the patent office on 2020-10-01 for controlling bacterial biofilms.
The applicant listed for this patent is DDP SPECIALTY ELECTRONIC MATERIALS US, INC., PENN STATE RESEARCH FOUNDATION. Invention is credited to Thomas K. Wood, Bei Yin.
Application Number | 20200308592 16/795726 |
Document ID | / |
Family ID | 1000004871162 |
Filed Date | 2020-10-01 |
United States Patent
Application |
20200308592 |
Kind Code |
A1 |
Yin; Bei ; et al. |
October 1, 2020 |
CONTROLLING BACTERIAL BIOFILMS
Abstract
Methods of controlling bacteria cells are disclosed. These
methods comprise upregulating expression of a DVU2956 sigma
54-dependent enhancer-binding protein (EBP) in bacteria cells,
resulting in (i) dispersing a biofilm of the cells or reducing
biofilm formation by the cells, and/or (ii) reducing hydrogen
sulfide formation by the cells. Further disclosed are methods of
identifying compounds for controlling bacteria cells as in (i)
and/or (ii) above. Polynucleotides and cells are disclosed that can
optionally be used to practice compound identification methods.
Inventors: |
Yin; Bei; (WILMINGTON,
DE) ; Wood; Thomas K.; (Port Matilda, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DDP SPECIALTY ELECTRONIC MATERIALS US, INC.
PENN STATE RESEARCH FOUNDATION |
Collegeville
UNIVERSITY PARK |
PA
PA |
US
US |
|
|
Family ID: |
1000004871162 |
Appl. No.: |
16/795726 |
Filed: |
February 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62808786 |
Feb 21, 2019 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/18 20130101; C12Q
1/025 20130101; C12N 15/74 20130101 |
International
Class: |
C12N 15/74 20060101
C12N015/74; C12Q 1/02 20060101 C12Q001/02; C12Q 1/18 20060101
C12Q001/18 |
Claims
1. A method of controlling bacteria cells, said method comprising:
upregulating expression of a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) in the bacteria cells, thereby (i)
dispersing a biofilm of the cells or reducing biofilm formation by
the cells, and/or (ii) reducing hydrogen sulfide formation by the
cells.
2. The method of claim 1, wherein the cells are sulfate-reducing
bacteria (sulfide-producing bacteria) cells.
3. The method of claim 2, wherein the cells are of the order
Desulfovibrionales.
4. The method of claim 3, wherein the cells are of the genus
Desulfovibrio.
5. The method of claim 1, wherein said DVU2956 sigma 54-dependent
EBP is endogenous to the cells.
6. The method of claim 1, wherein the cells are comprised within a
biofilm, and the biofilm is dispersed following the upregulation
step.
7. The method of claim 6, wherein expression of said DVU2956 sigma
54-dependent EBP, prior to the upregulation step, is repressed by
the cells.
8. The method of claim 1, wherein the cells are treated with at
least one compound to induce the upregulated expression of said
DVU2956 sigma 54-dependent EBP.
9. The method of claim 1, wherein the cells are on one or more
surfaces of industrial equipment or are otherwise present in an
industrial process.
10. A method of identifying a candidate compound for controlling
bacteria cells, said method comprising: (a) providing bacteria
cells comprising a polynucleotide that comprises (i) a DVU2956
sigma 54-dependent enhancer-binding protein (EBP) regulatory
sequence operably linked to (ii) a nucleotide sequence; (b)
contacting the bacteria cells of step (a) with at least one test
compound; and (c) determining whether expression of said nucleotide
sequence by the bacteria cells of step (b) is upregulated, wherein
such upregulation indicates that the test compound is a candidate
compound for (i) controlling biofilm maintenance or biofilm
formation by said bacteria cells or other bacteria cells, and/or
(ii) reducing hydrogen sulfide formation by said bacteria cells or
other bacteria cells.
11. The method of claim 10, further comprising: (d) contacting said
bacteria cells or other bacteria cells comprised in a biofilm with
the candidate compound identified in step (c), wherein dispersal of
the biofilm indicates that the candidate compound inhibits biofilm
maintenance; (e) contacting said bacteria cells or other bacteria
cells comprised in a liquid culture with the candidate compound
identified in step (c), wherein an inability of the bacteria cells
or other bacteria cells to form a biofilm indicates that the
candidate compound inhibits biofilm formation; or (f) contacting
said bacteria cells or other bacteria cells with the candidate
compound identified in step (c), wherein a reduction in hydrogen
sulfide production by the bacteria cells or other bacteria cells
indicates that the candidate compound inhibits hydrogen sulfide
production by the bacteria cells or other bacteria cells.
12. The method of claim 10, wherein said nucleotide sequence
comprises a sequence that encodes a reporter protein, wherein
increased expression of the reporter protein by the bacterial cells
of step (b) indicates upregulated expression of said nucleotide
sequence.
13. The method of claim 10, wherein the DVU2956 sigma 54-dependent
EBP regulatory sequence comprises a promoter sequence.
14. The method of claim 10, wherein said bacterial cells or other
bacteria cells are sulfate-reducing bacteria (sulfide-producing
bacteria) cells.
15. The method of claim 10, comprising screening a plurality of
test compounds by following steps (a)-(c) to identify one or more
candidate compounds for controlling bacteria cells.
16. A polynucleotide comprising (i) a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) regulatory sequence operably linked
to (ii) a nucleotide sequence, wherein the regulatory sequence and
the nucleotide sequence are heterologous to each other, optionally
wherein the regulatory sequence includes a promoter sequence.
17. A cell comprising the polynucleotide of claim 16, optionally
wherein the nucleotide sequence is capable of being expressed by
the cell, and preferably wherein the cell is a bacterial cell.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/808,786 (filed Feb. 21, 2019), which is
incorporated herein by reference in its entirety.
FIELD
[0002] The present disclosure is in the field of molecular biology
and microbiology. The disclosure pertains to methods of controlling
bacterial biofilms, for example.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0003] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named 20200219_DI83116USNP_SequenceListing.txt created
on Feb. 19, 2020 and having a size of about 27 kilobytes and is
filed concurrently with the specification. The sequence listing
contained in this ASCII-formatted document is part of the
specification and is herein incorporated by reference in its
entirety.
BACKGROUND
[0004] Metabolic activity of microbes can cause problems in a broad
array of industries. For example, bacteria can create
microbiologically influenced corrosion (MIC) on metal surfaces of
equipment and cause degradation of polymer additives. Also,
biofilms formed by both aerobic and anaerobic bacteria can
physically plug oil and gas pipelines and water purification
systems, as well as reduce the efficiency of pumps and heat
transfer systems. Although aerobic and anaerobic bacteria coexist
in many environments, contaminant aerobic bacteria are more often
found topside (i.e., near the surface) in injection water, produced
water, and functional water-based fluids such as drilling muds,
completion or workover fluids, stimulation fluids, fracturing
fluids, and hydrotest fluids. Contaminant anaerobic bacteria, on
the other hand, are most commonly found downhole (i.e.,
underground) in oil or gas reservoirs, produced fluids, deaeration
towers, transmission pipelines, the water bottoms of oil and gas
storage tanks, and near bore areas.
[0005] A particular type of bacteria known as sulfate-reducing
bacteria (SRB) produce hydrogen sulfide, which can sour oil and
gas, and corrode pipelines and storage tanks. SRB, which are the
major cause of biocorrosion of iron and other metals used in
industry, incur enormous global economic costs (Enning and
Garrelfs, 2014, Appl. Environ. Microbiol. 80:1226-1236). Hence,
controlling SRB biofilms by preventing their formation and
promoting their dispersal is important.
[0006] SRB biofilms contain protein (Clark et al., 2007, Environ.
Microbiol. 9:2844-2854) and exopolysaccharide (EPS) containing
polymers of mannose, N-acetyl-beta-D-galactosamine (GalNAc) and
fucose (Poosarla et al., 2017, Environ. Microbiol. Rep. 9:779-787).
Based on this structure, dispersal of SRB biofilms has been shown
by treatment with proteases (Clark et al., 2007) and glycoside
hydrolases (Zhu et al., 2018, Environ. Microbiol.
20:2026-2037).
[0007] In regard to regulation of SRB biofilm formation, gene
expression in D. vulgaris biofilms growing on steel was studied
using microarrays, finding that some discontinuous distributed EPS
biosynthesis genes were induced (Zhang et al., 2007, Appl.
Microbiol. Biotechnol. 76:447-457). In addition, gene and protein
expression profiles of SRB biofilms as examined by microarrays and
iTRAQ.RTM. identified some unknown extracellular proteins as
important for biofilm formation (Clark et al., 2012, BMC Genomics
13:138). Another report focused on differential gene expression in
biofilm cells and planktonic cells at the single cell level (Qi et
al., 2016, Front. Microbiol. 7:597); that study found that EPS
biosynthesis gene dvu0281 and ferric iron uptake and storage genes
dvu1340 and dvu1397 were induced in biofilms, while certain genes
including those involved in energy metabolism (dvu0434 and
dvu0588), stress response (dvu2410), and iron transportation
(dvu2571) were repressed in biofilms.
[0008] Despite this work, little is known with respect to
regulation of biofilm formation and dispersal by bacteria such as
SRB. In providing further insights in this area, the instant
disclosure provides new modes of controlling bacterial
biofilms.
SUMMARY
[0009] In one embodiment, the present disclosure concerns a method
of controlling bacteria cells. This method comprises upregulating
expression of a DVU2956 sigma 54-dependent enhancer-binding protein
(EBP) in the bacteria cells, thereby (i) dispersing a biofilm of
the cells or reducing biofilm formation by the cells, and/or (ii)
reducing hydrogen sulfide formation by the cells.
[0010] In another embodiment, the present disclosure concerns a
method of identifying a candidate compound for controlling bacteria
cells. This method comprises: (a) providing bacteria cells
comprising a polynucleotide that comprises (i) a DVU2956 sigma
54-dependent enhancer-binding protein (EBP) regulatory sequence
operably linked to (ii) a nucleotide sequence; (b) contacting the
bacteria cells of step (a) with at least one test compound; and (c)
determining whether expression of the nucleotide sequence by the
bacteria cells of step (b) is upregulated, wherein such
upregulation indicates that the test compound is a candidate
compound for (i) controlling biofilm maintenance or biofilm
formation by the bacteria cells or other bacteria cells, and/or
(ii) reducing hydrogen sulfide formation by the bacteria cells or
other bacteria cells.
[0011] In another embodiment, the present disclosure concerns a
polynucleotide comprising (i) a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) regulatory sequence operably linked
to (ii) a nucleotide sequence, wherein the regulatory sequence and
the nucleotide sequence are heterologous to each other, optionally
wherein the regulatory sequence includes a promoter sequence. The
present disclosure also concerns a cell (e.g., bacterial cell)
comprising such a polynucleotide, optionally wherein the nucleotide
sequence is capable of being expressed by the cell.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES
[0012] FIG. 1: Upregulation of DVU2956 (SEQ ID NO:2), DVU2960 (SEQ
ID NO:4), DVU2962 (SEQ ID NO:8) and DVU2964 (SEQ ID NO:10) proteins
inhibits D. vulgaris biofilm formation, and DVU2956 protein (SEQ ID
NO:2) upregulation inhibits D. desulfuricans biofilm formation.
Error bars indicate one standard deviation. Refer to Example 2.
[0013] FIG. 2: Upregulation of DVU2956 protein (SEQ ID NO:2)
inhibits H.sub.2S formation by D. vulgaris biofilm cells.
Normalized H.sub.2S production (concentration [ppm]/OD.sub.620 nm)
is respectively shown relative to negative control D.
vulgaris/pVLT33-Pdvu0304 (for D. vulgaris/pVLT33-Pdvu0304-dvu2956),
negative control wild type D. vulgaris (for D. vulgaris
(dvu2956.sup.-)), and negative control D. vulgaris
(dvu2956.sup.-)/pMQ70-Pdvu2956 (for D. vulgaris
(dvu2956.sup.-)/pMQ70-Pdvu2956-dvu2956). Data collected from the
96-well plate and sealed vial protocols are indicated by straight
lines and dashed lines, respectively. The symbols * (P<0.05) and
** (P<0.01) indicate significant differences, per one-way ANOVA
analysis, between a test and its respective control. Error bars
indicate one standard deviation. Refer to Example 3.
[0014] FIG. 3: Normalized fluorescence of D.
vulgaris/pMQ70-Pdvu2956-mNeonGreen and D. vulgaris/pMQ70 planktonic
cells at excitation of 425 nm and emission of 517 nm. Refer to
Example 4.
[0015] FIG. 4: Normalized fluorescence of E.
coli/pMQ70-Pdvu2956-mNeonGreen/pET27b-dvu2956 and E.
coli/pMQ70/pET27b-dvu2956 planktonic cells at excitation of 425 nm
and emission of 517 nm, following treatment for 90 minutes with 0,
0.1, 0.5, or 1 mM IPTG. Refer to Example 4.
TABLE-US-00001 [0016] TABLE 1 Summary of Nucleic Acid and Protein
SEQ ID Numbers.sup.a Nucleic acid Protein Description SEQ ID NO.
SEQ ID NO. DVU2956 sigma 54-dependent EBP, D. vulgaris 1 2 (345 aa)
Hildenborough. DVU2960, D. vulgaris Hildenborough. 3 4 (474 aa)
DVU2961, D. vulgaris Hildenborough. 5 6 (115 aa) DVU2962, D.
vulgaris Hildenborough. 7 8 (577 aa) DVU2964, D. vulgaris
Hildenborough. 9 10 (219 aa) dvu2956 gene regulatory sequence, D.
vulgaris 12 Hildenborough comprising promoter and 5'-UTR sequences.
dvu2956 gene promoter sequence (Pdvu2956), D. vulgaris 13
Hildenborough. dvu2956 gene 5'-UTR sequence, D.
vulgarisHildenborough. 14 Synthetic ribosome binding site (sRBS).
15 Monomeric yellow-green fluorescent protein (mNeonGreen .TM., 16
GenBank .RTM. Accession No. KC295282), codon-optimized. Pdvu2956-5'
UTR-sRBS-mNeonGreen .TM. cassette. 17 Conserved motif GAFTGA of
sigma 54 interaction domain of 18 (6 aa) DVU2956 protein, D.
vulgaris Hildenborough. Helix-turn-helix (HTH) domain of
DNA-binding domain of 19 (41 aa) DVU2956 protein, D. vulgaris
Hildenborough. Motif within HTH domain of DVU2956 protein,
D.vulgaris 20 (9 aa) Hildenborough. Upstream activating sequence
(UAS) consensus sequence. 21 .sup.aSEQ ID NO: 11 is intentionally
not included in this table and merely serves as a placeholder.
DETAILED DESCRIPTION
[0017] The disclosures of all cited patent and non-patent
literature are incorporated herein by reference in their
entirety.
[0018] Unless otherwise disclosed, the terms "a" and "an" as used
herein are intended to encompass one or more (i.e., at least one)
of a referenced feature.
[0019] Where present, all ranges are inclusive and combinable,
except as otherwise noted.
[0020] For example, when a range of "1 to 5" (i.e., 1-5) is
recited, the recited range should be construed as including ranges
"1 to 4", "1 to 3", "1-2", "1-2 & 4-5", "1-3 & 5", and the
like.
[0021] The terms "sigma factor" (.sigma. factor), "sigma subunit",
"specificity factor" and the like herein refer to bacterial
proteins that serve as transcription initiation factors enabling
specific binding of RNA polymerase (RNAP) to a gene promoter. Sigma
factors have been described, for example, by Feklistov et al.
(2014, Annu. Rev. Microbiol. 68:357-376), which is incorporated
herein by reference. Examples of sigma factors include "sigma 54"
(alternatively "sigma factor 54", ".sigma..sup.54", "sigma 54
subunit", "sigma-N" and like terms; encoded by the rpoN gene),
which is a sigma factor that (i) binds a gene promoter (a "sigma
54-dependent promoter") at certain conserved nucleotide sequences
typically located -24 (GG) and -12 (TGC) with respect to (i.e., 24
and 12 base pairs [bp] upstream of) the transcription start site
(+1) of the gene, (ii) requires binding of a sigma 54-dependent
enhancer-binding protein (EBP) at an upstream activating sequence
(UAS) typically located about 100 bp or more upstream from the
sigma 54-dependent promoter, and (iii) requires interaction between
the EBP and sigma 54 to initiate transcription by sigma 54/RNAP
complex. Sigma 54 factors have been described, for example, by Buck
et al. (2000, J. Bacteriol. 182:4129-4136), which is incorporated
herein by reference.
[0022] The terms "sigma 54-dependent enhancer-binding protein",
"sigma 54-dependent EBP", "sigma 54-dependent transcriptional
regulator" and the like herein refer to a bacterial protein that
can bind a UAS typically (but not always) located about 100 bp or
more upstream from a sigma 54-dependent promoter and activate
transcriptional initiation by a sigma 54/RNAP complex that is bound
to the sigma 54-dependent promoter. While a sigma 54-dependent EBP
minimally requires a central ATPase (AAA+) domain that orchestrates
ATP hydrolysis, EBP oligomerization and binding to sigma 54 ("sigma
54 interaction domain" herein), it typically also has a C-terminal
DNA-binding domain for binding UAS and optionally also has an
N-terminal regulatory domain. Sigma 54-dependent EBPs have been
described, for example, by Bush et al. (2012, Microbiol. Mol. Biol.
Rev. 76:497-529) and Kazakov et al. (2015, BMC Genomics 16:919),
which are incorporated herein by reference.
[0023] Examples of sigma 54-dependent EBPs include "DVU2956 sigma
54-dependent EBP" (or "DVU2956 protein" and other like terms).
While having a sigma 54 interaction domain, a DVU2956 sigma
54-dependent EBP herein also has a C-terminal DNA-binding domain,
but lacks an N-terminal regulatory domain. In addition to the above
features, the sigma 54 interaction domain of a DVU2956 sigma
54-dependent EBP typically comprises conserved motif GAFTGA (SEQ ID
NO:18) and is about 150-200 (e.g. 160-170) amino acid residues in
length. The DNA-binding domain of this EBP typically comprises a
helix-turn-helix (HTH) domain comprising a sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to SEQ ID NO:19 with motif KGEAARLLG (SEQ ID NO:20), and
binds UAS with a consensus sequence of GCGGNNNNNNNNNGNCNN (SEQ ID
NO:21). Such a UAS typically (but not always) is located about 100
bp or more upstream from a sigma 54-dependent promoter. DVU2956
sigma 54-dependent EBPs have been described, for example, by
Kazakov et al. (2015).
[0024] The terms "biofilm", "bacterial biofilm", "surface-attached
community of bacteria" and the like herein refer to a
collective/assemblage/population of one or more types of bacteria
cells associated with a surface. The cells in a biofilm usually are
comprised within a matrix/scaffold of protein and extracellular
polymeric substance(s) (EPS) such as polysaccharide material. A
biofilm matrix can also comprise noncellular materials such as
mineral crystals, corrosion particles, clay or silt particles,
and/or other components; a biofilm of sulfate-reducing bacteria
herein can, in some aspects, contain elemental sulfur and/or metal
sulfide (e.g., FeS, CuS, NiS, ZnS, TiS.sub.2, MoS.sub.2,
Cr.sub.2S.sub.3). Absent any change in protein expression as
presently disclosed and/or some other treatment that alters
bacterial cell physiology and/or non-cellular material in the
biofilm, a bacterial biofilm typically is resistant to removal by
otherwise gentle or moderate means (e.g., mild rinsing with an
aqueous composition [mild fluid shear] or application of a mild
agent). Biofilms typically adhere to surfaces submerged in, or
subjected to, aquatic environments. Biofilms have been described,
for example, by Davey and O'Toole (2000, Microbiol. Mol. Biol. Rev.
64:847-867), Donlan (2002, Emerg. Infect. Dis. 8:881-890), Satpathy
et al. (2016, Biocatal. Agric. Biotechnol. 7:56-66), and Beech and
Cheung (1995, Int. Biodeter. Biodegr. 35:59-72), which are
incorporated herein by reference.
[0025] The term "planktonic cells" and like terms herein refer to
bacteria cells floating as single cells in a liquid medium. As
opposed to biofilm cells, planktonic cells typically live freely
and are not associated with other cells in a matrix. A single type
of bacteria can exist either in a planktonic or biofilm state,
depending on environmental cues and/or gene expression, for
example.
[0026] The terms biofilm "dispersal", "dispersion" and the like
herein refer to the detachment of cells from a biofilm; such
detached cells typically then exist in a planktonic state. Biofilm
dispersal herein is active dispersal, which is driven in response
to the protein upregulation introduced by the presently disclosed
method. While the mechanism of dispersal herein is active, passive
dispersal (e.g., via abrasion or liquid shear) can be applied, if
desired, along with active dispersal.
[0027] The terms "sulfate-reducing bacteria" (SRB),
"sulfide-producing bacteria", and the like herein refer to bacteria
that can obtain energy by oxidizing organic compounds or molecular
hydrogen while reducing sulfate (SO.sub.4.sup.2-) and/or other
terminal sulfur-containing electron acceptors (e.g., inorganic
sulfur compounds such as sulfite [SO.sub.3.sup.2-], dithionite
[S.sub.2O.sub.4.sup.2-], thiosulfate (S.sub.2O.sub.3.sup.2-],
trithionate [S.sub.3O.sub.6.sup.2-], tetrathionate
[S.sub.4O.sub.6.sup.2-], elemental sulfur [S.sub.8], and
polysulfides [S.sub.n.sup.2-]) to hydrogen sulfide (H.sub.2S). In
general, reference to SRB herein is not intended to refer to
sulfate-reducing archaea. Sulfate-reducing bacteria have been
described, for example, by Muyzer and Stams (2008, Nature Reviews
Microbiology 6:441-454), Youssef et al. (2009, Adv. Appl.
Microbiol. 66:141-251), and Hamilton (1985, Ann. Rev. Microbiol.
39:195-217), which are incorporated herein by reference. Any
reference herein to SRB can likewise be with respect to
"sulfide-producing bacteria".
[0028] The terms "compound", "small molecule", "small-molecule
compound" and the like in some aspects herein refer a low molecular
weight (e.g., <1000, 900, 800, 700, 600, 500 daltons) compound.
Such a compound, for example, can be organic and/or of a size on
the order of about 0.8-1.2 nm (e.g., about 1 nm).
[0029] The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid molecule" and the like are used interchangeably
herein. These terms encompass nucleotide sequences and the like. A
polynucleotide may be a polymer of DNA or RNA that is single- or
double-stranded, that optionally contains synthetic, non-natural or
altered nucleotide bases. A polynucleotide may be comprised of one
or more segments of cDNA, genomic DNA, synthetic DNA, or
combinations thereof.
[0030] The term "gene" as used herein refers to a DNA
polynucleotide sequence that expresses an RNA (RNA is transcribed
from the DNA polynucleotide sequence) from a coding region, which
RNA can be a messenger RNA (encoding a protein) or a
non-protein-coding RNA. A gene may refer to the coding region
alone, or may further include regulatory sequences upstream and/or
downstream to the coding region (e.g., promoters, 5'-untranslated
regions, 3'-transcription terminator regions). A coding region
encoding a protein can alternatively be referred to herein as an
"open reading frame" (ORF). A gene that is "native" or "endogenous"
refers to a gene as found in nature with its own regulatory
sequences; such a gene is located in its natural location in the
genome of a host cell. A "chimeric" gene refers to any gene that is
not a native gene, comprising regulatory and coding sequences that
are not found together in nature (i.e., the regulatory and coding
regions are heterologous with each other). Accordingly, a chimeric
gene may comprise regulatory sequences and coding sequences that
are derived from different sources, or regulatory sequences and
coding sequences derived from the same source, but arranged in a
manner different than that found in nature. A "foreign" or
"heterologous" gene can refer to a gene that is introduced into a
host organism by gene transfer. A foreign/heterologous gene herein
can be (i) a native gene that is inserted into a different organism
with respect to where the native gene was derived, (ii) a native
gene introduced into a new location within the native host, or
(iii) a chimeric gene. Polynucleotide sequences in certain
embodiments disclosed herein are heterologous. A "transgene" is a
gene that has been introduced into the genome by a gene delivery
procedure (e.g., transformation), and therefore typically is
heterologous. A "codon-optimized" open reading frame has its
frequency of codon usage designed to mimic the frequency of
preferred codon usage of a host cell.
[0031] The term "heterologous" means not naturally found in the
location of interest. For example, a heterologous gene can be one
that is not naturally found in a host organism, but that is
introduced into the host organism by gene transfer. As another
example, a nucleic acid molecule that is present in a chimeric gene
can be characterized as being heterologous, as such a nucleic acid
molecule is not naturally associated with the other segments of the
chimeric gene (e.g., a promoter can be heterologous to a coding
sequence).
[0032] A "non-native" amino acid sequence or polynucleotide
sequence comprised in a cell or organism herein does not occur in a
native (natural) counterpart of such cell or organism. Such an
amino acid sequence or polynucleotide sequence can also be referred
to as being heterologous to the cell or organism.
[0033] The terms "polypeptide", "peptide", "protein" and the like
herein refer to a chain of amino acid residues, usually having a
defined sequence. Typical amino acids contained in polypeptides
herein include (respective three- and one-letter codes shown
parenthetically): alanine (Ala, A), arginine (Arg, R), asparagine
(Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid
(Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H),
isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine
(Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser,
S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y),
valine (Val, V).
[0034] A "regulatory sequence" as used herein refers to a (i)
nucleotide sequence located upstream of a gene's transcription
start site (e.g., promoter), (ii) 5' untranslated region, (iii)
intron, or (iv) 3' non-coding region, and may influence the
transcription, processing or stability, and/or translation of an
RNA sequence transcribed from the gene. Regulatory sequences herein
include promoters, enhancers, silencers, 5' untranslated leader
sequences, introns, polyadenylation recognition sequences, RNA
processing sites, effector binding sites, stem-loop structures, and
other elements involved in regulation of gene expression (from
transcription through to translation). One or more regulatory
elements herein may be heterologous to a coding region herein.
[0035] A "promoter" as used herein refers to a DNA sequence capable
of controlling the transcription of RNA from a gene. In general, a
promoter sequence is upstream of the transcription start site of a
gene. Promoters may be derived in their entirety from a native
gene, or be composed of different elements derived from different
promoters found in nature, or even comprise synthetic DNA segments.
Promoters that cause a gene to be expressed in a cell at most times
under all circumstances are commonly referred to as "constitutive
promoters". A promoter may alternatively be inducible. One or more
promoters herein may be heterologous to a coding region herein.
[0036] An "inducible promoter" as used herein refers to a promoter
capable of controlling the transcription of RNA from a gene under
certain specific conditions (i.e., by the presence or absence of
biotic or abiotic factors). These types of promoters typically have
no, or very low, activity under conditions in which inducing
conditions are not present.
[0037] A "strong promoter" as used herein refers to a promoter that
can direct a relatively large number of productive initiations per
unit time, and/or is a promoter driving a higher level of gene
transcription than the average transcription level of the genes in
a cell.
[0038] The terms "3' non-coding sequence", "transcription
terminator", "terminator" and the like as used herein refer to DNA
sequences located downstream of a coding sequence. This includes
polyadenylation recognition sequences and other sequences encoding
regulatory signals capable of affecting mRNA processing or gene
expression.
[0039] As used herein, a first nucleic acid sequence is
"hybridizable" to a second nucleic acid sequence when a
single-stranded form of the first nucleic acid sequence can anneal
to the second nucleic acid sequence under suitable annealing
conditions (e.g., temperature, solution ionic strength).
Hybridization and washing conditions are exemplified in Sambrook J,
Fritsch E F and Maniatis T, Molecular Cloning: A Laboratory Manual,
2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.
(1989), which is incorporated herein by reference, particularly
Chapter 11 and Table 11.1.
[0040] The terms "upstream" and "downstream" as used herein with
respect to polynucleotides refer to "5' of" and "3' of",
respectively.
[0041] The term "cassette" as used herein refers to a promoter
operably linked to a DNA sequence encoding a protein-coding RNA or
non-protein-coding RNA. A cassette may optionally be operably
linked to (further comprise) a 3' non-coding sequence. Herein, a
cassette as it is comprised in a plasmid construct can optionally
be denoted as shown with the following example: with construct
pVLT33-Pdvu0304-dvu2956, "pVLT33" refers to the plasmid backbone of
the construct, "Pdvu0304" refers to the promoter, and "dvu2956"
refers the nucleotide sequence (here, encoding protein DVU2956)
targeted for transcription by the promoter. In some aspects, a
cassette can refer to a promoter plus transcribed sequence
(typically an ORF) as, for example, promoter::transcribed sequence
(e.g., the foregoing example can be referred to as
Pdvu0304::dvu2956).
[0042] The term "expression" as used herein refers to (i)
transcription of RNA (e.g., mRNA or a non-protein-coding RNA) from
a coding region, and/or (ii) translation of a polypeptide from
mRNA. Expression of a coding region of a polynucleotide sequence
can be up-regulated or down-regulated in certain embodiments.
[0043] The term "operably linked" ("operatively linked") as used
herein refers to the association of two or more nucleic acid
sequences such that the function of one is affected by the other.
For example, a regulatory sequence (e.g., promoter) is operably
linked with a coding sequence when it is capable of affecting the
expression of that coding sequence. A coding sequence can be
operably linked to one (e.g., promoter) or more (e.g., promoter and
terminator) regulatory sequences, for example.
[0044] The term "recombinant" when used herein to characterize a
DNA sequence such as a plasmid, vector, or construct refers to an
artificial combination of two otherwise separated segments of
sequence, e.g., by chemical synthesis and/or by manipulation of
isolated segments of nucleic acids by genetic engineering
techniques.
[0045] The term "transformation" as used herein refers to the
transfer of a nucleic acid molecule into a host organism or host
cell by any method. A nucleic acid molecule that has been
transformed into an organism/cell may be one that (i) replicates
autonomously in the organism/cell, (ii) integrates into the genome
of the organism/cell, or (iii) exists transiently in the cell
without replicating or integrating. Non-limiting examples of
nucleic acid molecules suitable for transformation are disclosed
herein, such as plasmids and linear DNA molecules. Host
organisms/cells herein containing a transforming nucleic acid
sequence can be referred to as "transgenic", "recombinant",
"transformed", "engineered", as a "transformant", and/or as being
"modified for exogenous gene expression", for example.
[0046] The terms "sequence identity", "identity" and the like as
used herein with respect to polynucleotide or polypeptide sequences
refer to the nucleic acid residues or amino acid residues in two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window. Thus, "percentage of sequence
identity", "percent identity" and the like refer to the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide or
polypeptide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the results by 100 to yield
the percentage of sequence identity. It would be understood that,
when calculating sequence identity between a DNA sequence and an
RNA sequence, T residues of the DNA sequence align with, and can be
considered "identical" with, U residues of the RNA sequence. For
purposes of determining "percent complementarity" of first and
second polynucleotides, one can obtain this by determining (i) the
percent identity between the first polynucleotide and the
complement sequence of the second polynucleotide (or vice versa),
for example, and/or (ii) the percentage of bases between the first
and second polynucleotides that would create canonical Watson and
Crick base pairs.
[0047] Percent identity can be readily determined by any known
method, including but not limited to those described in: (i)
Computational Molecular Biology (Lesk, A. M., Ed.) Oxford
University: NY (1988); (ii) Biocomputing: Informatics and Genome
Projects (Smith, D. W., Ed.) Academic: NY (1993); (iii) Computer
Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
G., Eds.) Humana: NJ (1994); (iv) Sequence Analysis in Molecular
Biology (von Heinje, G., Ed.) Academic (1987); and (v) Sequence
Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY
(1991), all of which are incorporated herein by reference.
[0048] Preferred methods for determining percent identity are
designed to give the best match between the sequences tested.
Methods of determining identity and similarity are codified in
publicly available computer programs, for example. Sequence
alignments and percent identity calculations can be performed using
the MEGALIGN program of the LASERGENE bioinformatics computing
suite (DNASTAR Inc., Madison, Wis.), for example. Multiple
alignment of sequences can be performed, for example, using the
Clustal method of alignment which encompasses several varieties of
the algorithm including the Clustal V method of alignment
(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins,
D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in
the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing
suite (DNASTAR Inc.). For multiple alignments, the default values
can correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default
parameters for pairwise alignments and calculation of percent
identity of protein sequences using the Clustal method can be
KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For
nucleic acids, these parameters can be KTUPLE=2, GAP PENALTY=5,
WINDOW=4 and DIAGONALS SAVED=4. Additionally, the Clustal W method
of alignment can be used (described by Higgins and Sharp, CABIOS.
5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci.
8:189-191(1992); Thompson, J. D. et al, Nucleic Acids Research, 22
(22): 4673-4680, 1994) and found in the MEGALIGN v8.0 program of
the LASERGENE bioinformatics computing suite (DNASTAR Inc.).
Default parameters for multiple alignment (protein/nucleic acid)
can be: GAP PENALTY=10/15, GAP LENGTH PENALTY=0.2/6.66, Delay
Divergent Seqs(%)=30/30, DNA Transition Weight=0.5, Protein Weight
Matrix=Gonnet Series, DNA Weight Matrix=IUB.
[0049] Various polypeptide amino acid sequences and polynucleotide
sequences are disclosed herein as features of certain embodiments.
Variants of these sequences that are at least about 70-85%, 85-90%,
or 90%-95% identical to the sequences disclosed herein can be used
or referenced. Alternatively, a variant amino acid sequence or
polynucleotide sequence can have at least about 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
99.5% identity with a sequence disclosed herein. A variant amino
acid sequence or polynucleotide sequence has the same
function/activity of the disclosed sequence, or at least about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the
disclosed sequence. Any polypeptide amino acid sequence disclosed
herein not beginning with a methionine can typically further
comprise at least a start-methionine at the N-terminus of the amino
acid sequence. In contrast, any polypeptide amino acid sequence
disclosed herein beginning with a methionine can optionally lack
such a methionine residue.
[0050] The terms "control cell", "suitable control cell" and the
like herein can be referenced with respect to a cell in which a
particular modification (e.g., over-expression of a polynucleotide,
down-regulation of a polynucleotide) has been made (i.e., an
"experimental cell"). A control cell can be any cell that does not
have or does not express the particular modification of the
experimental cell. Thus, a control cell can be an untransformed
wild type cell or can be genetically transformed but does not
express the genetic transformation. For example, a control cell can
be a direct parent of the experimental cell, which direct parent
cell does not have the particular modification of the experimental
cell. Alternatively, a control cell can be a parent of the
experimental cell that is removed by one or more generations.
Alternatively still, a control cell can be a sibling of the
experimental cell, which sibling does not comprise the particular
modification that is present in the experimental cell.
[0051] The term "isolated" means a composition (or process) in a
form or environment that does not occur in nature. Non-limiting
examples of isolated compositions include (1) any non-naturally
occurring composition (e.g., a polypeptide, polynucleotide, or cell
herein), (2) any composition including, but not limited to, any
cell, polypeptide, polynucleotide, cofactor, or
carbohydrate/saccharide that is at least partially removed from one
or more of, or all of, the naturally occurring constituents with
which it is associated in nature; (3) any composition modified by
the hand of man relative to that composition found in nature; or
(4) any composition modified by increasing or decreasing the amount
of the composition relative to other components with which it is
naturally associated. It is believed that embodiments, compositions
and methods disclosed herein are synthetic/man-made (could not have
been made except for human intervention/involvement), and/or have
properties that are not naturally occurring.
[0052] The term "increased" as used herein can refer to a quantity
or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%,
100%, or 200% more than the quantity or activity for which the
increased quantity or activity is being compared. The terms
"increased", "elevated", "enhanced", "greater than", "improved" and
the like are used interchangeably herein. These terms can be used
to characterize the "over-expression" or "up-regulation" of a
polynucleotide encoding a protein, for example.
[0053] Embodiments of the present disclosure concern a method of
controlling bacteria cells. This method comprises upregulating
expression of a DVU2956 sigma 54-dependent enhancer-binding protein
(EBP) in the bacteria cells, thereby (i) dispersing a biofilm of
the cells or reducing biofilm formation by the cells, and/or (ii)
reducing hydrogen sulfide formation by the cells. Thus, the term
"controlling" as used herein is generally intended to refer to
inhibiting/reducing bacterial biofilm formation and/or maintenance,
and/or reducing bacterial hydrogen sulfide formation.
[0054] Bacteria cells controlled by a method herein can be
sulfate-reducing bacteria (SRB) cells, for example. SRB cells in
some aspects include those of the taxonomic order
Desulfovibrionales, Desulfobacterales, Syntrophobacterales,
Nitrospirales, Clostridiales, Selenomonadales,
Thermodesulfobacteriales, Desulfurellales, or
Thermoanaerobacterales. Examples of Desulfovibrionales genera (and
species) herein include Desulfovibrio (e.g., D. vulgaris, D.
sulfuricans, D. termitidis, D. gigas, D. aminophilus, D. africanus,
D. putealis, D. cuneatus, D. mexicanus, D. magneticus, D. piger, D.
alaskensis, D. salexigens, D. ferrophilus, D. senezii, D.
fairfieldensis), Desulfomicrobium (e.g., D. escambiense, D.
apsheronum, D. baculatum, D. norvegicum, D. orale, D. macestii),
Desulfohalobium (e.g., D. retbaense, D. utahense), and Lawsonia
(e.g., L. intracellularis). Examples of Desulfobacterales genera
(and species) herein include Desulfobacter (e.g., D. postgatei),
Desulfobulbus, Desulfobacula, Desulfotignum (e.g., D. toluenicum),
Desulfobacterium (e.g., D. cetonicum), and Desulfococcus (e.g., D.
multivorans). Examples of Nitrospirales genera (and species) herein
include Nitrospira (e.g., N. moscoviensis, N. marina). Examples
of
[0055] Syntrophobacterales genera (and species) herein include
Syntrophobacter (e.g., S. fumaroxidans). Examples of Clostridiales
genera (and species) herein include Desulfotomaculum (e.g., D.
australicum). Examples of Thermodesulfobacteriales genera (and
species) herein include Thermodesulfobacterium (e.g., T. commune)
and Thermodesulfatator (e.g., T. autotrophicus). Yet, in some
aspects, SRB cells can be those of Desulfatitalea tepidiphila,
Desulfosporosinus lacus, Thermodesulfovibrio aggregans, or
Desulfotalea psychrophila. Other examples of SRB of the present
disclosure include any of those disclosed in Muyzer and Stams
(2008, Nature Reviews Microbiology 6:441-454), which is
incorporated herein by reference.
[0056] In some aspects, bacteria cells controlled by a method
herein are those that are capable of forming a biofilm. Examples of
such bacteria include any SRB cell type as disclosed herein. Other
examples of bacteria of the present disclosure that are capable of
forming a biofilm include the following genera (and species):
Clostridium (e.g., C. acetobutylicum, C. baratii, C. bifermentans,
C. botulinum, C. butyricum, C. celerecrescens, C. cellulolyticum,
C. clostridioforme, C. difficile, C. drakei, C. fallax, C.
ljungdahlii, C. malenominatum, C. perfringens, C. phytofermentans,
C. sordelli, C. thermocellum, C. magnum, C. tetani), Shigella
(e.g., S. flexneri, S. dysenteriae, S. sonnei), Escherichia (e.g.,
E. coli, E. albertii, E. fergusonii, E. hermannii, E. vulneris),
Bacillus (e.g., B. subtilis, B. licheniformis, B. coagulans, B.
cereus, B. pumilus, B. ligniniphilus, B. sphaericus, B. alvei, B.
laterosporus, B. megaterium, B. anthracis), Pseudomonas (e.g., P.
aeruginosa, P. putida, P. syringae, P. tolaasii, P. agarici, P.
oryzihabitans, P. plecoglossicida, P. hussainii), Klebsiella (e.g.,
K. pneumoniae, K. planticola, K. oxytoca, K. aerogenes, K.
granulomatis, K. variicola), Staphylococcus (e.g., S. aureus, S.
epidermidis), Streptococcus (e.g., S. pyogenes, S. viridans, S.
agalactiae, S. bovis, S. pneumoniae), Enterococcus, Neisseria (N.
gonorrhoeae, N. meningitidis), Propionibacterium (e.g., P. acnes),
Corynebacterium (e.g., C. diphtheriae), Listeria (e.g., L.
monocytogenes), Enterobacter, Enterococcus, Salmonella (e.g., S.
typhimurium, S. enterica), Campylobacter (e.g., C. jejuni). Still
other examples of bacteria herein are any of those disclosed in
U.S. Pat. Nos. 9,192,598 and 9,675,736, and Davey and O'Toole
(2000, Microbiol. Mol. Biol. Rev. 64:847-867), Donlan (2002, Emerg.
Infect. Dis. 8:881-890), and Satpathy et al. (2016, Biocatal.
Agric. Biotechnol. 7:56-66), all of which references are
incorporated herein by reference.
[0057] In some aspects, bacteria cells controlled by a method
herein are (i) anaerobic or aerobic, and/or (ii) Gram-negative or
Gram-positive. For instance, bacteria cells such as SRB cells can
be anaerobic and/or Gram-negative, although SRB cells in some cases
can be aerobic and/or Gram-positive. Bacteria cells in some aspects
can be thermophilic, thermotolerant, or non-thermotolerant;
mesophilic; and/or psychrophilic, psychrotolerant, or
non-psychrotolerant. Bacteria cells in some aspects can be
halophilic, halotolerant, or non-halotolerant; acidophilic,
acidotolerant, or non-acidotolerant; and/or alkaliphilic,
alkalitolerant, or non-alkalitolerant. Bacteria cells in some
aspects can be aquatic (e.g., marine and/or fresh water) or
semi-aquatic (not restricted to living in or alongside water). A
population of bacteria cells herein, such those in a biofilm or a
planktonic state, can be comprised of one or more (e.g., at least
2, 3, 4, 5, 6) different species of bacteria. In some aspects, a
population of bacteria cells herein comprises at least about 95%,
96%, 97%, 98%, 99%, or 99.9% (of cells) of one type of bacteria
species. While a biofilm here typically comprises only or mostly
(e.g., >99% of biofilm cells) bacteria as its cell type, it can
also comprise other types of microbial cells in some cases, such as
those of archaea, protozoa, fungi/yeast, and/or algae.
[0058] A method of controlling bacteria cells herein comprises
upregulating expression of a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) in bacteria cells. Examples of a
DVU2956 protein herein are those that are expressed by a bacteria
as presently disclosed. A DVU2956 protein can be of an SRB herein
in some cases; examples include a Desulfovibrionales,
Desulfovibrio, D. vulgaris, Desulfomicrobium, Desulfohalobium,
Lawsonia, Desulfobacter, Desulfococcus, Nitrospira, or
Syntrophobacter DVU2956 protein.
[0059] It is contemplated that the amino acid sequence of a DVU2956
protein herein can comprise or consist of, for example, any of the
amino acid sequences disclosed in GenBank Acc. Nos. AAS97427.1,
WP_010940215.1, WP_007524656.1, WP_012612883.1, RXF76019.1,
WP_035067106.1, EPR37456.1, WP_035042928.1, WP_021759108.1,
WP_084630783.1, WP_027368917.1, WP_014260494.1, WP_005989720.1,
WP_028576688.1, WP_043647008.1, WP_092375156.1, PKN42718.1,
WP_015774434.1, WP_092188840.1, PKN09949.1, WP_034637971.1,
OGR38622.1, WP_089273750.1, WP_075353577.1, OIN98835.1,
WP_043635439.1, WP_015862636.1, QAZ66189.1, EKO38189.1,
WP_024825447.1, OEU52209.1, OEU60302.1, RLC09126.1, RLB96743.1,
RLB90370.1, KPK14763.1, PY025948.1, PYN09170.1, PYN24203.1,
RPH84916.1, PYM87057.1, WP_020876998.1, PYM29887.1, PYN47838.1,
RME62715.1, PYN79803.1, OGG45084.1, KPK31454.1, RP154032.1,
WP_105329578.1, OGP61280.1, PYN88734.1, OLD39282.1, OLA97374.1,
PYM70540.1, OEU44690.1, WP_013629548.1, PYN32973.1, RMF68781.1,
OFV88829.1, PYN46639.1, WP_105358245.1, WP_029909485.1, PYN15987.1,
WP_006522732.1, OLB08282.1, RMG02185.1, WP_129125732.1, OLD78639.1,
PYM43921.1, PYM98548.1, MF47173.1, RLB82706.1, OLC13927.1,
EIQ51949.1, WP_014811363.1, WP_014780627.1, KPK33783.1, OGQ36645.1,
WP_000968686.1, EGJ01125.1, CSH68239.1, WP_026687822.1, RPH79962.1,
WP_008686581.1, WP_035107244.1, RLB01088.1, KPJ59864.1,
WP_001350708.1, WP_112058104.1, WP_053381039.1, WP_115625741.1,
WP_084270987.1, WP_028455140.1, WP_080887765.1, WP_122653646.1,
PYM94244.1, WP_093394811.1, RKX30710.1, PLX96612.1, and SPW53012.1,
which are incorporated herein by reference. A variant of any of
these amino acid sequences may be used, but should have some of
(e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%
of), or all of, the function (refer to above definitions) of its
corresponding non-variant reference. Such a variant DVU2956 protein
can comprise, or consist of, an amino acid sequence that is at
least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to
the amino acid sequence of its corresponding non-variant
reference.
[0060] In some aspects, a DVU2956 protein herein can comprise, or
consist of, the amino acid sequence of SEQ ID NO:2 (a D. vulgaris
DVU2956 protein). Alternatively, a DVU2956 protein as presently
disclosed can comprise, or consist of, an amino acid sequence that
is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%
identical to SEQ ID NO:2, for example. Such a variant DVU2956
protein should have some of (e.g., at least about 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 95% of), or all of, the function (refer to
above definitions) of the DVU2956 protein of SEQ ID NO:2.
[0061] Typically, a DVU2956 protein that is upregulated in bacteria
cells herein is endogenous/native to the cells. However,
upregulation by exogenously (ectopically) introducing a DVU2956
protein to bacteria cells is also envisaged. Such an exogenous
DVU2956 protein can be autologous (i.e., corresponding, without
amino acid variation, to the DVU2956 protein expressed endogenously
by the cells) or heterologous to the cells. In some aspects, any of
the foregoing DVU2956 proteins are non-native DVU2956 proteins, and
thus are not 100% identical to any of the above reference
sequences.
[0062] Upregulation of a DVU2956 protein in a bacteria cell herein
results in an increased level of DVU2956 protein in the cell.
DVU2956 protein upregulation in certain aspects can be through
upregulation of a polynucleotide sequence encoding any DVU2956
protein (or variant thereof) as presently disclosed, for example. A
polynucleotide sequence in some aspects comprises a nucleotide
sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%
identical to SEQ ID NO:1 or a nucleotide sequence that encodes any
of the foregoing DVU2956 proteins. Upregulation of a polynucleotide
sequence encoding a DVU2956 protein can be done by one or more of a
variety of methods. For example, it is contemplated that a bacteria
cell herein can be treated with (contacted with, exposed to) at
least one compound or agent (e.g., small molecule) that directly or
indirectly induces/stimulates/activates transcription of a
polynucleotide sequence encoding a DVU2956 protein; such a
polynucleotide sequence typically is endogenous to the cell. As
another example, a DVU2956 protein-encoding polynucleotide can be
provided in multi-copy to a cell; such a polynucleotide sequence is
operably linked to at least a promoter sequence that is functional
in the cell. Providing a polynucleotide sequence in multi-copy can
be accomplished by providing one or more copies of the
polynucleotide (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, or 50 copies) to a cell. As another example, a DVU2956
protein-encoding polynucleotide sequence can be upregulated through
operable linkage to a constitutive promoter, strong promoter, or
inducible promoter, any of which can be heterologous. Another mode
of upregulating a polynucleotide sequence herein is via increasing
the half-life/stability of DVU2956 protein-encoding mRNA
transcripts (e.g., potentially via treatment of cells with at least
one compound or agent having this effect).
[0063] Other modes of upregulating expression of a DVU2956 protein
in bacteria herein include increasing translation of mRNA encoding
DVU2956 protein, and/or increasing the half-life/stability of
DVU2956 protein. Another mode of upregulating DVU2956 protein
herein involves enhancing a function/activity of the protein,
thereby enhancing the ability of DVU2956 protein to allow sigma
54-dependent transcription. It is contemplated that one or more of
these additional modes of upregulation can be effected by treating
bacteria with at least one compound or agent (e.g., small
molecule).
[0064] Upregulation of a DVU2956 protein in a bacteria cell herein
can optionally be considered with respect to a suitable control
cell. For example, the increased level of a DVU2956 protein (or an
RNA transcript encoding it) in a cell can be characterized to be
about, or at least about, 25%, 50%, 100%, 150%, 200%, 250%, 500%,
1000%, 1500%, 2000%, 2500%, 3000%, 4000%, 5000%, or 10000% above
the expression of the DVU2956 protein (or an RNA transcript
encoding it) in a suitable control cell. An example of a suitable
control cell is a cell as it existed before it was modified to have
upregulated DVU2956 protein expression. An example of a control
bacteria cell for the purposes of this disclosure is one that is in
a biofilm. Thus, for example, DVU2956 protein upregulation as
determined in a cell that has been dispersed from a biofilm
following the disclosed method can be as compared to DVU2956
protein expression as it existed in the cell when it was in the
biofilm.
[0065] DVU2956 protein expression in bacteria cells of a biofilm,
prior to upregulation, is repressed by the cells in some aspects.
Since repression herein is contemplated to generally be due to, at
least in part, repressed transcription of a polynucleotide encoding
DVU2956 protein, repression can also be determined by measuring the
level of DVU2956 protein-encoding mRNA transcripts. Repression can
mean, for example, that there is no DVU2956 protein expressed by
the cells, or its level is below detection limits. In some aspects,
repression of DVU2956 protein expression (or of its encoding mRNA
transcripts) in bacteria cells of a biofilm can mean that the
expression is about, or at least about, 1-, 1.5-, 2-, 2.5-, 5-,
10-, 15-, 20-, 25-, 30-, 40-, 50-, or 100-fold less than the
DVU2956 protein expression (or of its encoding mRNA transcripts) of
cells that have been dispersed from a biofilm (e.g., are now
planktonic) following the disclosed method.
[0066] A comparison of DVU2956 protein (or RNA) expression between
biofilm cells and cells that have dispersed from the biofilm
following DVU2956 protein upregulation herein can optionally be
made at about, or at least about, 0, 1, 2, 4, 6, 8, 10, 12, 15, 18,
21, or 24 hours following dispersal. Protein expression comparisons
herein can be made using any suitable protein expression analysis
such as spectrometry (e.g., high-performance liquid chromatography
[HPLC], liquid chromatography-mass spectrometry [LC/MS]) or an
antibody-dependent method (e.g., enzyme-linked immunosorbent assay
[ELISA], western blotting, immunoprecipitation). Expression
comparisons as determined at the transcriptional level herein can
be made using a transcriptional expression analysis such as
northern blotting, quantitative reverse transcription polymerase
chain reaction (qRT-PCR), microarray analysis, serial analysis of
gene expression (SAGE), or comparative transcriptomic analysis
(RNA-Seq, also referred to as whole transcriptome shotgun
sequencing).
[0067] In some aspects, DVU2956 protein upregulation is done to
inhibit/reduce biofilm formation by bacteria (e.g., prevent
planktonic bacteria cells from forming a biofilm), while in some
aspects DVU2956 protein upregulation is done to inhibit/reduce
biofilm maintenance (e.g., cause the dispersal of bacteria cells
from the biofilm). Still, in some aspects, DVU2956 protein
upregulation is done to inhibit/reduce sulfide (e.g., hydrogen
sulfide) formation by bacteria cells; this aspect can optionally
also characterize dispersing biofilm cells (typically SRB) using
the disclosed method.
[0068] Dispersal of biofilms herein typically refers to, at a
minimum, dispersing live bacteria cells from a biofilm. In some
aspects, biofilm dispersal comprises only dispersal of live
bacteria cells, or dispersal of live cells and other biofilm
components (e.g., dead cells, one or more biofilm matrix
components). The percentage of live bacteria cells in a biofilm
that disperse upon upregulating a DVU2956 protein herein can be
about, or at least about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 99%, or 100%. A bacteria cell herein that has been
dispersed from a biofilm typically is planktonic, and/or dies in
some aspects. An entire biofilm or most of it (e.g., over 90%, 95%
or 99% by weight), including both its live and non-living
components, can be dispersed in some embodiments. Biofilm dispersal
can be measured following the disclosure of Guilhen et al. (2017,
Mol. Microbiol. 105:188-210, incorporated herein by reference), for
example. While no passive dispersal action is necessary for
achieving dispersal in typical embodiments, such an action can be
combined with the presently disclosed method if desired.
[0069] A reduction of biofilm formation by bacteria herein
resulting from upregulating a DVU2956 protein can be by about, or
at least about, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, for
example, as compared to the amount or rate of biofilm formation
that would have occurred if DVU2956 protein upregulation was not
performed. In some aspects, upregulating a DVU2956 protein herein
can reduce (e.g., by over 80%, 90%, or 95%) or completely block the
rate of growth/spreading of an established bacterial biofilm as
compared to the rate of growth/spreading that would have occurred
if DVU2956 protein upregulation was not performed.
[0070] A reduction of hydrogen sulfide (H.sub.2S) formation by
bacteria cells herein (typically SRB) resulting from upregulating a
DVU2956 protein can be by about, or at least about, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, for example, as
compared to the hydrogen sulfide formation that would have occurred
if DVU2956 protein upregulation was not performed. Hydrogen sulfide
can be measured following the disclosure in the below Examples or
as disclosed in Rabinowitz (1978, Methods Enzymol. 53:275-277,
incorporated herein by reference), for example. Since aspects of
the disclosed method are drawn to controlling hydrogen sulfide
formation, these aspects can alternatively be characterized as a
method of controlling souring such as that occurring in some
industrial settings (see below), and thus also as a method of
controlling corrosion brought on by hydrogen sulfide. Based on this
latter point, such a method can optionally be characterized as a
method of controlling metal sulfide production (such as that formed
during corrosion of metal), examples of which include iron sulfide
(FeS), copper sulfide (CuS), nickel sulfide (NiS), zinc sulfide
(ZnS), tin sulfide (TiS.sub.2), molybdenum sulfide (MoS.sub.2) and
chromium sulfide (Cr.sub.2S.sub.3).
[0071] Upregulation of a DVU2956 protein in bacteria cells herein
can be performed for about, or at least about, 3, 6, 8, 10, 12, 15,
18, 21, 24, 30, 36, 42, 48, 60, 72, 84, or 96 hours, for example,
to effectively control the bacterial cells as disclosed herein. The
temperature in which bacteria cells herein are induced for DVU2956
protein upregulation can be about, at least about, or up to about,
-1, 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,
75, 80, 85, 15-40, 15-70, 15-85, or 70-85.degree. C., for
example.
[0072] Upregulation of a DVU2956 protein in bacteria cells herein
can be performed by treating the cells to upregulate expression of
the DVU2956 protein. For example, such upregulation can be induced
by treating (or exposing or contacting) bacteria cells herein with
at least one compound or agent (e.g., small molecule). Examples of
a compound for upregulating DVU2956 protein expression herein are
those that can induce transcriptional activation of a dvu2956
promoter as identified by a method of the present disclosure (see
below). In some aspects, such as transcriptional activation of a
dvu2956 promoter, a compound acts directly by interacting with a
factor that itself is directly involved in orchestrating regulation
of DVU2956 protein expression (e.g., a transcription factor or
polymerase that regulates a dvu2956 promoter), while in other
aspects the compound acts indirectly on factors that are
mechanistically upstream of a factor that directly regulates
DVU2956 protein expression.
[0073] A compound for upregulating DVU2956 protein expression in
some aspects can be comprised in an aqueous composition or
non-aqueous composition, typically depending on the nature of the
compound itself (e.g., hydrophilic or hydrophobic) and/or the
environment in which the bacteria are treated. Such a composition
can be a solution or a dispersion/emulsion, for example. The
solvent of a liquid composition comprising a compound herein can
comprise about, at least about, or less than about, 0, 10, 20, 30,
40, 50, 60, 70, 80, 90, or 100 wt % water, for example. In some
aspects, the composition can be formulated as a liquid, lotion,
cream, spray, gel, ointment, washing powder, or cleaning agent such
as a cleaning solution, cleaning liquid, cleaning lotion, cleaning
cream, cleaning spray, cleaning gel and the like. In some aspects,
a compound for upregulating DVU2956 protein expression can be
present in a composition (formulation) that further comprises one
or more of a surfactant/detergent, solubilizing agent (typically
for solubilizing the compound), buffer, salt, viscosity/rheology
modifier, lubricant, or metal chelator, and/or any of these or
other ingredients or formulations as disclosed in Worthington et
al. (2012, Org. Biomol. Chem. 10:7457-7474), Zain et al. (2018,
Int. J. Corrosion vol. 2018, pp. 1-7, article ID 3567569), Cheung
and Beech (1996, Biofouling 9:231-249), or U.S. Patent Appl. Publ.
Nos. 2013/0029884, 2005/0238729, 2010/0298275, 2013/0052250,
2015/009891, 2016/0152495, or 2016/0152495, or U.S. Pat. Nos.
4,552,591, 4,925,582, 6,478,972, 6,514,458, 6,395,189, 7,927,496,
or 8784659, which are incorporated herein by reference. It is
contemplated that upregulation of a DVU2956 protein in bacteria
cells, such as by treatment with a compound, generally is not
biocidal. For example, all of, or at least 95% or 99% of, bacteria
cells are not killed when induced to upregulate DVU2956 protein
expression. A compound or treatment herein that upregulates DVU2956
protein expression generally is not biocidal against bacteria such
as SRB. For example, a compound that upregulates DVU2956 protein
expression typically is not one as disclosed in any of the
foregoing references. In some aspects, a compound for upregulating
DVU2956 protein expression herein, while generally not biocidal
itself, can be used in conjunction with a biocidal compound such as
disclosed in any of the foregoing references.
[0074] In some aspects, bacteria cells that are targeted for
upregulating DVU2956 protein expression can be on, or adjacent to,
a surface, such as would be the case if the cells are in a biofilm.
A surface can be abiotic, inert, and/or biotic (of life or derived
from life), for example. An abiotic or inert surface can comprise a
metal (e.g., iron, copper, nickel, zinc, titanium, molybdenum,
chromium), for example, and optionally be a metal alloy (e.g.,
steel, stainless steel). An abiotic or inert surface in some other
aspects can comprise plastic, rubber, porcelain/ceramic,
silica/glass, and/or mineral material (e.g.,
stone/rock/concrete).
[0075] An abiotic or inert surface in some aspects can be of a
device/component/equipment and/or system/process of an industrial
setting. Examples of industrial settings herein include those of an
energy (e.g., fossil fuel such as petroleum), water (e.g., water
treatment and/or purification, industrial water, wastewater
treatment), agriculture (e.g., grain, fruits/vegetables, fishing,
aquaculture, dairy, animal farming, timber, plants), chemical
(e.g., pharmaceutical, chemical processing), food
processing/manufacturing, mining, or transportation (e.g., fresh
water and/or maritime shipping, train or truck container)
industry.
[0076] An abiotic or inert surface in some aspects can be of a
device/component/equipment and/or system/process of a water
treatment, water storage, and/or other water-bearing system (e.g.,
piping/conduits, heat exchangers, condensers, filters/filtration
systems, storage tanks, water cooling towers, pasteurizers,
boilers). An abiotic or inert surface in some aspects can be of a
device/component/equipment and/or system/process of a fossil fuel
(e.g., oil/petroleum or natural gas) extraction, storage, delivery,
or processing/refining operation (e.g., oil drilling pipes, oil
pipelines, oil storage tanks, gas drilling pipes, gas pipelines,
offshore rig, wellbore, wellhead, shipping containers, oil
well/down hole). Such an extraction (recovery) operation can be
based on land or offshore. An abiotic or inert surface in some
aspects can be of a ship (e.g., hull, ballast tank).
[0077] An abiotic or inert surface in some aspects can be of a
device/component/equipment and/or system/process of a
medical/dental/healthcare setting (e.g., hospital, clinic,
examination room, nursing home), food service setting (e.g.,
restaurant, commissary kitchen, cafeteria), retail setting (e.g.,
grocery, soft drink machine/dispenser), hospitality/travel setting
(e.g., hotel/motel), sports/recreational setting (e.g., gym,
exercise equipment, locker room, aquatics/tubs, spa), or
office/home setting (e.g., bathroom, tub/shower, kitchen, air
vents, appliances [e.g., fridge, freezer], sprinkler system).
Examples of medical devices include contact lenses, intravenous
catheters and connectors, endotracheal tubes, intrauterine devices,
mechanical heart valves, pacemakers, peritoneal dialysis catheters,
prosthetic joints, tympanostomy tubes, urinary catheters, voice
prostheses, and instruments. Another abiotic or inert surface
herein can be of a water (optionally potable water) installation
(e.g., water storage tank, water heater) or where there is standing
or condensed water (e.g., as sometimes found in an air/ventilation
duct).
[0078] In some aspects, bacteria cells that are targeted for
upregulating DVU2956 protein expression can be comprised in any of
the foregoing settings (e.g., industrial,
medical/dental/healthcare, food service, retail,
hospitality/travel, sports/recreational, office/home) in a
planktonic state. Biofilm formation is inhibited or reduced in
these settings by following the disclosed upregulation method.
[0079] In some aspects, bacteria cells that are targeted for
upregulating DVU2956 protein expression can be comprised on a
biotic surface (e.g., biofilm) or otherwise in a biotic setting
(e.g., planktonic state). A biotic surface or setting can
optionally be associated with any of the foregoing settings, such
as an industrial setting (e.g., petroleum, wood/pulp processing,
animal meat processing, food processing) or
medical/dental/healthcare setting (e.g., teeth, skin/nails/wounds,
body orifice [e.g., nasal, oral, genitourinary],
gastrointestinal/alimentary canal, eye/conjunctiva, ear/ear canal,
pulmonary, cardiovascular). A biotic surface or setting can
optionally be associated with food (e.g., fruit/vegetable,
meat/fish [e.g., frozen, cured], pre-prepared food/meal [e.g.,
frozen, fresh], dairy product, grain product).
[0080] Accordingly, the presently disclosed methods and
compositions can be adapted for controlling bacteria in various
applications, such as those associated with any of the above
aspects. Just to illustrate, they can be used in various phases of
oil or natural gas production, transmission, and storage, both
topside and downhole, such as in aeration towers, de-aeration
towers, storage tanks, injection water, production water, pigging
operations, drilling muds, completion or workover fluids,
stimulation fluids, packing fluids, fracturing fluids, and
hydrotest fluids. As other examples, the presently disclosed
methods and compositions can be used in water treatment and
purification processes/systems (e.g., membranes and other system
components that are susceptible to fouling); paper and pulp
production; ballast water disinfection; preventing bacterial
contamination of water-based fluids and systems used in cooling
and/or heating processes; and preventing bacterial contamination of
water-based slurry, ink and tape-joint compounds, water-based
household products and personal care products, latex, paint, and
coatings. As other examples, the presently disclosed methods and
compositions can be used for any application, system/process,
apparatus, and/or surface as disclosed in any of U.S. Patent Appl.
Publ. Nos. 2013/0029884, 2005/0238729, 2010/0298275, 2016/0152495,
2013/0052250, 2015/009891, or 2016/0152495, or U.S. Pat. Nos.
4,552,591, 4,925,582, 6,478,972, 6,514,458, 6,395,189, 7,927,496,
or 8784659, which are all incorporated herein by reference.
[0081] Embodiments of the present disclosure concern other methods
of controlling bacteria cells. These other methods comprise
upregulating expression of a DVU2960 protein, DVU2962 protein,
and/or a DVU2964 protein in bacteria cells, thereby (i) dispersing
a biofilm of the cells or reducing biofilm formation by the cells,
and/or (ii) reducing hydrogen sulfide formation by the cells. In
some aspects, a DVU2960 protein herein can comprise, or consist of,
the amino acid sequence of SEQ ID NO:4. Alternatively, a DVU2960
protein herein can comprise, or consist of, an amino acid sequence
that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%
identical to SEQ ID NO:4, for example. In some aspects, a DVU2962
protein herein can comprise, or consist of, the amino acid sequence
of SEQ ID NO:8. Alternatively, a DVU2962 protein herein can
comprise, or consist of, an amino acid sequence that is at least
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ
ID NO:8, for example. In some aspects, a DVU2964 protein herein can
comprise, or consist of, the amino acid sequence of SEQ ID NO:10.
Alternatively, a DVU2964 protein herein can comprise, or consist
of, an amino acid sequence that is at least about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:10, for
example. A variant DVU2960, DVU2962, or DVU2964 protein should have
some of (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
95% of), or all of, the function of its respective non-variant
DVU2960, DVU2962, or DVU2964 protein. It is contemplated that any
embodiment disclosed herein of upregulating expression of a DVU2956
protein to control bacterial cell biofilm formation and/or
maintenance, as reasonably appropriate, can alternatively employ
upregulating expression of a DVU2960, DVU2962, and/or DVU2964
protein to effect such bacterial cell control. Upregulating
expression of a DVU2960, DVU2962, and/or DVU2964 protein in
bacteria cells herein can optionally be in addition to upregulating
expression of a DVU2956 protein in the cells.
[0082] Embodiments of the present disclosure also concern a method
of identifying a candidate compound for controlling bacteria cells.
This method can comprise: (a) providing bacteria cells comprising a
polynucleotide that comprises (i) a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) regulatory nucleotide sequence
operably linked to (ii) a nucleotide sequence ("reporter nucleotide
sequence"); (b) contacting the bacteria cells of step (a) with at
least one test compound (small molecule); and (c) determining
whether expression of the nucleotide sequence by the bacteria cells
of step (b) is upregulated, wherein such upregulation indicates
that the test compound is a candidate compound for (i) controlling
biofilm maintenance or biofilm formation by the bacteria cells or
other bacteria cells, and/or (ii) reducing hydrogen sulfide
formation by the bacteria cells or other bacteria cells. Such a
method can optionally be characterized as a screening method. Steps
(a) and (b) of a screening method herein can be performed
separately or together; step (b) in-and-of-itself encompasses step
(a). A candidate compound identified by a screening method herein
can optionally be characterized as a compound (putative compound)
for controlling bacteria as presently disclosed. Typically, a
polynucleotide of a screening method herein is heterologous, in
that it is heterologous with respect to the bacteria cells, and/or
its DVU2956 protein regulatory sequence is heterologous to its
reporter nucleotide sequence.
[0083] Bacteria cells employed in steps (a)-(c) of a screening
method in some aspects can be any type of bacteria cell disclosed
herein, such as above or in the below Examples. For example, the
bacteria cells can be SRB (e.g., Desulfovibrio species), E. coli,
or Bacillus cells.
[0084] A DVU2956 protein regulatory nucleotide sequence in some
aspects of a screening method herein can comprise one or more
regulatory sequences. For example, a regulatory sequence can
comprise a promoter sequence, upstream activating sequence (and/or
other transcription factor binding sequence), and/or
5'-untranslated region (5'-UTR) sequence, all of which are
typically derivable from a dvu2956 gene/locus. In some aspects, a
regulatory sequence herein can comprise (i) a promoter or (ii) a
promoter that is operably linked to a 5'-UTR sequence. Typically, a
reporter nucleotide sequence of a heterologous polynucleotide
herein is operably linked downstream of a regulatory sequence. If
the reporter nucleotide sequence contains an open reading frame and
is intended to be expressible as a protein, a ribosome binding site
(RBS, Shine-Dalgarno sequence) (native, synthetic, and/or
heterologous) typically is included upstream of the start codon; a
spacer sequence located between the 3'-end of an RBS and the start
codon, if present, can be about 2-10 bp (e.g., 5-7 bp) long, for
example. Any suitable RBS sequence can be employed, such as one
disclosed herein. In some aspects, a regulatory sequence (e.g.,
promoter and/or 5'-UTR) is located immediately upstream of a start
codon or RBS, or about, or within about, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 1-50, 1-25, 1-20, 1-15, 1-10, or 1-5 bp upstream.
[0085] Examples of a promoter for use in a regulatory sequence
herein can comprise or consist of a nucleotide sequence that is at
least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% to
identical to SEQ ID NO:13. Examples of a 5'-UTR for use in a
regulatory sequence herein can comprise or consist of a nucleotide
sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5%, or 100% to identical to SEQ ID NO:14. In aspects in which a
regulatory sequence comprises both promoter and 5'-UTR sequences,
such a regulatory sequence can comprise or consist of a nucleotide
sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5%, or 100% to identical to SEQ ID NO:12, for example. In some
aspects, a DVU2956 protein regulatory nucleotide sequence is
derivable from any type of bacteria as disclosed herein. While a
DVU2956 protein regulatory nucleotide sequence of a screening
method herein can be heterologous to the bacteria cells used in the
method (e.g., assaying an SRB regulatory sequence in a commonly
used lab strain such as E. coli), it can be autologous to the
bacteria cells in some other aspects (e.g., assaying a D. vulgaris
regulatory sequence in a D. vulgaris strain).
[0086] A reporter nucleotide sequence of a heterologous
polynucleotide in a screening method herein can encode a protein
(i.e., comprise an open reading frame). Such a protein can
optionally be characterized as a reporter protein. Increased
expression of reporter protein by bacterial cells of step (b) of a
screening method herein indicates upregulated expression of the
reporter nucleotide sequence. Increased reporter protein expression
can be discerned by detecting the reporter protein directly (e.g.,
using an antibody-dependent method or spectrometry method as
disclosed above) and/or by detecting activity/function of the
reporter protein (e.g., enzymatic activity, fluorescence). Examples
of a reporter protein herein include glutathione-5-transferase
(GST), horseradish peroxidase (HRP), chloramphenicol
acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase
(GUS), luciferase, green fluorescent protein (GFP), HcRed, DsRed,
cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),
blue fluorescent protein (BFP), and yellow-green fluorescent
protein. In some aspects, a reporter nucleotide sequence of a
heterologous polynucleotide does not encode a protein, in which
case its upregulation can be measured by detecting RNA transcripts
of the reporter nucleotide sequence (e.g., using any RNA detection
method herein).
[0087] In some aspects, upregulated expression of a reporter
nucleotide sequence in a screening method herein is increased
expression of the nucleotide sequence by about, or at least about,
25%, 50%, 100%, 150%, 200%, 250%, 500%, 1000%, 1500%, 2000%, 2500%,
3000%, 4000%, 5000%, or 10000% above its expression by a suitable
control (e.g., the bacteria cells prior to contacting them with a
test compound). Increased reporter nucleotide sequence expression
can be, for example, based on measuring levels of encoded protein,
protein activity, and/or RNA transcripts of the reporter nucleotide
sequence.
[0088] A heterologous polynucleotide of a screening method herein
can optionally be characterized as a reporter or reporter
construct, for example. Examples of such a reporter construct can
be selected from a plasmid, cosmid, phagemid, bacterial artificial
chromosome (BAC), virus/phage, or linear DNA (e.g., linear PCR
product). A reporter construct in some aspects can be capable of
existing transiently (i.e., not integrated into the genome) or
stably (i.e., integrated into the genome) in a bacterial cell. A
reporter construct in some aspects can comprise, or lack, one or
more suitable marker sequences (e.g., selection or phenotype
marker).
[0089] Upregulated expression of a reporter nucleotide sequence by
a DVU2956 protein regulatory sequence in a screening method herein
indicates that (1) a test compound is potentially able to
upregulate dvu2956 gene expression in the bacteria from which the
regulatory sequence was derived (or is derivable from), and/or (2)
a test compound is potentially able to upregulate dvu2956 gene
expression in another type of bacteria. This in turn indicates that
the test compound is a candidate compound for upregulating DVU2956
protein expression, and therefore also that it is a candidate
compound for (i) controlling biofilm maintenance or biofilm
formation by bacteria cells, and/or (ii) reducing hydrogen sulfide
formation by bacteria cells. These effects (i and ii) can be any of
those as described herein regarding upregulating DVU2956 protein
expression.
[0090] A screening method can further comprise one or more of
following steps (d)-(f) to determine whether a candidate compound
(identified in step c) is suitable for controlling bacteria:
[0091] (d) contacting the bacteria cells or other bacteria cells
comprised in a biofilm with the candidate compound identified in
step (c), wherein dispersal of the biofilm indicates that the
candidate compound inhibits biofilm maintenance;
[0092] (e) contacting the bacteria cells or other bacteria cells
comprised in a liquid culture with the candidate compound
identified in step (c), wherein an inability of the bacteria cells
or other bacteria cells to form a biofilm indicates that the
candidate compound inhibits biofilm formation; and/or
[0093] (f) contacting the bacteria cells or other bacteria cells
with the candidate compound identified in step (c), wherein a
reduction in H.sub.2S production by the bacteria cells or other
bacteria cells indicates that the candidate compound inhibits
H.sub.2S production by the bacteria cells or other bacteria
cells.
Step (d) typically further comprises a step of determining or
measuring biofilm dispersal by bacteria contacted with the
candidate compound. The degree of dispersal can be as disclosed
above. Step (e) typically further comprises a step of determining
or measuring biofilm formation by bacteria contacted with the
candidate compound. The degree of reduction of biofilm formation
can be as disclosed above. Step (f) typically further comprises a
step of determining or measuring H.sub.2S production by bacteria
contacted with the candidate compound. The degree of reduction of
H.sub.2S production can be as disclosed above.
[0094] In some aspects, the bacteria used in steps (a)-(c) of a
screening method is the same as the bacteria from which the DVU2956
protein regulatory sequence was derived (or is derivable from),
while in other aspects the bacteria used in steps (a)-(c) is the
different from the bacteria from which the DVU2956 protein
regulatory sequence was derived (or is derivable from). In some
aspects, the bacteria used in steps (a)-(c) of a screening method
is the same as the bacteria used in step(s) (d), (e), and/or (f),
while in other aspects the bacteria used in steps (a)-(c) is
different from the bacteria used in step(s) (d), (e), and/or (f).
In some aspects, step(s) (d), (e), and/or (f) is/are conducted with
the same type of bacteria from which the DVU2956 protein regulatory
sequence was derived (or is derivable from). In some less typical
aspects, a screening method employs a bacteria in which the
bacteria's endogenous dvu2956 gene is used as the reporter
polynucleotide sequence for assaying whether a candidate compound
can control bacteria cells.
[0095] Test/candidate compounds of a screening method herein can
optionally be formulated as described above for formulating a
compound for upregulating DVU2956 protein expression in
applications of controlling bacteria. Steps of contacting a
test/candidate compound with bacteria cells can be performed for
about, or at least about, 3, 6, 8, 10, 12, 15, 18, 21, 24, 30, 36,
42, 48, 60, 72, 84, or 96 hours, for example. The temperature in
which bacteria cells in a screening method are contacted with a
test/candidate compound can be about, at least about, or up to
about, -1, 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
60, 70, 75, 15-40, or 15-70.degree. C., for example. Bacteria cells
in a screening method herein can be grown in any media and/or
conditions suitable for growing the bacteria cells (e.g., below
Examples), and/or in conditions that are similar to or the same as
those in which the bacteria live in the field.
[0096] A screening method in some aspects comprises screening a
plurality of test compounds by following steps (a)-(c) to identify
one or more candidate compounds for controlling bacteria cells. A
plurality of test compounds can optionally be characterized as a
library of compounds. A library herein can comprise about, or at
least about, 5000, 10000, 25000, 50000, 100000, 250000, 500000,
750000, or 1000000 compounds, for example. Test compounds can be
applied in a screening method individually or in combination with
other test compounds; the latter approach eventually leads to
screening compounds on an individual basis after identifying one or
more pools of compounds that upregulate the reporter nucleotide
sequence.
[0097] Embodiments of the present disclosure also concern a
polynucleotide comprising (i) a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) regulatory sequence operably linked
to (ii) a nucleotide sequence, wherein the regulatory sequence and
the nucleotide sequence are heterologous to each other, optionally
wherein the regulatory sequence includes a promoter sequence. A
DVU2956 protein regulatory sequence and/or nucleotide sequence of
such a polynucleotide can be any of those disclosed herein (e.g.,
as in the above screening method or below Examples). In some
alternative aspects, the regulatory sequence and the nucleotide
sequence are autologous to each other.
[0098] A polynucleotide herein comprising a DVU2956 protein
regulatory sequence and nucleotide sequence can be a vector or
construct useful for transferring a nucleotide sequence into a cell
and/or testing activity of the regulatory sequence (as above), for
example. Examples of a suitable vector/construct can be selected
from a plasmid, yeast artificial chromosome (YAC), cosmid,
phagemid, bacterial artificial chromosome (BAC), virus/phage, or
linear DNA (e.g., linear PCR product). A polynucleotide sequence in
some aspects can be capable of existing transiently (i.e., not
integrated into the genome) or stably (i.e., integrated into the
genome) in a cell. A polynucleotide sequence in some aspects can
comprise, or lack, one or more suitable marker sequences (e.g.,
selection or phenotype marker).
[0099] Some aspects herein are drawn to a cell comprising a
polynucleotide sequence as presently disclosed. Such a cell can be
any bacterial cell as disclosed herein (e.g., E. coli, Bacillus,
SRB [e.g., Desulfovibrio species]), for example. In some aspects, a
cell can be a eukaryotic cell such as a fungus (e.g., yeast),
insect, or mammalian cell. A cell can optionally be capable of
using the DVU2956 protein regulatory sequence to express the
nucleotide sequence of the polynucleotide. In some aspects, the
polynucleotide sequence exists transiently (i.e., not integrated
into the genome) or stably (i.e., integrated into the genome) in
the cell.
[0100] Non-limiting examples of compositions and methods disclosed
herein include:
1. A method of controlling bacteria cells, the method comprising:
upregulating expression of a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) in the bacteria cells, thereby (i)
dispersing a biofilm of the cells or reducing biofilm formation by
the cells, and/or (ii) reducing hydrogen sulfide formation by the
cells. 2. The method of embodiment 1, wherein the cells are
sulfate-reducing bacteria (sulfide-producing bacteria) cells. 3.
The method of embodiment 2, wherein the cells are of the order
Desulfovibrionales. 4. The method of embodiment 3, wherein the
cells are of the genus Desulfovibrio. 5. The method of embodiment
1, 2, 3, or 4, wherein the DVU2956 sigma 54-dependent EBP is
endogenous to the cells. 6. The method of embodiment 1, 2, 3, 4, or
5, wherein the cells are comprised within a biofilm, and the
biofilm is dispersed following the upregulation step. 7. The method
of embodiment 1, 2, 3, 4, 5, or 6, wherein expression of the
DVU2956 sigma 54-dependent EBP prior to the upregulation step is
repressed by the cells. 8. The method of embodiment 1, 2, 3, 4, 5,
6, or 7, wherein the cells are treated with at least one compound
to induce the upregulated expression of the DVU2956 sigma
54-dependent EBP. 9. The method of embodiment 1, 2, 3, 4, 5, 6, 7,
or 8, wherein the cells are on one or more surfaces of industrial
equipment (one or more surfaces of a piece of industrial equipment)
or are otherwise present in an industrial process. 10. A method of
identifying a candidate compound for controlling bacteria cells,
the method comprising: (a) providing bacteria cells comprising a
polynucleotide that comprises (i) a DVU2956 sigma 54-dependent
enhancer-binding protein (EBP) regulatory sequence operably linked
to (ii) a nucleotide sequence; (b) contacting the bacteria cells of
step (a) with at least one test compound; and (c) determining
whether expression of the nucleotide sequence by the bacteria cells
of step (b) is upregulated, wherein such upregulation indicates
that the test compound is a candidate compound for (i) controlling
biofilm maintenance or biofilm formation by the bacteria cells or
other bacteria cells, and/or (ii) reducing hydrogen sulfide
formation by the bacteria cells or other bacteria cells. 11. The
method of embodiment 10, further comprising: (d) contacting the
bacteria cells or other bacteria cells comprised in a biofilm with
the candidate compound identified in step (c), wherein dispersal of
the biofilm indicates that the candidate compound inhibits biofilm
maintenance; (e) contacting the bacteria cells or other bacteria
cells comprised in a liquid culture with the candidate compound
identified in step (c), wherein an inability of the bacteria cells
or other bacteria cells to form a biofilm indicates that the
candidate compound inhibits biofilm formation; or (f) contacting
the bacteria cells or other bacteria cells with the candidate
compound identified in step (c), wherein a reduction in hydrogen
sulfide production by the bacteria cells or other bacteria cells
indicates that the candidate compound inhibits hydrogen sulfide
production by the bacteria cells or other bacteria cells. 12. The
method of embodiment 10 or 11, wherein the nucleotide sequence
comprises a sequence that encodes a reporter protein, wherein
increased expression of the reporter protein by the bacterial cells
of step (b) indicates upregulated expression of the nucleotide
sequence. 13. The method of embodiment 10, 11, or 12, wherein the
DVU2956 sigma 54-dependent EBP regulatory sequence comprises a
promoter sequence. 14. The method of embodiment 10, 11, 12, or 13,
wherein the bacterial cells or other bacteria cells are
sulfate-reducing bacteria (sulfide-producing bacteria) cells. 15.
The method of embodiment 10, 11, 12, 13, or 14, comprising
screening a plurality of test compounds by following steps (a)-(c)
to identify one or more candidate compounds for controlling
bacteria cells. 16. A polynucleotide comprising (i) a DVU2956 sigma
54-dependent enhancer-binding protein (EBP) regulatory sequence
operably linked to (ii) a nucleotide sequence, wherein the
regulatory sequence and the nucleotide sequence are heterologous to
each other, optionally wherein the regulatory sequence includes a
promoter sequence. 17. A cell comprising the polynucleotide of
embodiment 16, optionally wherein the nucleotide sequence is
capable of being expressed by the cell, and preferably wherein the
cell is a bacterial cell.
EXAMPLES
[0101] The present disclosure is further exemplified in the below
Examples. It should be understood that these Examples, while
indicating certain aspects herein, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of the
disclosed embodiments, and without departing from the spirit and
scope thereof, can make various changes and modifications to adapt
the disclosed embodiments to various uses and conditions.
MATERIALS AND METHODS
Bacterial Strains and Growth Conditions
TABLE-US-00002 [0102] Strains Features.sup.a Source D. vulgaris
Hildenborough Wild-type, ATCC 29579 American Type Culture
Collection (ATCC) D. desulfuricans Wild-type, isolated from
sulfidic mud, Deutsche Sammlung von DSM 12129 Mikroorganismen und
Zellkulturen GmbH (DSMZ) E. coli TG1 K-12 supE thi-1 .DELTA.(lac-
J. Minshull proAB) .DELTA.(mcrB- hsdSM)5,
(r.sub.K.sup.-m.sub.K.sup.-) E. coli BL21(DE3) F.sup.- ompT
hsdS.sub.B(r.sub.B.sup.-m.sub.B.sup.-) gal M. Nomura dcm (DE3)
[0103] Desulfovibrio vulgaris Hildenborough (ATCC 29579) was grown
anaerobically at 30.degree. C. in 25-mL screwcap tubes containing
10 mL of modified Baar's medium (ATCC.RTM. Medium 1249; .about.166
mM MgSO.sub.4, .about.19 mM sodium citrate, .about.7 mM CaSO.sub.4,
.about.19 mM NH.sub.4Cl, .about.3 mM K.sub.2HPO.sub.4, .about.31 mM
sodium lactate, .about.1 g/L yeast extract, pH 7.5) with 0.025%
sodium sulfide (as an oxygen scavenger). Initial cultures were
grown from glycerol stocks stored at -80.degree. C.; all
subcultures were grown from a 5% inoculum from the initial culture
and incubated without shaking; 400 ng/.mu.L Geneticin.RTM. (G418)
was used to maintain plasmids. E. coli strains were cultured at
37.degree. C. with shaking at 250 rpm using LB medium with 50
ng/.mu.L kanamycin to maintain broad host vectors based on plasmid
pVLT33.
Plasmids and Recombinant Work
TABLE-US-00003 [0104] Plasmids Features.sup.a Source pVLT33
broad-host-range expression vector de Lorenzo et al., 1993, (IncQ,
RSF1010 replicon), Km.sup.r, Ptac, Gene 123: 17-24..sup.b laclq
tra.sup.- mob.sup.+ pBluescriptII .RTM. (SK-) E. coli vector, pUC
ori, f1 (-) ori, Amp.sup.r, Stratagene Plac pBSKan E. coli vector,
pUC ori, f1 (-) ori, Km.sup.r, Plac Canada et al., 2002, J.
Bacteriol. 184: 344-349..sup.b pET-27b(+) PT7, pBR322 ori, Km.sup.r
Novagen .RTM. pMQ70 PBAD, Car.sup.r, shuttle vector for inducible
Shanks et al., 2006, expression using BAD promoter (PBAD) Appl.
Environ. Microbiol. 72: 5027-5036..sup.b pVLT33-Pdvu0304-dvu2956
Pdvu0304::dvu2956, RSF1010 replicon, Herein Km.sup.r, Ptac, laclq
tra.sup.- mob.sup.+ (for expressing DVU2956 [SEQ ID NO: 2] in
biofilm cells) pVLT33-Pdvu0304-dvu2960 Pdvu0304::dvu2960, RSF1010
replicon, Herein Km.sup.r, Ptac, laclq tra.sup.- mob.sup.+ (for
expressing DVU2960 [SEQ ID NO: 4] in biofilm cells)
pVLT33-Pdvu0304-dvu2961 Pdvu0304::dvu2961, RSF1010 replicon, Herein
Km.sup.r, Ptac, laclq tra.sup.- mob.sup.+ (for expressing DVU2961
[SEQ ID NO: 6] in biofilm cells) pVLT33-Pdvu0304-dvu2962
Pdvu0304::dvu2962, RSF1010 replicon, Herein Km.sup.r, Ptac, laclq
tra.sup.- mob.sup.+ (for expressing DVU2962 [SEQ ID NO: 8] in
biofilm cells) pVLT33-Pdvu0304-dvu2964 Pdvu0304::dvu2964, RSF1010
replicon, Herein Km.sup.r, Ptac, laclq tra.sup.- mob.sup.+ (for
expressing DVU2964 [SEQ ID NO: 10] in biofilm cells)
pVLT33-Ptac-dvu2956 Ptac::dvu2956, RSF1010 replicon, Km.sup.r,
Herein laclq tra.sup.- mob.sup.+ pBluescriptII .RTM.
(SK-)-.DELTA.dvu2956 pBluescriptII (SK-)- .DELTA.dvu2956 .OMEGA.
Km.sup.r Herein (for disrupting dvu2956 in D. vulgaris)
.sup.aKm.sup.r, Amp.sup.r and Car.sup.r indicate kanamycin-,
ampicillin- and carbenicillin-resistance, respectively.
.sup.bReference is incorporated herein by reference.
[0105] A nucleotide sequence encoding D. vulgaris DVU2956 sigma
54-dependent EBP (SEQ ID NO:2) was cloned into broad-host range
plasmid pVLT33 under the control of the biofilm phase promoter
Pdvu0304 (D. vulgaris) and ribosome binding site "AAGGAG" to render
plasmid pVLT33-Pdvu0304-dvu2956. Similar constructs were made for
individually expressing D. vulgaris DVU2960 (SEQ ID NO:4), DVU2961
(SEQ ID NO:6), DVU2962 (SEQ ID NO:8) and DVU2964 (SEQ ID NO:10)
proteins. The DVU2956-encoding sequence was also cloned into
plasmid pMQ70, which was used for inducing DVU2956 (SEQ ID NO:2)
expression via inducible promotor PBAD.
[0106] The native dvu2956 gene in D. vulgaris was knocked out using
plasmid pBluescriptII.RTM. (SK-)-.DELTA.dvu2956, which does not
replicate. This construct contains upstream (1008 bp) and
downstream (915 bp) sequences of dvu2956 that flank a
kanamycin-resistance gene derived from vector pBSKan. For
complementing D. vulgaris (dvu2956.sup.-) phenotypes, a regulatory
sequence (SEQ ID NO:12) comprising dvu2956 promoter (SEQ ID NO:13),
herein denoted as "Pdvu2956") and dvu2956 5'-untranslated region
(SEQ ID NO:14) sequences of D. vulgaris was cloned into broad-host
range plasmid pMQ70 (replacing PBAD) to render plasmid
pMQ70-Pdvu2956. Then, ribosome binding site "AAGGAG" and nucleotide
sequence encoding DVU2956 (SEQ ID NO:2) were inserted downstream of
Pdvu2956 to render plasmid pMQ70-Pdvu2956-dvu2956. Successful
preparation of the plasmid constructs used herein was confirmed by
sequencing.
[0107] Competent D. vulgaris and D. desulfuricans cells (turbidity
of OD.sub.600 nm .about.0.3) were prepared by washing twice
anaerobically with pre-chilled, sterile 10% glycerol. Plasmid DNA
(0.5 to 1 .mu.g) was added to the competent cells (50 .mu.L) by
mixing gently, and this preparation was transferred to a
pre-chilled (0.degree. C.), 1-mm electroporation cuvette in an
anaerobic chamber. Electroporation (25 .rho.F, 200 .OMEGA., 1.5
kV/cm) was then performed aerobically, and the cuvette was moved
back to the anaerobic chamber immediately, where Modified Baar's
medium (1 mL) was added. The electroporated cells were mixed gently
and transferred to a 1.5-mL Eppendorf.RTM. tube where they
recovered overnight at 30.degree. C. Recovered cell preparation (50
.mu.L) was then inoculated into either 10 mL of Modified Baar's
medium (0.2% yeast extract) or 1% agar plates; both these media
contained either G418 (400 .mu.g/mL for D. vulgaris, 800 .mu.g/mL
for D. desulfuricans) or 300 .mu.g/mL carbenicillin for D.
vulgaris. Genomic DNA from 1-2 mL culture or a colony was isolated
using UltraClean.RTM. Microbial DNA isolation kit (MO BIO cat. no.
12224) and used for PCR verification of the presence of the correct
plasmid in transformed cells.
Biofilm Formation and Maintenance Assays
[0108] D. vulgaris, D. desulfuricans and plasmid-transformed
strains of both of these bacteria were grown anaerobically in
Modified Baar's medium (300 .mu.L) in 96-well micro-titer plates
(Fisher Scientific, cat no. 07-200-656) for 24 hours at 30.degree.
C. without shaking, after which time the planktonic cell turbidity
in each well was measured spectrophotometrically at 620 nm using a
Sunrise.TM. microplate reader (TECAN, Switzerland). The plates were
then incubated under the same conditions for an additional 24 to 48
hours, after which biofilm formation was measured by crystal violet
staining. For biofilm dispersal assays, 24 hours after seeding the
plates for biofilm growth, arabinose (10 mM) was added to strains
harboring plasmids allowing arabinose-based induction of DVU2956
(SEQ ID NO:2). The arabinose-treated strains were then incubated
anaerobically for another 24 hours without shaking, after which
time biofilm formation was measured by crystal violet staining.
[0109] For crystal violet staining, the liquid portion of the
cultures was discarded and the wells were washed by dipping the
plates into 1 L of distilled water, after which the plates were
dried with paper towel. Crystal violet (0.1%, 300 .mu.L) was added
to each well and the plates were incubated for 20 minutes at room
temperature. After discarding the bulk of the staining solution,
the wells were washed three times with distilled water as above to
remove unbound crystal violet. The wells were then soaked for 5
minutes in 95% ethanol (300 .mu.L) to dissolve the remaining
(bound) crystal violet, which was then measured
spectrophotometrically at 540 nm using the Sunrise.TM. microplate
reader. Normalized biofilm formation percentage values were
calculated based on the ratio of a sample's respective OD 620 nm
reading (initial planktonic cell turbidity) to its OD 540 nm
reading (bound crystal violet) to compare the degree of biofilm
formation or dispersal.
RNA Analyses by RNA-Seq and qRT-PCR
[0110] D. vulgaris biofilm and planktonic cells were first grown,
after which RNA was isolated from each cell type for further
analysis.
[0111] Baar's modified medium (300 mL) was inoculated with a D.
vulgaris culture. The culture was anaerobically incubated at
30.degree. C. for 3 days to an OD.sub.600 nm .gtoreq.0.3.
Subsequently, 3.times.400 mL of Baar's modified medium was
inoculated with this culture for a starting OD.sub.600 nm=0.1. Each
of these cultures was then added to a 1-L beaker. Autoclaved glass
wool (10 g) was also included in each beaker. The cultures were
kept anaerobically standing at 30.degree. C. for 16-24 hours until
OD.sub.600 nm=.about.0.2. RNA was then separately isolated from the
biofilm and planktonic cells as described below, and used for RNA
analysis.
[0112] RNA-Seq Comparative transcriptomic analysis of RNA samples
(RNA-Seq) was based on normalizing gene transcript sequencing
results to Transcripts Per Kilobase Million (TPM). TPM was
calculated by first dividing the read counts by the length of each
gene in kilobases to yield reads per kilobase (RPK) for each gene.
Then the total RPK values of all the genes in a sample were added
together and divided by 1000000 to yield a per-million scaling
factor. Finally, the RPK value of each gene was divided by this
scaling factor to obtain the TPM value of each gene, and so the sum
of all the TPMs in each sample was 1000000. This method thus
allowed a direct comparison of the transcription of each gene
between different samples.
[0113] qRT-PCR Prior to performing qRT-PCR, regular PCR with D.
vulgaris genomic DNA was performed to ensure that only a single
band was produced by the primers. The qRT-PCR thermocycling
protocol was: 95.degree. C. for 5 min; 40 cycles of 95.degree. C.
for 15 s, 60.degree. C. for 1 min (annealing temperature was
60.degree. C. for all primers). Two replicate qRT-PCR reactions
were performed for each sample/primer pair. Components from the
iTaq.TM. universal SYBR.RTM. Green One-Step kit (Bio-Rad) were used
for each reaction.
Bioinformatics
[0114] Protein domain analysis was performed using the Conserved
Domain Search Service of the National Center for Biotechnology
Information (NCBI) website (ncbi.nlm.nih.gov). Sequence alignment
was performed using the Basic Local Alignment Search Tool (BLAST)
at the NCBI website. Multiple amino acid sequence alignment
analysis was conducted using Clustal X2.0 (Larkin et al., 2007,
Bioinformatics 23: 2947-2948).
Hydrogen Sulfide Production Assay
[0115] Hydrogen sulfide (H.sub.2S) by D. vulgaris strains was
measured via a methylene blue spectrophotometric assay (Rabinowitz,
1978, Methods Enzymol. 53:275-277; incorporated herein by
reference). Briefly, in this assay, N,N-dimethyl-p-phenylenediamine
dihydrochloride is converted into methylthioninium chloride
(methylene blue) by reacting with H.sub.2S dissolved in
hydrochloride acid in the presence of ferric chloride. For a
96-well screening format, D. vulgaris was anaerobically grown in
Modified Baar's medium in a 96-well plate (starting turbidity/OD of
0.05 at 600 nm, 150-.mu.L culture volume) and incubated for 48
hours for biofilm formation. Anaerobically, 25 .mu.L of each
biofilm culture was transferred to another 96-well plate containing
225 .mu.L deoxygenated water per well to render a 10-fold dilution
of the transferred culture. Next, 5%
N,N-dimethyl-p-phenylenediamine dihydrochloride (24.5 .mu.L,
prepared in 5.5 N HCl) (Sigma-Aldrich, cat. no. 536-46-9) was added
to each well. After mixing the wells gently by pipetting and
anaerobically incubating the mixes at room temperature for 3
minutes, the 96-well plate was removed from the anaerobic chamber
and the absorbance at 670 nm of each well was measured using a
Sunrise.TM. microplate reader.
[0116] For a more rigorous H.sub.2S assay, sealed glass vials were
used to prevent H.sub.2S loss. D. vulgaris strains were grown
anaerobically for 7 to 10 days in 10 mL of Modified Baar's medium
with 2.5 g of sterilized glass wool to promote biofilm formation.
Supernatants from these cultures were transferred by syringe to
another sealed vial for dilution with 10 mL of PBS buffer.
Anaerobically, 1 mL of diluted sample was then transferred by
syringe to another sealed bottle with 125 .mu.L of 12% sodium
hydroxide and 3.25 mL of 1% zinc acetate (to fix the sulfide from
H.sub.2S by forming ZnS precipitates). After mixing gently and
incubating for 30 minutes at room temperature, 625 .mu.L of 5%
N,N-dimethyl-p-phenylenediamine dihydrochloride and 125 .mu.L 0.023
M FeCl.sub.3 were added and the mixture was shaken for 20 minutes
at 300 rpm at room temperature. Water (2.125 mL) was added and
mixed, and then at least 1 mL of each reaction was removed to
determine absorbance at 670 nm.
Example 1
DVU2956 Sigma 54-Dependent Enhancer-Binding Protein (EBP) Gene
Expression is Repressed in Bacteria in a Biofilm
[0117] This Example describes identifying a gene encoding sigma
54-dependent EBP as being repressed by bacteria comprised within a
biofilm. In particular, a biofilm of Desulfovibrio vulgaris, which
is a sulfate-reducing bacteria, was found to exhibit repressed
expression of DVU2956 sigma 54-dependent EBP.
[0118] To identify genes involved in bacteria biofilm maintenance
and/or formation, RNA-Seq analysis (whole transcriptome shotgun
sequencing analysis) was performed to compare the transcription of
biofilm cells versus planktonic cells (i.e., floating as single
cells) of Desulfovibrio vulgaris Hildenborough (American Type
Culture Collection [ATCC.RTM.] No. 29579). To prepare each of these
types of cells, D. vulgaris was inoculated into Modified Baar's
medium and incubated anaerobically at 30.degree. C. for 16-24 hours
in beakers containing glass wool until OD.sub.600 nm=.about.0.2.
The glass wool was collected from the beakers and quickly washed
with an RNase-free 0.85% NaCl solution. Biofilm cells were released
from the glass wool by sonication and then collected in a
centrifuge tube by centrifugation at -2.degree. C. for 2 minutes
(8200 rpm). Planktonic cells, which were left behind in the medium
after glass wool removal and at exponential growth phase, were also
collected using centrifugation. Each set of collected cells was
individually resuspended in pre-chilled RNA/ater.TM. solution
(Thermo Fisher Scientific) and chilled for 5 seconds in a dry
ice/ethanol bath. The chilled cells were then centrifuged for 15
seconds at 13000 rpm, after which the collected cells were
flash-frozen in the dry ice/ethanol bath. Total RNA samples of the
biofilm and planktonic cells (each in triplicate) were prepared
using the High Pure RNA Isolation Kit (Roche).
[0119] The total RNA samples were then entered into RNA-Seq for
transcriptional quantification and comparison. Transcript
sequencing results were normalized on a Transcripts Per Kilobase
Million (TPM) basis, thereby allowing a direct comparison of the
transcription of each gene in the D. vulgaris biofilm cells versus
the D. vulgaris planktonic cells. This comparison showed, for
example, that expression of the sigma 54-dependent EBP, DVU2956
(SEQ ID NO:2), is repressed in biofilm cells relative to expression
of this EBP in planktonic cells (Table 2). The average TPM for
DVU2956 in biofilm cells was about 8.9, thereby reflecting about a
25-fold reduction in DVU2956 expression as compared to its
expression in planktonic cells (TPM=222.9).
TABLE-US-00004 TABLE 2 Highly Repressed D. vulgaris Genes in
Biofilm Cells Relative to Expression by Planktonic Cells Gene
Average TPM in Fold Difference in Protein Gene ID Name Planktonic
Cells Biofilm Cells.sup.a Length (aa) DVUA0144 5523.7 -.infin. 47
DVUA0134 4552.2 -.infin. 343 DVUA0014 3189.2 -.infin. 108 DVUA0122
2988.9 -.infin. 216 DVUA0010 2680.3 -.infin. 100 DVUA0009 2233.7
-.infin. 568 DVUA0128 2054.2 -.infin. 48 DVUA0012 2038.2 -.infin.
542 DVUA0119 1782.7 -.infin. 438 DVUA0065 1773.8 -.infin. 678
DVUA0096 1731.1 -.infin. 403 DVUA0121 1695.3 -.infin. 484 DVUA0060
1503.5 -.infin. 461 DVUA0125 1492.9 -.infin. 450 DVUA0083 1460.8
-.infin. 42 DVUA0045 1427.7 -.infin. 538 DVUA0102 1392.8 -.infin.
271 DVUA0044 1076.4 -.infin. 325 DVUA0113 1074.0 -.infin. 602
DVUA0149 1062.9 -.infin. 594 DVUA0046 1004.2 -.infin. 482 DVUA0008
898.1 -.infin. 504 DVU3321 398.1 -.infin. 30 DVU2605 360.6 -.infin.
144 DVU1297 318.9 -.infin. 50 DVU2700 292.2 -.infin. 139 DVU0205
291.6 -.infin. 55 DVU2248 287.7 -.infin. 34 DVU3081 271.4 -.infin.
300 DVU1275 270.2 -.infin. 186 DVU0667 263.5 -.infin. 329 DVU2528
257.4 -.infin. 65 DVU3361 252.1 -.infin. 341 DVU0230 250.5 -.infin.
154 DVU1731 247.2 -.infin. 56 DVU1127 246.1 -.infin. 137 DVU3155
235.1 -.infin. 963 DVU2207 224.3 -.infin. 57 DVU0001 221.9 -.infin.
437 DVU2596 218.3 -.infin. 259 DVU1725 211.2 -.infin. 202 DVU3166
205.5 -.infin. 218 DVU3058 205.1 -.infin. 1223 DVU1790 193.1
-.infin. 771 DVU1162 191.8 -.infin. 63 DVU1693 185.8 -.infin. 324
DVU1052 185.7 -.infin. 354 DVU1590 183.2 -.infin. 456 DVU0833 182.2
-.infin. 134 DVU2790 181.8 -.infin. 46 DVU2768 80.3 -.infin. 298
DVU1128 205.7 -75.6 221 DVU2202 223.8 -9.6 505 DVU2239 disH 231.9
-4.0 481 DVU2699 121.9 -95.0 215 DVU3205 294.2 -5.6 481 DVU0310
fliI 272.6 -3.3 437 DVU0311 191.9 -1.5 250 DVU0312 fliG 340.9 -1.6
338 DVU0313 fliF 429.3 -2.7 538 DVU0512 384.7 -2.0 260 DVU0513 flgG
258.1 -1.5 260 DVU0514 205.9 -3.4 310 DVU0516 flgI 320.6 -2.6 378
DVU0517 228.5 -2.4 610 DVU3231 217.9 -2.8 365 DVU3232 flhA 330.4
-7.0 703 DVU3233 flhB 255.6 -5.3 357 DVU3234 477.9 -2.6 263 DVU0043
fliQ 5110.0 -1.7 89 DVU0044 fliP 87.6 -4.1 235 DVU0045 149.9 -1.6
173 DVU0048 186.8 -5.8 246 DVU0050 motA-1 185.5 -23.4 252 DVU0086
951.9 -40.2 73 DVU2269 3760.9 -16.4 55 DVU2687 4449.5 -11.3 176
DVU2167 1010.9 -8.7 69 DVU1733 988.3 -7.4 73 DVU0019 ngr 1214.2
-6.7 202 DVU2956 222.9 -25.0 345 DVU1803 141.9 -5.2 362 DVU2732
128.3 -5.2 66 DVU2733 305.9 -14.3 249 .sup.aProvided is the
fold-difference (each value is negative) in TPM for each listed
gene in D. vulgaris biofilm cells as compared to the TPM for the
same gene in D. vulgaris planktonic cells.
To confirm this RNA-Seq result indicating DVU2956 repression,
qRT-PCR was performed with independently prepared RNA samples
(above isolation protocol) of D. vulgaris biofilm and planktonic
cells. The results showed that expression of DVU2956 (SEQ ID NO:2)
and DVU2960, which is regulated by DVU2956 (Kazakov et al., 2015,
BMC Genomics 16:919), were repressed 184-fold and 26.6-fold in
biofilm cells, respectively, as compared to planktonic cells.
[0120] Amino acid sequence alignment analysis showed that homologs
of D. vulgaris DVU2956 protein (SEQ ID NO:2) are widely present in
sulfate-reducing bacteria, and also present in some other bacteria
(Table 3). Based on these observations, it is contemplated that
DVU2956 sigma 54-dependent EBP plays a role in biofilm regulation
across a wide spectrum of bacterial species.
TABLE-US-00005 TABLE 3 Percent Amino Acid Identity of DVU2956 Sigma
54-Dependent EBP across Sulfate-Reducing Bacteria and Other
Bacteria DVU2956 Percent Bacteria Amino Acid Identity.sup.a
Desulfovibrio vulgaris str. Hildenborough 100% Desulfovibrio
vulgaris DP4 100% Desulfovibrio vulgaris RCH1 100% Desulfovibrio
vulgaris str. `Miyazaki F` 71% Desulfomicrobium baculatum DSM 4028
57% Desulfobacterales bacterium C00003104 48% Desulfococcus
multivorans 45% Desulfobacula sp. RIFOXYB2_FULL_45_6 43%
Desulfobacter postgatei 44% Desulfatitalea tepidiphila 44%
Desulfotomaculum australicum 47% Desulfosporosinus lacus 46%
Thermodesulfovibrio aggregans 43% Thermodesulfobacterium commune
43% Thermodesulfatator autotrophicus 44% Desulfotalea psychrophila
42% Syntrophobacter fumaroxidans 44% Clostridium magnum 43%
Shigella dysenteriae 45% Escherichia coli 45% Bacillus
ligniniphilus 47% Candidatus Moduliflexus flocculans 45%
Pseudomonas hussainii 45% Brevibacillus sp. NRRL NRS-603 44%
Klebsiella pneumoniae KCTC 2242 45% .sup.aPercent identity was
determined with respect to the DVU2956 sigma 54- dependent EBP of
SEQ ID NO: 2.
Example 2
Upregulated Expression of DVU2956 Sigma 54-Dependent EBP in
Bacteria Inhibits Biofilm Formation and Maintenance
[0121] This Example describes that upregulating expression of
DVU2956 sigma 54-dependent EBP in bacteria inhibits biofilm
formation by planktonic cells and disperses cells that have already
formed a biofilm. In particular, upregulated expression of DVU2956
sigma 54-dependent EBP in D. vulgaris was found to induce these
features. This upregulated expression was also shown to inhibit
biofilm formation by Desulfovibrio desulfuricans. This Example
further describes that upregulated expression of DVU2960, DVU2962
and DVU2964 proteins similarly inhibits bacterial biofilm
formation.
[0122] D. vulgaris, D. desulfuricans and certain
plasmid-transformed strains of both of these bacteria were grown
and analyzed for biofilm formation and maintenance as described
above (Materials and Methods). By utilizing a promoter from gene
dvu0304 that is active only in biofilms, it was found that biofilm
formation (at 48 hours) by D. vulgaris harboring construct
pVLT33-Pdvu0304-dvu2956 (which drives expression of DVU2956 [SEQ ID
NO:2] in biofilm cells) was inhibited by .about.70% as compared to
negative controls (wild type D. vulgaris and D.
vulgaris/pVLT33-Pdvu0304) (FIG. 1). This experiment was performed
four times with consistent results. Further work demonstrated that
knocking out the native dvu2956 gene in D. vulgaris, which rendered
D. vulgaris (dvu2956), increased biofilm formation by 30.1.+-.0.6%
compared to wild type D. vulgaris (FIG. 1). When D. vulgaris
(dvu2956.sup.-) was complemented with plasmid
pMQ70-Pdvu2956-dvu2956, biofilm formation (at 24 hours) was
inhibited by 48.+-.11% as compared to control strain D. vulgaris
(dvu2956.sup.-)/pMQ70-Pdvu2956 (FIG. 1). Further, by using broad
host range vector pMQ70 to express DVU2956 (SEQ ID NO:2) from an
inducible promoter (PBAD), induction of DVU2956 expression in
established D. vulgaris biofilms was shown to disperse the biofilms
after 24 hours by 42.+-.4% as compared to negative control (D.
vulgaris/pMQ70).
[0123] Because DVU2956 sigma 54-dependent EBP regulates the genes
dvu2957-dvu2964, which constitute the target operon of DVU2956,
direct expression of some of these genes (dvu2960, dvu2961,
dvu2962, dvu2964) was tested for their effect on biofilm formation
by D. vulgaris. It was found that biofilm-specific production (via
using pVLT33-Pdvu0304-based constructs) of DVU2960 (SEQ ID NO:4),
DVU2962 (SEQ ID NO:8) and DVU2964 (SEQ ID NO:10) proteins in D.
vulgaris led to 95%, 90% and 45% inhibition of biofilm formation
(at 48 hours), respectively, as compared to negative controls (wild
type D. vulgaris and D. vulgaris/pVLT33-Pdvu0304) (FIG. 1).
[0124] The effect of DVU2956 sigma 54-dependent EBP in another
sulfate-reducing bacteria, Desulfovibrio desulfuricans (DSM 12129),
was tested. It was found that, similar to the effects observed in
D. vulgaris, DVU2956 upregulation inhibited biofilm formation (at
24 hours) by 78.+-.9% in D. desulfuricans/pVLT33-Pdvu0304-dvu2956
as compared to negative control (D. desulfuricans/pVLT33-Pdvu0304)
(FIG. 1). Hence, DVU2956 functions in different species of
sulfate-reducing bacteria.
Example 3
Upregulated Expression of DVU2956 Sigma 54-Dependent EBP in
Bacteria Controls Hydrogen Sulfide Production
[0125] This Example describes that upregulation of DVU2956 sigma
54-dependent EBP expression controls hydrogen sulfide (H.sub.2S)
production in sulfate-reducing bacteria. In particular, this
feature was observed in D. vulgaris with upregulated expression of
DVU2956 sigma 54-dependent EBP.
[0126] H.sub.2S production assays were performed with D. vulgaris
strains as described above (Materials and Methods). As observed in
96-well culture conditions, D. vulgaris biofilm-specific expression
of DVU2956 (SEQ ID NO:2) (D. vulgaris/pVLT33-Pdvu0304-dvu2956)
decreased H.sub.2S production by 51.+-.2% as compared to negative
control (D. vulgaris/pVLT33-Pdvu0304), and knock-out of the native
dvu2956 gene increased H.sub.2S production by 131.+-.5% as compared
to negative control (wild type D. vulgaris) (FIG. 2). Verifying
these results using sealed vials, it was found that
biofilm-specific expression of DVU2956 (SEQ ID NO:2) inhibited
H.sub.2S production by 34.6.+-.0.6%, and knock-out of the native
dvu2956 gene increased H.sub.2S production by 136.+-.3% (FIG. 2).
This latter phenotype of D. vulgaris (dvu2956.sup.-) could be
complemented via ectopic production of DVU2956 (SEQ ID NO:2):
H.sub.2S production in D. vulgaris (dvu2956)pMQ70-Pdvu2956-dvu2956
was decreased by 45.+-.13% compared to negative control D. vulgaris
(dvu2956.sup.-)/pMQ70-Pdvu2956 (FIG. 2). These results all
consistently demonstrate that DVU2956 sigma 54-dependent EBP
expression reduces H.sub.2S production.
Example 4
dvu2956 Gene Regulatory Sequence can be Used to Assay for Modes of
Upregulating DVU2956 Sigma 54-Dependent EBP Expression in
Bacteria
[0127] This Example demonstrates that a regulatory sequence of gene
dvu2956 can be used to assay for conditions that upregulate
expression of DVU2956 sigma 54-dependent EBP in bacteria. A dvu2956
promoter sequence from D. vulgaris was tested for this purpose.
This tool will be useful for identifying small molecules, and/or
other compositions and conditions, that upregulate dvu2956 gene
expression in bacteria, thereby controlling bacterial biofilm
formation and H.sub.2S production.
[0128] A D. vulgaris dvu2956 gene regulatory sequence (SEQ ID
NO:12), which comprises (in 5' to 3' direction) predicted promoter
(SEQ ID NO:13) and 5'-untranslated region (5'-UTR) sequences (SEQ
ID NO:14), was linked upstream of a synthetic ribosome binding site
(RBS, SEQ ID NO:15) and a codon-optimized nucleotide sequence (SEQ
ID NO:16) encoding a monomeric yellow-green fluorescent protein
(mNeonGreen.TM., GenBank.RTM. Accession No. KC295282). This
nucleotide sequence (provided as SEQ ID NO:17) was constructed in
E. coli TG1 to render plasmid construct pMQ70-Pdvu2956-mNeonGreen
(8164 bp) and then transferred into D. vulgaris (wild type) and E.
coli strain BL21(DE3), the latter of which was already transformed
with plasmid pET27b-dvu2956 (pET27b-dvu2956 allows for IPTG-induced
expression of ectopic DVU2956 [SEQ ID NO:2]). PCR analyses
confirmed the presence of plasmids pMQ70-Pdvu2956-mNeonGreen (in
both strains) and pET27b-dvu2956 (in E. coli). Additional D.
vulgaris and E. coli strains that were transformed with plasmid
pMQ70 were prepared, and served as negative controls in the
fluorescence assays described below.
[0129] A comparison of the fluorescence signal of 10-day cultures
of planktonic D. vulgaris cells showed that D.
vulgaris/pMQ70-Pdvu2956-mNeonGreen had a fluorescence that was 2.4
fold-higher than that of D. vulgaris/pMQ70 (negative control) at
425 nm excitation and 517 nm emission (FIG. 3). This result
indicates that Pdvu2956 can heterologously drive gene expression in
sulfate-reducing bacterial cells, and therefore is contemplated to
be useful as a means for assaying compositions (e.g., small
molecules) and/or conditions that upregulate expression of DVU2956
sigma 54-dependent EBP in these cells.
[0130] A similar analysis was conducted to test whether Pdvu2956
can heterologously drive gene expression in bacteria other than
sulfate-reducing bacteria. A culture of E.
co/i/pMQ70-Pdvu2956-mNeonGreen/pET27b-dvu2956 exhibited several
fold higher fluorescence as compared to negative control (E.
co/i/pMQ70/pET27b-dvu2956) (FIG. 4, 0 mM IPTG). This result
indicates that Pdvu2956 can heterologously drive gene expression in
other types of bacterial cells. Pdvu2956 therefore is contemplated
to be a useful tool for assaying compositions (e.g., small
molecules) and/or conditions that upregulate expression of DVU2956
sigma 54-dependent EBP in sulfate-reducing bacteria. It was further
observed that ectopic expression of DVU2956 (SEQ ID NO:2) as
induced by IPTG exposure for 90 minutes resulted in up to about a
14-fold higher fluorescence signal in E.
co/i/pMQ70-Pdvu2956-mNeonGreen/pET27b-dvu2956 as compared to
negative control (FIG. 4, 1 mM IPTG). This result shows that
DVU2956 protein expression possibly upregulates Pdvu2956 activity.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 21 <210> SEQ ID NO 1 <211> LENGTH: 1038
<212> TYPE: DNA <213> ORGANISM: Desulfovibrio vulgaris
Hildenborough <400> SEQUENCE: 1 atgagcggca aaaaattacc
gcatagcggc aattctcttc ccattgaggg gacggtatcg 60 ccccagcgcg
aaacgcttca gaacggaacg gacttcctgc ttctgtcagg cgtcatgcaa 120
aggttgcggg tgcttgcgtc gcaagtcgcc tcctccgatg cgccagtgct cattcatggt
180 gagacgggaa cgggcaagga actcttcgcc cgtctcatcc acgatatggg
cattcgagcc 240 aaaaagcctt ttgtggcagt caattgcgga gtccacgagg
gtgagctctt cgccgacaag 300 ttcttcggcc atgagcaggg ggcgttcacc
ggggcgcaca gaatgagtca gggatgtttc 360 gagctcgctt cggaggggac
gctcttcctt gacgaagtgg gcgagatacc cggtgccaat 420 caggcggatt
tcctgcgggt gctggaagag aagcgtttca ggcgcatcgg cgggcagcgt 480
gacatcccgt ttcaggcgcg tatcgtcgcc gcctcgaacc gtgatttgca ggagatggtg
540 gggcaggggc agttcagggc cgacctcttc tacaggctca atgtcattcc
cgtggtcctt 600 ccgcccttgc gtgcccgcaa ggaggaggtc gtgccgctgg
cacggcattt cctgacccat 660 tatggcgaca agtaccatcg gcccggtgtc
cgtttcgccc cggagacgga acaggcgctg 720 gcggcgtacc agtggccggg
gaacgtgcgc gagctcaaga atcttgtgga acgcatcgcg 780 ttgctcgcgc
cggaaggcgt tctcgggcct gaacatctgc cgcttgagtt gcgttcagcc 840
gctgcggtgg aggtgggggt gccccacgag gtgccggaag acctgagcct cgaccgggcc
900 agacgagagg ccgaggtgcg cgtcatcctc aaggccatgc gcgccactgg
cggcaacaag 960 ggcgaggcgg cccgcctgct gggagtgagc ccgcgtaccc
tgcgctacaa gtttgcggag 1020 tatgcgttgc gattctga 1038 <210> SEQ
ID NO 2 <211> LENGTH: 345 <212> TYPE: PRT <213>
ORGANISM: Desulfovibrio vulgaris Hildenborough <400>
SEQUENCE: 2 Met Ser Gly Lys Lys Leu Pro His Ser Gly Asn Ser Leu Pro
Ile Glu 1 5 10 15 Gly Thr Val Ser Pro Gln Arg Glu Thr Leu Gln Asn
Gly Thr Asp Phe 20 25 30 Leu Leu Leu Ser Gly Val Met Gln Arg Leu
Arg Val Leu Ala Ser Gln 35 40 45 Val Ala Ser Ser Asp Ala Pro Val
Leu Ile His Gly Glu Thr Gly Thr 50 55 60 Gly Lys Glu Leu Phe Ala
Arg Leu Ile His Asp Met Gly Ile Arg Ala 65 70 75 80 Lys Lys Pro Phe
Val Ala Val Asn Cys Gly Val His Glu Gly Glu Leu 85 90 95 Phe Ala
Asp Lys Phe Phe Gly His Glu Gln Gly Ala Phe Thr Gly Ala 100 105 110
His Arg Met Ser Gln Gly Cys Phe Glu Leu Ala Ser Glu Gly Thr Leu 115
120 125 Phe Leu Asp Glu Val Gly Glu Ile Pro Gly Ala Asn Gln Ala Asp
Phe 130 135 140 Leu Arg Val Leu Glu Glu Lys Arg Phe Arg Arg Ile Gly
Gly Gln Arg 145 150 155 160 Asp Ile Pro Phe Gln Ala Arg Ile Val Ala
Ala Ser Asn Arg Asp Leu 165 170 175 Gln Glu Met Val Gly Gln Gly Gln
Phe Arg Ala Asp Leu Phe Tyr Arg 180 185 190 Leu Asn Val Ile Pro Val
Val Leu Pro Pro Leu Arg Ala Arg Lys Glu 195 200 205 Glu Val Val Pro
Leu Ala Arg His Phe Leu Thr His Tyr Gly Asp Lys 210 215 220 Tyr His
Arg Pro Gly Val Arg Phe Ala Pro Glu Thr Glu Gln Ala Leu 225 230 235
240 Ala Ala Tyr Gln Trp Pro Gly Asn Val Arg Glu Leu Lys Asn Leu Val
245 250 255 Glu Arg Ile Ala Leu Leu Ala Pro Glu Gly Val Leu Gly Pro
Glu His 260 265 270 Leu Pro Leu Glu Leu Arg Ser Ala Ala Ala Val Glu
Val Gly Val Pro 275 280 285 His Glu Val Pro Glu Asp Leu Ser Leu Asp
Arg Ala Arg Arg Glu Ala 290 295 300 Glu Val Arg Val Ile Leu Lys Ala
Met Arg Ala Thr Gly Gly Asn Lys 305 310 315 320 Gly Glu Ala Ala Arg
Leu Leu Gly Val Ser Pro Arg Thr Leu Arg Tyr 325 330 335 Lys Phe Ala
Glu Tyr Ala Leu Arg Phe 340 345 <210> SEQ ID NO 3 <211>
LENGTH: 1425 <212> TYPE: DNA <213> ORGANISM:
Desulfovibrio vulgaris Hildenborough <400> SEQUENCE: 3
atgaagcaag catccctgcc agcaggtgcc cggatagccg ggctcgtcgg gcgcgaactc
60 gcgctcggcc cggtcttcga cgccatcccc accgggttgg cggtgctcga
tgccgacctg 120 cgcatatgcc tgatgaaccg cgcccttgag accatgacgg
ggttcaccac cgccgaggtg 180 gcgggcatcc cctgccggca tgtccttcgg
gccagcgtat gcctgcaccg ctgccctacc 240 cgcaccgctg tctgcgatgc
caccggcggc aacgcgccat gccatgcaca agaaggcgac 300 ctgctcaacc
gccatcgccg ccgcattccc gtgcgcctga ccctcgcccc tctccatgat 360
gcgcaggggc agctgtgcgg ctggctgcat accgccgaag acctttcgct cgtccgtgag
420 cttgaagaac ggtgcagcaa ggggcaggca tccgggccac tggtggggcg
cagcgtggtg 480 atggaggaac tcttccgtac cgtgcaggcc cttgcccaga
ccgagacccc cgtgctcatc 540 acgggcgaga cgggcacagg caaggacgcc
gtggccgaga ccatccacaa ggcatcaccg 600 cgcggtcgcg aaccgttcgt
caaggccagc ctgtcatccc ttcccgattt tctggtggag 660 tccgaactgt
tcggccaccg caagggcgcg ttccccgggg ccgaagagga caagcccggc 720
cgtttcagga tggcacaggg cggtacgttg tgcctgtccg agttgggcga cctgcccccc
780 gccatgcagg gacggctcgt gacgttcctc gacgagggac tggtctggcc
cgtgggcgcc 840 acagaccccg tcaggtgcga cgtgcgcctg ctcgttgcca
gcaacctcga cctggaggcc 900 atggtcgctt ccggaaggct gcgcgaagac
ctctacagca gactggcagc cgtgcgcatc 960 cacctgcccc cgctacgcga
acggggcgaa gacctcgagt tcctgctcag ccactacctt 1020 gcccatttcg
ccgccagatt gcgcaagacc atacacgggt tctccggcaa atctctgcgc 1080
gtgctgcttg cctacggctt tccgggcaac gtgcgtgagc tcaagaacat cgtcgagtac
1140 gccgcgaccc tgtgtacggg cgaggtcgtc atgccctcgc acctgccggc
ctacctcttc 1200 cacacgcgtc cggcagccgc acccccgcgt gccatcgaac
gcgacacccc caagggtgcc 1260 gccgaagacg aggcatcagc ccgcagcagc
gtcgtcgacc tcgaacgccg tctcatcgtg 1320 gacgcactgg cacgcgccgg
aaaccgcaag ggcgaggccg cccgcatcct cggctggggc 1380 agaagcaccc
tgtggcgcaa gatgaaacag ttcggactgg agtga 1425 <210> SEQ ID NO 4
<211> LENGTH: 474 <212> TYPE: PRT <213> ORGANISM:
Desulfovibrio vulgaris Hildenborough <400> SEQUENCE: 4 Met
Lys Gln Ala Ser Leu Pro Ala Gly Ala Arg Ile Ala Gly Leu Val 1 5 10
15 Gly Arg Glu Leu Ala Leu Gly Pro Val Phe Asp Ala Ile Pro Thr Gly
20 25 30 Leu Ala Val Leu Asp Ala Asp Leu Arg Ile Cys Leu Met Asn
Arg Ala 35 40 45 Leu Glu Thr Met Thr Gly Phe Thr Thr Ala Glu Val
Ala Gly Ile Pro 50 55 60 Cys Arg His Val Leu Arg Ala Ser Val Cys
Leu His Arg Cys Pro Thr 65 70 75 80 Arg Thr Ala Val Cys Asp Ala Thr
Gly Gly Asn Ala Pro Cys His Ala 85 90 95 Gln Glu Gly Asp Leu Leu
Asn Arg His Arg Arg Arg Ile Pro Val Arg 100 105 110 Leu Thr Leu Ala
Pro Leu His Asp Ala Gln Gly Gln Leu Cys Gly Trp 115 120 125 Leu His
Thr Ala Glu Asp Leu Ser Leu Val Arg Glu Leu Glu Glu Arg 130 135 140
Cys Ser Lys Gly Gln Ala Ser Gly Pro Leu Val Gly Arg Ser Val Val 145
150 155 160 Met Glu Glu Leu Phe Arg Thr Val Gln Ala Leu Ala Gln Thr
Glu Thr 165 170 175 Pro Val Leu Ile Thr Gly Glu Thr Gly Thr Gly Lys
Asp Ala Val Ala 180 185 190 Glu Thr Ile His Lys Ala Ser Pro Arg Gly
Arg Glu Pro Phe Val Lys 195 200 205 Ala Ser Leu Ser Ser Leu Pro Asp
Phe Leu Val Glu Ser Glu Leu Phe 210 215 220 Gly His Arg Lys Gly Ala
Phe Pro Gly Ala Glu Glu Asp Lys Pro Gly 225 230 235 240 Arg Phe Arg
Met Ala Gln Gly Gly Thr Leu Cys Leu Ser Glu Leu Gly 245 250 255 Asp
Leu Pro Pro Ala Met Gln Gly Arg Leu Val Thr Phe Leu Asp Glu 260 265
270 Gly Leu Val Trp Pro Val Gly Ala Thr Asp Pro Val Arg Cys Asp Val
275 280 285 Arg Leu Leu Val Ala Ser Asn Leu Asp Leu Glu Ala Met Val
Ala Ser 290 295 300 Gly Arg Leu Arg Glu Asp Leu Tyr Ser Arg Leu Ala
Ala Val Arg Ile 305 310 315 320 His Leu Pro Pro Leu Arg Glu Arg Gly
Glu Asp Leu Glu Phe Leu Leu 325 330 335 Ser His Tyr Leu Ala His Phe
Ala Ala Arg Leu Arg Lys Thr Ile His 340 345 350 Gly Phe Ser Gly Lys
Ser Leu Arg Val Leu Leu Ala Tyr Gly Phe Pro 355 360 365 Gly Asn Val
Arg Glu Leu Lys Asn Ile Val Glu Tyr Ala Ala Thr Leu 370 375 380 Cys
Thr Gly Glu Val Val Met Pro Ser His Leu Pro Ala Tyr Leu Phe 385 390
395 400 His Thr Arg Pro Ala Ala Ala Pro Pro Arg Ala Ile Glu Arg Asp
Thr 405 410 415 Pro Lys Gly Ala Ala Glu Asp Glu Ala Ser Ala Arg Ser
Ser Val Val 420 425 430 Asp Leu Glu Arg Arg Leu Ile Val Asp Ala Leu
Ala Arg Ala Gly Asn 435 440 445 Arg Lys Gly Glu Ala Ala Arg Ile Leu
Gly Trp Gly Arg Ser Thr Leu 450 455 460 Trp Arg Lys Met Lys Gln Phe
Gly Leu Glu 465 470 <210> SEQ ID NO 5 <211> LENGTH: 348
<212> TYPE: DNA <213> ORGANISM: Desulfovibrio vulgaris
Hildenborough <400> SEQUENCE: 5 atgcccgcga cactgctcat
ccccctctac cgcgacgagg tcgcaccacg cttcgacctc 60 gccggtgaag
tgctgctcgt gaccctcgac gccgaaggga ccgagacgga acgctccagc 120
gtcgtgcttg cccatgcctc ttccgaggac atctgccgca tggcactgga agagaaggtc
180 cgcaccgtca tatgcagcgg cattgacgag gaattctggc agtacctgcg
ctggaagcgt 240 attgaggtca tcgacaacgt catcggcccg gtcgaggagg
cgttgcgacg tcatgcggcc 300 ggaatgctcc gttcaggcga catcctcttc
cacagggagg gggcatga 348 <210> SEQ ID NO 6 <211> LENGTH:
115 <212> TYPE: PRT <213> ORGANISM: Desulfovibrio
vulgaris Hildenborough <400> SEQUENCE: 6 Met Pro Ala Thr Leu
Leu Ile Pro Leu Tyr Arg Asp Glu Val Ala Pro 1 5 10 15 Arg Phe Asp
Leu Ala Gly Glu Val Leu Leu Val Thr Leu Asp Ala Glu 20 25 30 Gly
Thr Glu Thr Glu Arg Ser Ser Val Val Leu Ala His Ala Ser Ser 35 40
45 Glu Asp Ile Cys Arg Met Ala Leu Glu Glu Lys Val Arg Thr Val Ile
50 55 60 Cys Ser Gly Ile Asp Glu Glu Phe Trp Gln Tyr Leu Arg Trp
Lys Arg 65 70 75 80 Ile Glu Val Ile Asp Asn Val Ile Gly Pro Val Glu
Glu Ala Leu Arg 85 90 95 Arg His Ala Ala Gly Met Leu Arg Ser Gly
Asp Ile Leu Phe His Arg 100 105 110 Glu Gly Ala 115 <210> SEQ
ID NO 7 <211> LENGTH: 1734 <212> TYPE: DNA <213>
ORGANISM: Desulfovibrio vulgaris Hildenborough <400>
SEQUENCE: 7 atgaagaacc cgctaggcac catgctctcg cgtctgcgct cggtgttcga
cgttccggat 60 gagatcgcgc cggaacgcta ccgcatgctg cggcgcaaga
taacgctgct catgacggcg 120 gtgtcggttc tgccgctgct catcctcacc
gccgtaagct accaccagta ccagagcacc 180 ctcacccgcg aaatcgtcac
ccccgtgcgg gcgctggtga acaagacccg ccactcgttc 240 gaactgttcc
ttgccgaacg gtcgtccacc gtgagccttc tcgccaagac ctattccatg 300
gcggaacttt ccgacgagaa gaacctcaac cgcatcttcc tcgcgctcaa gggcgagttc
360 cccggcttcg tcgacctcgg ggtcatcgac ggcaggggcg tgcaggtggg
ctatgtcggg 420 ccgtacgacg tacgggggaa gaactactcc gaggcggact
ggtacaaccg cacccgcgtg 480 aagggtgtct acatcagcga cgtgttcatg
ggtttcaggc gctttccgca catcgccatc 540 gccgtgcagc gcatgaaccc
cgacggcagt tcgtggatgc ttcgcgccac catcgagacc 600 acgcagttcg
acaggctcat cgcctccatg gggctcgacc cggagagtga cgccttcctc 660
atcaacaccg ccggagtact ccagaccaac tcgcgcttct acggcaatgt gctcgacgtg
720 atgcccatgc cggtgcccca cctgagctac gagccctcga tcatcgacac
ggaagatccc 780 gagggcaggc agattttcct ctcttcagcc tttctgcaga
atgccgactt cgccatcgtg 840 gcggtcaagc ccaagaccga gatcctgcgc
ccgtggacat cgctgcgcag cgaccttctg 900 attttcgtcg ccttcagtgt
cgccctcatc atctcagcgg ccttcggctt caccgacatg 960 ctcgtacgac
gcatgcgcga cagcgacgaa cggcgcatcg ccgccttcgt gcagatagag 1020
cacacccaga aactctcctc catcggcagg ctggcggcgg gcgtcgccca cgaaatcaac
1080 aaccccctcg ccatcatcaa cgaaaaggcg ggccttgcgg cagacctcat
cgcgctttcg 1140 caggactttc cgcagaaaga acgcttctcg gccatcgtcg
aggccatctc gcgttctgtc 1200 gaccgttgcc ggtccatcac gcacaggctt
ctcggcttct cgcgccgcat ggacgccacc 1260 tacgagcaac tcgacgtcaa
cggcatcctc aaagagacca tgagcttcct ggaacaggag 1320 gccgtgcacc
gttccatcac catcggcacc agtctcgacg cggggctgcc acgcatcacc 1380
tccgacaggg ggcagttgca gcaggtcttc ctcaacatca tcaacaacgc cttcgccgca
1440 gtgcaggacg gtggttccgt gacgctcacc acgttcgcag cggacggcgg
catggtgggc 1500 gtctcgatac aggacaacgg aaagggcatg tctgaagaag
tgcagcggca catcttcgaa 1560 ccgttcttca ccaccaagaa gacggcgggg
acgggcctag gcatgttcat cacctacggc 1620 atcatcaagc gactgggcgg
agagatcggc atcaacagca gggagggcgt tggcaccacc 1680 gtcaccgtct
acctgccgca ggacgcgccc gcaccgcaat cgctggagaa ctag 1734 <210>
SEQ ID NO 8 <211> LENGTH: 577 <212> TYPE: PRT
<213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 8 Met Lys Asn Pro Leu Gly Thr Met Leu Ser Arg
Leu Arg Ser Val Phe 1 5 10 15 Asp Val Pro Asp Glu Ile Ala Pro Glu
Arg Tyr Arg Met Leu Arg Arg 20 25 30 Lys Ile Thr Leu Leu Met Thr
Ala Val Ser Val Leu Pro Leu Leu Ile 35 40 45 Leu Thr Ala Val Ser
Tyr His Gln Tyr Gln Ser Thr Leu Thr Arg Glu 50 55 60 Ile Val Thr
Pro Val Arg Ala Leu Val Asn Lys Thr Arg His Ser Phe 65 70 75 80 Glu
Leu Phe Leu Ala Glu Arg Ser Ser Thr Val Ser Leu Leu Ala Lys 85 90
95 Thr Tyr Ser Met Ala Glu Leu Ser Asp Glu Lys Asn Leu Asn Arg Ile
100 105 110 Phe Leu Ala Leu Lys Gly Glu Phe Pro Gly Phe Val Asp Leu
Gly Val 115 120 125 Ile Asp Gly Arg Gly Val Gln Val Gly Tyr Val Gly
Pro Tyr Asp Val 130 135 140 Arg Gly Lys Asn Tyr Ser Glu Ala Asp Trp
Tyr Asn Arg Thr Arg Val 145 150 155 160 Lys Gly Val Tyr Ile Ser Asp
Val Phe Met Gly Phe Arg Arg Phe Pro 165 170 175 His Ile Ala Ile Ala
Val Gln Arg Met Asn Pro Asp Gly Ser Ser Trp 180 185 190 Met Leu Arg
Ala Thr Ile Glu Thr Thr Gln Phe Asp Arg Leu Ile Ala 195 200 205 Ser
Met Gly Leu Asp Pro Glu Ser Asp Ala Phe Leu Ile Asn Thr Ala 210 215
220 Gly Val Leu Gln Thr Asn Ser Arg Phe Tyr Gly Asn Val Leu Asp Val
225 230 235 240 Met Pro Met Pro Val Pro His Leu Ser Tyr Glu Pro Ser
Ile Ile Asp 245 250 255 Thr Glu Asp Pro Glu Gly Arg Gln Ile Phe Leu
Ser Ser Ala Phe Leu 260 265 270 Gln Asn Ala Asp Phe Ala Ile Val Ala
Val Lys Pro Lys Thr Glu Ile 275 280 285 Leu Arg Pro Trp Thr Ser Leu
Arg Ser Asp Leu Leu Ile Phe Val Ala 290 295 300 Phe Ser Val Ala Leu
Ile Ile Ser Ala Ala Phe Gly Phe Thr Asp Met 305 310 315 320 Leu Val
Arg Arg Met Arg Asp Ser Asp Glu Arg Arg Ile Ala Ala Phe 325 330 335
Val Gln Ile Glu His Thr Gln Lys Leu Ser Ser Ile Gly Arg Leu Ala 340
345 350 Ala Gly Val Ala His Glu Ile Asn Asn Pro Leu Ala Ile Ile Asn
Glu 355 360 365 Lys Ala Gly Leu Ala Ala Asp Leu Ile Ala Leu Ser Gln
Asp Phe Pro 370 375 380 Gln Lys Glu Arg Phe Ser Ala Ile Val Glu Ala
Ile Ser Arg Ser Val 385 390 395 400 Asp Arg Cys Arg Ser Ile Thr His
Arg Leu Leu Gly Phe Ser Arg Arg 405 410 415 Met Asp Ala Thr Tyr Glu
Gln Leu Asp Val Asn Gly Ile Leu Lys Glu 420 425 430 Thr Met Ser Phe
Leu Glu Gln Glu Ala Val His Arg Ser Ile Thr Ile 435 440 445 Gly Thr
Ser Leu Asp Ala Gly Leu Pro Arg Ile Thr Ser Asp Arg Gly 450 455 460
Gln Leu Gln Gln Val Phe Leu Asn Ile Ile Asn Asn Ala Phe Ala Ala 465
470 475 480 Val Gln Asp Gly Gly Ser Val Thr Leu Thr Thr Phe Ala Ala
Asp Gly 485 490 495 Gly Met Val Gly Val Ser Ile Gln Asp Asn Gly Lys
Gly Met Ser Glu 500 505 510 Glu Val Gln Arg His Ile Phe Glu Pro Phe
Phe Thr Thr Lys Lys Thr 515 520 525 Ala Gly Thr Gly Leu Gly Met Phe
Ile Thr Tyr Gly Ile Ile Lys Arg 530 535 540 Leu Gly Gly Glu Ile Gly
Ile Asn Ser Arg Glu Gly Val Gly Thr Thr 545 550 555 560 Val Thr Val
Tyr Leu Pro Gln Asp Ala Pro Ala Pro Gln Ser Leu Glu 565 570 575 Asn
<210> SEQ ID NO 9 <211> LENGTH: 660 <212> TYPE:
DNA <213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 9 atgcgcgaga acgcctgtgt gggatgtctc accgcatccg
catcgcacga actgcagaac 60 gccctcgcgg tcatccgcga atctgccggc
ctgatgcaag acctcatcgc ccttggcgga 120 gagaacatcc cccggcgtga
gcgcatcgtc gaactgctct ctctcatcca gcagcaggtg 180 gcgcgcggcg
gcgagttggc gggggggctc aacacccttg gtcacgcatg ggaagaggat 240
gacggcgacc tcgcccgcat cctcgaagag ttcgtcatcc ttgccgggcg catgggcagg
300 atgcgttccg tcaccgtggg actggcaccg ggcgaaagcg gcttgcgcgc
gccgagtgcg 360 gggcttgcct tgcgagtgct gctgttcgac cttttgcaga
cctgccttga agaggctccc 420 ggttgcgcgc tcaccttcgc cccggcaagg
cgcgacggca ttcccggaat acatctgcga 480 gtaggactgg cacgttgcga
cgcgactaca gacgtactgc gccgactcga cgcgcaactt 540 gcccgtctcg
gagcccgtaa ggagcccgac aacgggcacg gaacgcccgc aagggacgaa 600
ccggacggag tgttgtacta tttcacggcc tgtccctctg caacagggca cagggcatag
660 <210> SEQ ID NO 10 <211> LENGTH: 219 <212>
TYPE: PRT <213> ORGANISM: Desulfovibrio vulgaris
Hildenborough <400> SEQUENCE: 10 Met Arg Glu Asn Ala Cys Val
Gly Cys Leu Thr Ala Ser Ala Ser His 1 5 10 15 Glu Leu Gln Asn Ala
Leu Ala Val Ile Arg Glu Ser Ala Gly Leu Met 20 25 30 Gln Asp Leu
Ile Ala Leu Gly Gly Glu Asn Ile Pro Arg Arg Glu Arg 35 40 45 Ile
Val Glu Leu Leu Ser Leu Ile Gln Gln Gln Val Ala Arg Gly Gly 50 55
60 Glu Leu Ala Gly Gly Leu Asn Thr Leu Gly His Ala Trp Glu Glu Asp
65 70 75 80 Asp Gly Asp Leu Ala Arg Ile Leu Glu Glu Phe Val Ile Leu
Ala Gly 85 90 95 Arg Met Gly Arg Met Arg Ser Val Thr Val Gly Leu
Ala Pro Gly Glu 100 105 110 Ser Gly Leu Arg Ala Pro Ser Ala Gly Leu
Ala Leu Arg Val Leu Leu 115 120 125 Phe Asp Leu Leu Gln Thr Cys Leu
Glu Glu Ala Pro Gly Cys Ala Leu 130 135 140 Thr Phe Ala Pro Ala Arg
Arg Asp Gly Ile Pro Gly Ile His Leu Arg 145 150 155 160 Val Gly Leu
Ala Arg Cys Asp Ala Thr Thr Asp Val Leu Arg Arg Leu 165 170 175 Asp
Ala Gln Leu Ala Arg Leu Gly Ala Arg Lys Glu Pro Asp Asn Gly 180 185
190 His Gly Thr Pro Ala Arg Asp Glu Pro Asp Gly Val Leu Tyr Tyr Phe
195 200 205 Thr Ala Cys Pro Ser Ala Thr Gly His Arg Ala 210 215
<210> SEQ ID NO 11 <400> SEQUENCE: 11 000 <210>
SEQ ID NO 12 <211> LENGTH: 86 <212> TYPE: DNA
<213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(86) <223> OTHER INFORMATION: dvu2956 gene
regulatory sequence <400> SEQUENCE: 12 aatgcttttt gtcgtggact
tgtgacatgt ataacaagtt ccgtgccagc ggtgagcgct 60 tgccgttcca
cggattacaa gctatc 86 <210> SEQ ID NO 13 <211> LENGTH:
41 <212> TYPE: DNA <213> ORGANISM: Desulfovibrio
vulgaris Hildenborough <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(41) <223> OTHER
INFORMATION: predicted dvu2956 gene promoter sequence (Pdvu2956)
<400> SEQUENCE: 13 aatgcttttt gtcgtggact tgtgacatgt
ataacaagtt c 41 <210> SEQ ID NO 14 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: Desulfovibrio vulgaris
Hildenborough <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(45) <223> OTHER
INFORMATION: predicted dvu2956 gene 5'-UTR sequence <400>
SEQUENCE: 14 cgtgccagcg gtgagcgctt gccgttccac ggattacaag ctatc 45
<210> SEQ ID NO 15 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic ribosome binding site
(sRBS) <400> SEQUENCE: 15 gcgagataaa aaaaggagga cttt 24
<210> SEQ ID NO 16 <211> LENGTH: 711 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Codon-optimized sequence encoding
mNeonGreen <400> SEQUENCE: 16 atggtgagca agggcgagga
ggataacatg gcctctctcc cagcgacaca tgagttacac 60 atctttggct
ccatcaacgg tgtggacttt gacatggtgg gtcagggcac cggcaatcca 120
aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca gttctccccc
180 tggattctgg tccctcatat cgggtatggc ttccatcagt acctgcccta
ccctgacggg 240 atgtcgcctt tccaggccgc catggtagat ggctccggat
accaagtcca tcgcacaatg 300 cagtttgaag atggtgcctc ccttactgtt
aactaccgct acacctacga gggaagccac 360 atcaaaggag aggcccaggt
gaaggggact ggtttccctg ctgacggtcc tgtgatgacc 420 aactcgctga
ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc 480
atcatcagta cctttaagtg gagttacacc actggaaatg gcaagcgcta ccggagcact
540 gcgcggacca cctacacctt tgccaagcca atggcggcta actatctgaa
gaaccagccg 600 atgtacgtgt tccgtaagac ggagctcaag cactccaaga
ccgagctcaa cttcaaggag 660 tggcaaaagg cctttaccga tgtgatgggc
atggacgagc tgtacaagta a 711 <210> SEQ ID NO 17 <211>
LENGTH: 821 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Pdvu2956-5' UTR-sRBS-mNeonGreen cassette. <400> SEQUENCE: 17
aatgcttttt gtcgtggact tgtgacatgt ataacaagtt ccgtgccagc ggtgagcgct
60 tgccgttcca cggattacaa gctatcgcga gataaaaaaa ggaggacttt
atggtgagca 120 agggcgagga ggataacatg gcctctctcc cagcgacaca
tgagttacac atctttggct 180 ccatcaacgg tgtggacttt gacatggtgg
gtcagggcac cggcaatcca aatgatggtt 240 atgaggagtt aaacctgaag
tccaccaagg gtgacctcca gttctccccc tggattctgg 300 tccctcatat
cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt 360
tccaggccgc catggtagat ggctccggat accaagtcca tcgcacaatg cagtttgaag
420 atggtgcctc ccttactgtt aactaccgct acacctacga gggaagccac
atcaaaggag 480 aggcccaggt gaaggggact ggtttccctg ctgacggtcc
tgtgatgacc aactcgctga 540 ccgctgcgga ctggtgcagg tcgaagaaga
cttaccccaa cgacaaaacc atcatcagta 600 cctttaagtg gagttacacc
actggaaatg gcaagcgcta ccggagcact gcgcggacca 660 cctacacctt
tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt 720
tccgtaagac ggagctcaag cactccaaga ccgagctcaa cttcaaggag tggcaaaagg
780 cctttaccga tgtgatgggc atggacgagc tgtacaagta a 821 <210>
SEQ ID NO 18 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 18 Gly Ala Phe Thr Gly Ala 1 5 <210>
SEQ ID NO 19 <211> LENGTH: 41 <212> TYPE: PRT
<213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 19 Arg Arg Glu Ala Glu Val Arg Val Ile Leu
Lys Ala Met Arg Ala Thr 1 5 10 15 Gly Gly Asn Lys Gly Glu Ala Ala
Arg Leu Leu Gly Val Ser Pro Arg 20 25 30 Thr Leu Arg Tyr Lys Phe
Ala Glu Tyr 35 40 <210> SEQ ID NO 20 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Desulfovibrio vulgaris
Hildenborough <400> SEQUENCE: 20 Lys Gly Glu Ala Ala Arg Leu
Leu Gly 1 5 <210> SEQ ID NO 21 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Upstream activating
sequence (UAS) consensus sequence <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(13) <223>
OTHER INFORMATION: n = a, c, g, or t. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (15)..(15)
<223> OTHER INFORMATION: n = a, c, g, or t. <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(17)..(18) <223> OTHER INFORMATION: n = a, c, g, or t.
<400> SEQUENCE: 21 gcggnnnnnn nnngncnn 18
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 21 <210>
SEQ ID NO 1 <211> LENGTH: 1038 <212> TYPE: DNA
<213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 1 atgagcggca aaaaattacc gcatagcggc aattctcttc
ccattgaggg gacggtatcg 60 ccccagcgcg aaacgcttca gaacggaacg
gacttcctgc ttctgtcagg cgtcatgcaa 120 aggttgcggg tgcttgcgtc
gcaagtcgcc tcctccgatg cgccagtgct cattcatggt 180 gagacgggaa
cgggcaagga actcttcgcc cgtctcatcc acgatatggg cattcgagcc 240
aaaaagcctt ttgtggcagt caattgcgga gtccacgagg gtgagctctt cgccgacaag
300 ttcttcggcc atgagcaggg ggcgttcacc ggggcgcaca gaatgagtca
gggatgtttc 360 gagctcgctt cggaggggac gctcttcctt gacgaagtgg
gcgagatacc cggtgccaat 420 caggcggatt tcctgcgggt gctggaagag
aagcgtttca ggcgcatcgg cgggcagcgt 480 gacatcccgt ttcaggcgcg
tatcgtcgcc gcctcgaacc gtgatttgca ggagatggtg 540 gggcaggggc
agttcagggc cgacctcttc tacaggctca atgtcattcc cgtggtcctt 600
ccgcccttgc gtgcccgcaa ggaggaggtc gtgccgctgg cacggcattt cctgacccat
660 tatggcgaca agtaccatcg gcccggtgtc cgtttcgccc cggagacgga
acaggcgctg 720 gcggcgtacc agtggccggg gaacgtgcgc gagctcaaga
atcttgtgga acgcatcgcg 780 ttgctcgcgc cggaaggcgt tctcgggcct
gaacatctgc cgcttgagtt gcgttcagcc 840 gctgcggtgg aggtgggggt
gccccacgag gtgccggaag acctgagcct cgaccgggcc 900 agacgagagg
ccgaggtgcg cgtcatcctc aaggccatgc gcgccactgg cggcaacaag 960
ggcgaggcgg cccgcctgct gggagtgagc ccgcgtaccc tgcgctacaa gtttgcggag
1020 tatgcgttgc gattctga 1038 <210> SEQ ID NO 2 <211>
LENGTH: 345 <212> TYPE: PRT <213> ORGANISM:
Desulfovibrio vulgaris Hildenborough <400> SEQUENCE: 2 Met
Ser Gly Lys Lys Leu Pro His Ser Gly Asn Ser Leu Pro Ile Glu 1 5 10
15 Gly Thr Val Ser Pro Gln Arg Glu Thr Leu Gln Asn Gly Thr Asp Phe
20 25 30 Leu Leu Leu Ser Gly Val Met Gln Arg Leu Arg Val Leu Ala
Ser Gln 35 40 45 Val Ala Ser Ser Asp Ala Pro Val Leu Ile His Gly
Glu Thr Gly Thr 50 55 60 Gly Lys Glu Leu Phe Ala Arg Leu Ile His
Asp Met Gly Ile Arg Ala 65 70 75 80 Lys Lys Pro Phe Val Ala Val Asn
Cys Gly Val His Glu Gly Glu Leu 85 90 95 Phe Ala Asp Lys Phe Phe
Gly His Glu Gln Gly Ala Phe Thr Gly Ala 100 105 110 His Arg Met Ser
Gln Gly Cys Phe Glu Leu Ala Ser Glu Gly Thr Leu 115 120 125 Phe Leu
Asp Glu Val Gly Glu Ile Pro Gly Ala Asn Gln Ala Asp Phe 130 135 140
Leu Arg Val Leu Glu Glu Lys Arg Phe Arg Arg Ile Gly Gly Gln Arg 145
150 155 160 Asp Ile Pro Phe Gln Ala Arg Ile Val Ala Ala Ser Asn Arg
Asp Leu 165 170 175 Gln Glu Met Val Gly Gln Gly Gln Phe Arg Ala Asp
Leu Phe Tyr Arg 180 185 190 Leu Asn Val Ile Pro Val Val Leu Pro Pro
Leu Arg Ala Arg Lys Glu 195 200 205 Glu Val Val Pro Leu Ala Arg His
Phe Leu Thr His Tyr Gly Asp Lys 210 215 220 Tyr His Arg Pro Gly Val
Arg Phe Ala Pro Glu Thr Glu Gln Ala Leu 225 230 235 240 Ala Ala Tyr
Gln Trp Pro Gly Asn Val Arg Glu Leu Lys Asn Leu Val 245 250 255 Glu
Arg Ile Ala Leu Leu Ala Pro Glu Gly Val Leu Gly Pro Glu His 260 265
270 Leu Pro Leu Glu Leu Arg Ser Ala Ala Ala Val Glu Val Gly Val Pro
275 280 285 His Glu Val Pro Glu Asp Leu Ser Leu Asp Arg Ala Arg Arg
Glu Ala 290 295 300 Glu Val Arg Val Ile Leu Lys Ala Met Arg Ala Thr
Gly Gly Asn Lys 305 310 315 320 Gly Glu Ala Ala Arg Leu Leu Gly Val
Ser Pro Arg Thr Leu Arg Tyr 325 330 335 Lys Phe Ala Glu Tyr Ala Leu
Arg Phe 340 345 <210> SEQ ID NO 3 <211> LENGTH: 1425
<212> TYPE: DNA <213> ORGANISM: Desulfovibrio vulgaris
Hildenborough <400> SEQUENCE: 3 atgaagcaag catccctgcc
agcaggtgcc cggatagccg ggctcgtcgg gcgcgaactc 60 gcgctcggcc
cggtcttcga cgccatcccc accgggttgg cggtgctcga tgccgacctg 120
cgcatatgcc tgatgaaccg cgcccttgag accatgacgg ggttcaccac cgccgaggtg
180 gcgggcatcc cctgccggca tgtccttcgg gccagcgtat gcctgcaccg
ctgccctacc 240 cgcaccgctg tctgcgatgc caccggcggc aacgcgccat
gccatgcaca agaaggcgac 300 ctgctcaacc gccatcgccg ccgcattccc
gtgcgcctga ccctcgcccc tctccatgat 360 gcgcaggggc agctgtgcgg
ctggctgcat accgccgaag acctttcgct cgtccgtgag 420 cttgaagaac
ggtgcagcaa ggggcaggca tccgggccac tggtggggcg cagcgtggtg 480
atggaggaac tcttccgtac cgtgcaggcc cttgcccaga ccgagacccc cgtgctcatc
540 acgggcgaga cgggcacagg caaggacgcc gtggccgaga ccatccacaa
ggcatcaccg 600 cgcggtcgcg aaccgttcgt caaggccagc ctgtcatccc
ttcccgattt tctggtggag 660 tccgaactgt tcggccaccg caagggcgcg
ttccccgggg ccgaagagga caagcccggc 720 cgtttcagga tggcacaggg
cggtacgttg tgcctgtccg agttgggcga cctgcccccc 780 gccatgcagg
gacggctcgt gacgttcctc gacgagggac tggtctggcc cgtgggcgcc 840
acagaccccg tcaggtgcga cgtgcgcctg ctcgttgcca gcaacctcga cctggaggcc
900 atggtcgctt ccggaaggct gcgcgaagac ctctacagca gactggcagc
cgtgcgcatc 960 cacctgcccc cgctacgcga acggggcgaa gacctcgagt
tcctgctcag ccactacctt 1020 gcccatttcg ccgccagatt gcgcaagacc
atacacgggt tctccggcaa atctctgcgc 1080 gtgctgcttg cctacggctt
tccgggcaac gtgcgtgagc tcaagaacat cgtcgagtac 1140 gccgcgaccc
tgtgtacggg cgaggtcgtc atgccctcgc acctgccggc ctacctcttc 1200
cacacgcgtc cggcagccgc acccccgcgt gccatcgaac gcgacacccc caagggtgcc
1260 gccgaagacg aggcatcagc ccgcagcagc gtcgtcgacc tcgaacgccg
tctcatcgtg 1320 gacgcactgg cacgcgccgg aaaccgcaag ggcgaggccg
cccgcatcct cggctggggc 1380 agaagcaccc tgtggcgcaa gatgaaacag
ttcggactgg agtga 1425 <210> SEQ ID NO 4 <211> LENGTH:
474 <212> TYPE: PRT <213> ORGANISM: Desulfovibrio
vulgaris Hildenborough <400> SEQUENCE: 4 Met Lys Gln Ala Ser
Leu Pro Ala Gly Ala Arg Ile Ala Gly Leu Val 1 5 10 15 Gly Arg Glu
Leu Ala Leu Gly Pro Val Phe Asp Ala Ile Pro Thr Gly 20 25 30 Leu
Ala Val Leu Asp Ala Asp Leu Arg Ile Cys Leu Met Asn Arg Ala 35 40
45 Leu Glu Thr Met Thr Gly Phe Thr Thr Ala Glu Val Ala Gly Ile Pro
50 55 60 Cys Arg His Val Leu Arg Ala Ser Val Cys Leu His Arg Cys
Pro Thr 65 70 75 80 Arg Thr Ala Val Cys Asp Ala Thr Gly Gly Asn Ala
Pro Cys His Ala 85 90 95 Gln Glu Gly Asp Leu Leu Asn Arg His Arg
Arg Arg Ile Pro Val Arg 100 105 110 Leu Thr Leu Ala Pro Leu His Asp
Ala Gln Gly Gln Leu Cys Gly Trp 115 120 125 Leu His Thr Ala Glu Asp
Leu Ser Leu Val Arg Glu Leu Glu Glu Arg 130 135 140 Cys Ser Lys Gly
Gln Ala Ser Gly Pro Leu Val Gly Arg Ser Val Val 145 150 155 160 Met
Glu Glu Leu Phe Arg Thr Val Gln Ala Leu Ala Gln Thr Glu Thr 165 170
175 Pro Val Leu Ile Thr Gly Glu Thr Gly Thr Gly Lys Asp Ala Val Ala
180 185 190 Glu Thr Ile His Lys Ala Ser Pro Arg Gly Arg Glu Pro Phe
Val Lys 195 200 205 Ala Ser Leu Ser Ser Leu Pro Asp Phe Leu Val Glu
Ser Glu Leu Phe 210 215 220 Gly His Arg Lys Gly Ala Phe Pro Gly Ala
Glu Glu Asp Lys Pro Gly 225 230 235 240 Arg Phe Arg Met Ala Gln Gly
Gly Thr Leu Cys Leu Ser Glu Leu Gly 245 250 255 Asp Leu Pro Pro Ala
Met Gln Gly Arg Leu Val Thr Phe Leu Asp Glu 260 265 270 Gly Leu Val
Trp Pro Val Gly Ala Thr Asp Pro Val Arg Cys Asp Val 275 280 285 Arg
Leu Leu Val Ala Ser Asn Leu Asp Leu Glu Ala Met Val Ala Ser 290 295
300 Gly Arg Leu Arg Glu Asp Leu Tyr Ser Arg Leu Ala Ala Val Arg Ile
305 310 315 320 His Leu Pro Pro Leu Arg Glu Arg Gly Glu Asp Leu Glu
Phe Leu Leu 325 330 335
Ser His Tyr Leu Ala His Phe Ala Ala Arg Leu Arg Lys Thr Ile His 340
345 350 Gly Phe Ser Gly Lys Ser Leu Arg Val Leu Leu Ala Tyr Gly Phe
Pro 355 360 365 Gly Asn Val Arg Glu Leu Lys Asn Ile Val Glu Tyr Ala
Ala Thr Leu 370 375 380 Cys Thr Gly Glu Val Val Met Pro Ser His Leu
Pro Ala Tyr Leu Phe 385 390 395 400 His Thr Arg Pro Ala Ala Ala Pro
Pro Arg Ala Ile Glu Arg Asp Thr 405 410 415 Pro Lys Gly Ala Ala Glu
Asp Glu Ala Ser Ala Arg Ser Ser Val Val 420 425 430 Asp Leu Glu Arg
Arg Leu Ile Val Asp Ala Leu Ala Arg Ala Gly Asn 435 440 445 Arg Lys
Gly Glu Ala Ala Arg Ile Leu Gly Trp Gly Arg Ser Thr Leu 450 455 460
Trp Arg Lys Met Lys Gln Phe Gly Leu Glu 465 470 <210> SEQ ID
NO 5 <211> LENGTH: 348 <212> TYPE: DNA <213>
ORGANISM: Desulfovibrio vulgaris Hildenborough <400>
SEQUENCE: 5 atgcccgcga cactgctcat ccccctctac cgcgacgagg tcgcaccacg
cttcgacctc 60 gccggtgaag tgctgctcgt gaccctcgac gccgaaggga
ccgagacgga acgctccagc 120 gtcgtgcttg cccatgcctc ttccgaggac
atctgccgca tggcactgga agagaaggtc 180 cgcaccgtca tatgcagcgg
cattgacgag gaattctggc agtacctgcg ctggaagcgt 240 attgaggtca
tcgacaacgt catcggcccg gtcgaggagg cgttgcgacg tcatgcggcc 300
ggaatgctcc gttcaggcga catcctcttc cacagggagg gggcatga 348
<210> SEQ ID NO 6 <211> LENGTH: 115 <212> TYPE:
PRT <213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 6 Met Pro Ala Thr Leu Leu Ile Pro Leu Tyr Arg
Asp Glu Val Ala Pro 1 5 10 15 Arg Phe Asp Leu Ala Gly Glu Val Leu
Leu Val Thr Leu Asp Ala Glu 20 25 30 Gly Thr Glu Thr Glu Arg Ser
Ser Val Val Leu Ala His Ala Ser Ser 35 40 45 Glu Asp Ile Cys Arg
Met Ala Leu Glu Glu Lys Val Arg Thr Val Ile 50 55 60 Cys Ser Gly
Ile Asp Glu Glu Phe Trp Gln Tyr Leu Arg Trp Lys Arg 65 70 75 80 Ile
Glu Val Ile Asp Asn Val Ile Gly Pro Val Glu Glu Ala Leu Arg 85 90
95 Arg His Ala Ala Gly Met Leu Arg Ser Gly Asp Ile Leu Phe His Arg
100 105 110 Glu Gly Ala 115 <210> SEQ ID NO 7 <211>
LENGTH: 1734 <212> TYPE: DNA <213> ORGANISM:
Desulfovibrio vulgaris Hildenborough <400> SEQUENCE: 7
atgaagaacc cgctaggcac catgctctcg cgtctgcgct cggtgttcga cgttccggat
60 gagatcgcgc cggaacgcta ccgcatgctg cggcgcaaga taacgctgct
catgacggcg 120 gtgtcggttc tgccgctgct catcctcacc gccgtaagct
accaccagta ccagagcacc 180 ctcacccgcg aaatcgtcac ccccgtgcgg
gcgctggtga acaagacccg ccactcgttc 240 gaactgttcc ttgccgaacg
gtcgtccacc gtgagccttc tcgccaagac ctattccatg 300 gcggaacttt
ccgacgagaa gaacctcaac cgcatcttcc tcgcgctcaa gggcgagttc 360
cccggcttcg tcgacctcgg ggtcatcgac ggcaggggcg tgcaggtggg ctatgtcggg
420 ccgtacgacg tacgggggaa gaactactcc gaggcggact ggtacaaccg
cacccgcgtg 480 aagggtgtct acatcagcga cgtgttcatg ggtttcaggc
gctttccgca catcgccatc 540 gccgtgcagc gcatgaaccc cgacggcagt
tcgtggatgc ttcgcgccac catcgagacc 600 acgcagttcg acaggctcat
cgcctccatg gggctcgacc cggagagtga cgccttcctc 660 atcaacaccg
ccggagtact ccagaccaac tcgcgcttct acggcaatgt gctcgacgtg 720
atgcccatgc cggtgcccca cctgagctac gagccctcga tcatcgacac ggaagatccc
780 gagggcaggc agattttcct ctcttcagcc tttctgcaga atgccgactt
cgccatcgtg 840 gcggtcaagc ccaagaccga gatcctgcgc ccgtggacat
cgctgcgcag cgaccttctg 900 attttcgtcg ccttcagtgt cgccctcatc
atctcagcgg ccttcggctt caccgacatg 960 ctcgtacgac gcatgcgcga
cagcgacgaa cggcgcatcg ccgccttcgt gcagatagag 1020 cacacccaga
aactctcctc catcggcagg ctggcggcgg gcgtcgccca cgaaatcaac 1080
aaccccctcg ccatcatcaa cgaaaaggcg ggccttgcgg cagacctcat cgcgctttcg
1140 caggactttc cgcagaaaga acgcttctcg gccatcgtcg aggccatctc
gcgttctgtc 1200 gaccgttgcc ggtccatcac gcacaggctt ctcggcttct
cgcgccgcat ggacgccacc 1260 tacgagcaac tcgacgtcaa cggcatcctc
aaagagacca tgagcttcct ggaacaggag 1320 gccgtgcacc gttccatcac
catcggcacc agtctcgacg cggggctgcc acgcatcacc 1380 tccgacaggg
ggcagttgca gcaggtcttc ctcaacatca tcaacaacgc cttcgccgca 1440
gtgcaggacg gtggttccgt gacgctcacc acgttcgcag cggacggcgg catggtgggc
1500 gtctcgatac aggacaacgg aaagggcatg tctgaagaag tgcagcggca
catcttcgaa 1560 ccgttcttca ccaccaagaa gacggcgggg acgggcctag
gcatgttcat cacctacggc 1620 atcatcaagc gactgggcgg agagatcggc
atcaacagca gggagggcgt tggcaccacc 1680 gtcaccgtct acctgccgca
ggacgcgccc gcaccgcaat cgctggagaa ctag 1734 <210> SEQ ID NO 8
<211> LENGTH: 577 <212> TYPE: PRT <213> ORGANISM:
Desulfovibrio vulgaris Hildenborough <400> SEQUENCE: 8 Met
Lys Asn Pro Leu Gly Thr Met Leu Ser Arg Leu Arg Ser Val Phe 1 5 10
15 Asp Val Pro Asp Glu Ile Ala Pro Glu Arg Tyr Arg Met Leu Arg Arg
20 25 30 Lys Ile Thr Leu Leu Met Thr Ala Val Ser Val Leu Pro Leu
Leu Ile 35 40 45 Leu Thr Ala Val Ser Tyr His Gln Tyr Gln Ser Thr
Leu Thr Arg Glu 50 55 60 Ile Val Thr Pro Val Arg Ala Leu Val Asn
Lys Thr Arg His Ser Phe 65 70 75 80 Glu Leu Phe Leu Ala Glu Arg Ser
Ser Thr Val Ser Leu Leu Ala Lys 85 90 95 Thr Tyr Ser Met Ala Glu
Leu Ser Asp Glu Lys Asn Leu Asn Arg Ile 100 105 110 Phe Leu Ala Leu
Lys Gly Glu Phe Pro Gly Phe Val Asp Leu Gly Val 115 120 125 Ile Asp
Gly Arg Gly Val Gln Val Gly Tyr Val Gly Pro Tyr Asp Val 130 135 140
Arg Gly Lys Asn Tyr Ser Glu Ala Asp Trp Tyr Asn Arg Thr Arg Val 145
150 155 160 Lys Gly Val Tyr Ile Ser Asp Val Phe Met Gly Phe Arg Arg
Phe Pro 165 170 175 His Ile Ala Ile Ala Val Gln Arg Met Asn Pro Asp
Gly Ser Ser Trp 180 185 190 Met Leu Arg Ala Thr Ile Glu Thr Thr Gln
Phe Asp Arg Leu Ile Ala 195 200 205 Ser Met Gly Leu Asp Pro Glu Ser
Asp Ala Phe Leu Ile Asn Thr Ala 210 215 220 Gly Val Leu Gln Thr Asn
Ser Arg Phe Tyr Gly Asn Val Leu Asp Val 225 230 235 240 Met Pro Met
Pro Val Pro His Leu Ser Tyr Glu Pro Ser Ile Ile Asp 245 250 255 Thr
Glu Asp Pro Glu Gly Arg Gln Ile Phe Leu Ser Ser Ala Phe Leu 260 265
270 Gln Asn Ala Asp Phe Ala Ile Val Ala Val Lys Pro Lys Thr Glu Ile
275 280 285 Leu Arg Pro Trp Thr Ser Leu Arg Ser Asp Leu Leu Ile Phe
Val Ala 290 295 300 Phe Ser Val Ala Leu Ile Ile Ser Ala Ala Phe Gly
Phe Thr Asp Met 305 310 315 320 Leu Val Arg Arg Met Arg Asp Ser Asp
Glu Arg Arg Ile Ala Ala Phe 325 330 335 Val Gln Ile Glu His Thr Gln
Lys Leu Ser Ser Ile Gly Arg Leu Ala 340 345 350 Ala Gly Val Ala His
Glu Ile Asn Asn Pro Leu Ala Ile Ile Asn Glu 355 360 365 Lys Ala Gly
Leu Ala Ala Asp Leu Ile Ala Leu Ser Gln Asp Phe Pro 370 375 380 Gln
Lys Glu Arg Phe Ser Ala Ile Val Glu Ala Ile Ser Arg Ser Val 385 390
395 400 Asp Arg Cys Arg Ser Ile Thr His Arg Leu Leu Gly Phe Ser Arg
Arg 405 410 415 Met Asp Ala Thr Tyr Glu Gln Leu Asp Val Asn Gly Ile
Leu Lys Glu 420 425 430 Thr Met Ser Phe Leu Glu Gln Glu Ala Val His
Arg Ser Ile Thr Ile 435 440 445 Gly Thr Ser Leu Asp Ala Gly Leu Pro
Arg Ile Thr Ser Asp Arg Gly 450 455 460 Gln Leu Gln Gln Val Phe Leu
Asn Ile Ile Asn Asn Ala Phe Ala Ala 465 470 475 480 Val Gln Asp Gly
Gly Ser Val Thr Leu Thr Thr Phe Ala Ala Asp Gly 485 490 495 Gly Met
Val Gly Val Ser Ile Gln Asp Asn Gly Lys Gly Met Ser Glu 500 505 510
Glu Val Gln Arg His Ile Phe Glu Pro Phe Phe Thr Thr Lys Lys Thr 515
520 525
Ala Gly Thr Gly Leu Gly Met Phe Ile Thr Tyr Gly Ile Ile Lys Arg 530
535 540 Leu Gly Gly Glu Ile Gly Ile Asn Ser Arg Glu Gly Val Gly Thr
Thr 545 550 555 560 Val Thr Val Tyr Leu Pro Gln Asp Ala Pro Ala Pro
Gln Ser Leu Glu 565 570 575 Asn <210> SEQ ID NO 9 <211>
LENGTH: 660 <212> TYPE: DNA <213> ORGANISM:
Desulfovibrio vulgaris Hildenborough <400> SEQUENCE: 9
atgcgcgaga acgcctgtgt gggatgtctc accgcatccg catcgcacga actgcagaac
60 gccctcgcgg tcatccgcga atctgccggc ctgatgcaag acctcatcgc
ccttggcgga 120 gagaacatcc cccggcgtga gcgcatcgtc gaactgctct
ctctcatcca gcagcaggtg 180 gcgcgcggcg gcgagttggc gggggggctc
aacacccttg gtcacgcatg ggaagaggat 240 gacggcgacc tcgcccgcat
cctcgaagag ttcgtcatcc ttgccgggcg catgggcagg 300 atgcgttccg
tcaccgtggg actggcaccg ggcgaaagcg gcttgcgcgc gccgagtgcg 360
gggcttgcct tgcgagtgct gctgttcgac cttttgcaga cctgccttga agaggctccc
420 ggttgcgcgc tcaccttcgc cccggcaagg cgcgacggca ttcccggaat
acatctgcga 480 gtaggactgg cacgttgcga cgcgactaca gacgtactgc
gccgactcga cgcgcaactt 540 gcccgtctcg gagcccgtaa ggagcccgac
aacgggcacg gaacgcccgc aagggacgaa 600 ccggacggag tgttgtacta
tttcacggcc tgtccctctg caacagggca cagggcatag 660 <210> SEQ ID
NO 10 <211> LENGTH: 219 <212> TYPE: PRT <213>
ORGANISM: Desulfovibrio vulgaris Hildenborough <400>
SEQUENCE: 10 Met Arg Glu Asn Ala Cys Val Gly Cys Leu Thr Ala Ser
Ala Ser His 1 5 10 15 Glu Leu Gln Asn Ala Leu Ala Val Ile Arg Glu
Ser Ala Gly Leu Met 20 25 30 Gln Asp Leu Ile Ala Leu Gly Gly Glu
Asn Ile Pro Arg Arg Glu Arg 35 40 45 Ile Val Glu Leu Leu Ser Leu
Ile Gln Gln Gln Val Ala Arg Gly Gly 50 55 60 Glu Leu Ala Gly Gly
Leu Asn Thr Leu Gly His Ala Trp Glu Glu Asp 65 70 75 80 Asp Gly Asp
Leu Ala Arg Ile Leu Glu Glu Phe Val Ile Leu Ala Gly 85 90 95 Arg
Met Gly Arg Met Arg Ser Val Thr Val Gly Leu Ala Pro Gly Glu 100 105
110 Ser Gly Leu Arg Ala Pro Ser Ala Gly Leu Ala Leu Arg Val Leu Leu
115 120 125 Phe Asp Leu Leu Gln Thr Cys Leu Glu Glu Ala Pro Gly Cys
Ala Leu 130 135 140 Thr Phe Ala Pro Ala Arg Arg Asp Gly Ile Pro Gly
Ile His Leu Arg 145 150 155 160 Val Gly Leu Ala Arg Cys Asp Ala Thr
Thr Asp Val Leu Arg Arg Leu 165 170 175 Asp Ala Gln Leu Ala Arg Leu
Gly Ala Arg Lys Glu Pro Asp Asn Gly 180 185 190 His Gly Thr Pro Ala
Arg Asp Glu Pro Asp Gly Val Leu Tyr Tyr Phe 195 200 205 Thr Ala Cys
Pro Ser Ala Thr Gly His Arg Ala 210 215 <210> SEQ ID NO 11
<400> SEQUENCE: 11 000 <210> SEQ ID NO 12 <211>
LENGTH: 86 <212> TYPE: DNA <213> ORGANISM:
Desulfovibrio vulgaris Hildenborough <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(86)
<223> OTHER INFORMATION: dvu2956 gene regulatory sequence
<400> SEQUENCE: 12 aatgcttttt gtcgtggact tgtgacatgt
ataacaagtt ccgtgccagc ggtgagcgct 60 tgccgttcca cggattacaa gctatc 86
<210> SEQ ID NO 13 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(41) <223> OTHER INFORMATION: predicted
dvu2956 gene promoter sequence (Pdvu2956) <400> SEQUENCE: 13
aatgcttttt gtcgtggact tgtgacatgt ataacaagtt c 41 <210> SEQ ID
NO 14 <211> LENGTH: 45 <212> TYPE: DNA <213>
ORGANISM: Desulfovibrio vulgaris Hildenborough <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(45)
<223> OTHER INFORMATION: predicted dvu2956 gene 5'-UTR
sequence <400> SEQUENCE: 14 cgtgccagcg gtgagcgctt gccgttccac
ggattacaag ctatc 45 <210> SEQ ID NO 15 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
ribosome binding site (sRBS) <400> SEQUENCE: 15 gcgagataaa
aaaaggagga cttt 24 <210> SEQ ID NO 16 <211> LENGTH: 711
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Codon-optimized
sequence encoding mNeonGreen <400> SEQUENCE: 16 atggtgagca
agggcgagga ggataacatg gcctctctcc cagcgacaca tgagttacac 60
atctttggct ccatcaacgg tgtggacttt gacatggtgg gtcagggcac cggcaatcca
120 aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca
gttctccccc 180 tggattctgg tccctcatat cgggtatggc ttccatcagt
acctgcccta ccctgacggg 240 atgtcgcctt tccaggccgc catggtagat
ggctccggat accaagtcca tcgcacaatg 300 cagtttgaag atggtgcctc
ccttactgtt aactaccgct acacctacga gggaagccac 360 atcaaaggag
aggcccaggt gaaggggact ggtttccctg ctgacggtcc tgtgatgacc 420
aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc
480 atcatcagta cctttaagtg gagttacacc actggaaatg gcaagcgcta
ccggagcact 540 gcgcggacca cctacacctt tgccaagcca atggcggcta
actatctgaa gaaccagccg 600 atgtacgtgt tccgtaagac ggagctcaag
cactccaaga ccgagctcaa cttcaaggag 660 tggcaaaagg cctttaccga
tgtgatgggc atggacgagc tgtacaagta a 711 <210> SEQ ID NO 17
<211> LENGTH: 821 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Pdvu2956-5' UTR-sRBS-mNeonGreen cassette. <400>
SEQUENCE: 17 aatgcttttt gtcgtggact tgtgacatgt ataacaagtt ccgtgccagc
ggtgagcgct 60 tgccgttcca cggattacaa gctatcgcga gataaaaaaa
ggaggacttt atggtgagca 120 agggcgagga ggataacatg gcctctctcc
cagcgacaca tgagttacac atctttggct 180 ccatcaacgg tgtggacttt
gacatggtgg gtcagggcac cggcaatcca aatgatggtt 240 atgaggagtt
aaacctgaag tccaccaagg gtgacctcca gttctccccc tggattctgg 300
tccctcatat cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt
360 tccaggccgc catggtagat ggctccggat accaagtcca tcgcacaatg
cagtttgaag 420 atggtgcctc ccttactgtt aactaccgct acacctacga
gggaagccac atcaaaggag 480 aggcccaggt gaaggggact ggtttccctg
ctgacggtcc tgtgatgacc aactcgctga 540 ccgctgcgga ctggtgcagg
tcgaagaaga cttaccccaa cgacaaaacc atcatcagta 600 cctttaagtg
gagttacacc actggaaatg gcaagcgcta ccggagcact gcgcggacca 660
cctacacctt tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt
720 tccgtaagac ggagctcaag cactccaaga ccgagctcaa cttcaaggag
tggcaaaagg 780 cctttaccga tgtgatgggc atggacgagc tgtacaagta a 821
<210> SEQ ID NO 18 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 18 Gly Ala Phe Thr Gly Ala 1 5 <210>
SEQ ID NO 19 <211> LENGTH: 41 <212> TYPE: PRT
<213> ORGANISM: Desulfovibrio vulgaris Hildenborough
<400> SEQUENCE: 19 Arg Arg Glu Ala Glu Val Arg Val Ile Leu
Lys Ala Met Arg Ala Thr 1 5 10 15
Gly Gly Asn Lys Gly Glu Ala Ala Arg Leu Leu Gly Val Ser Pro Arg 20
25 30 Thr Leu Arg Tyr Lys Phe Ala Glu Tyr 35 40 <210> SEQ ID
NO 20 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Desulfovibrio vulgaris Hildenborough <400>
SEQUENCE: 20 Lys Gly Glu Ala Ala Arg Leu Leu Gly 1 5 <210>
SEQ ID NO 21 <211> LENGTH: 18 <212> TYPE: DNA
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Upstream activating sequence (UAS) consensus
sequence <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (5)..(13) <223> OTHER INFORMATION: n =
a, c, g, or t. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: n = a, c, g, or t. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (17)..(18) <223>
OTHER INFORMATION: n = a, c, g, or t. <400> SEQUENCE: 21
gcggnnnnnn nnngncnn 18
* * * * *