INFECTION AND IMMUNITY, June 2010, p. 2554–2570
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 6
vttRAand vttRBEncode ToxR Family Proteins That Mediate
Bile-Induced Expression of Type Three Secretion System
Genes in a Non-O1/Non-O139 Vibrio cholerae Strain?
Ashfaqul Alam,1Vincent Tam,2† Elaine Hamilton,1and Michelle Dziejman1*
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York1and
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts2
Received 21 September 2009/Returned for modification 15 November 2009/Accepted 31 March 2010
Strain AM-19226 is a pathogenic non-O1/non-O139 serogroup Vibrio cholerae strain that does not
encode the toxin-coregulated pilus or cholera toxin but instead causes disease using a type three secretion
system (T3SS). Two genes within the T3SS pathogenicity island, herein named vttRA(locus tag A33_1664)
and vttRB(locus tag A33_1675), are predicted to encode proteins that show similarity to the transcrip-
tional regulator ToxR, which is found in all strains of V. cholerae. Strains with a deletion of vttRAor vttRB
showed attenuated colonization in vivo, indicating that the T3SS-encoded regulatory proteins play a role
in virulence. lacZ transcriptional reporter fusions to intergenic regions upstream of genes encoding the
T3SS structural components identified growth in the presence of bile as a condition that modulates gene
expression. Under this condition, VttRAand VttRBwere necessary for maximal gene expression. In
contrast, growth in bile did not substantially alter the expression of a reporter fusion to the vopF gene,
which encodes an effector protein. Increased vttRBreporter fusion activity was observed in a ?vttRBstrain
background, suggesting that VttRBmay regulate its own expression. The collective results are consistent
with the hypothesis that T3SS-encoded regulatory proteins are essential for pathogenesis and control the
expression of selected T3SS genes.
Vibrio cholerae is a gram-negative, motile bacterium that is
found globally as a common inhabitant of brackish and estua-
rine waters. Strains exhibit extensive phenotypic and genetic
heterogeneity and can be classified according to several differ-
ent criteria. For example, serogroup designation is based on
the structure of the somatic O antigen, whereas the pathoge-
nicity of a strain is determined by its ability to colonize a
human host and cause the severe and potentially lethal diar-
rheal disease known as cholera (15, 26, 68). Importantly, more
than 200 different serogroups have been identified, and both
pathogenic and nonpathogenic strains of different serogroups
have been found to coexist in environmental reservoirs world-
wide (27, 77).
Only O1 and O139 serogroup strains are associated with
epidemic disease, and strains belonging to other serogroups
are collectively referred to as non-O1/non-O139 strains (14,
26). Pathogenicity is not serogroup specific, however, and iso-
lates of many different non-O1/non-O139 serogroups have
been associated with sporadic diarrheal disease, extraintestinal
infections, sepsis, and wound infections worldwide (3, 6, 7, 20,
39, 53, 54). Although unable to cause epidemic disease, non-
O1/non-O139 serogroup strains are viewed as an emerging
threat due to recent reports of limited outbreaks in indepen-
dent geographic locations and epidemiological data suggesting
an increased incidence of non-O1/non-O139 strain-associated
diarrheal disease in areas of endemicity such as India and
Southeast Asia (5, 19–21, 27, 66, 67).
Conventionally, pathogenic strains are identified by the
presence of horizontally acquired genes encoding the toxin-
coregulated pilus (TCP; essential for colonization) and chol-
era toxin (CT) (28, 65). In contrast to epidemic O1 and
O139 serogroup strains, which strictly employ TCP- and
CT-mediated mechanisms of pathogenesis, most non-O1/
non-O139 clinical isolates do not carry the genes encoding
TCP and CT (4, 27, 64). It is not well understood how strains
colonize the host in a TCP-independent manner, and al-
though other virulence factors have been identified (e.g., El
Tor hemolysins, a thermostable direct hemolysin), it is un-
clear whether such factors alone can recapitulate the clinical
similarity and severity of disease associated with CT-ex-
pressing strains (4, 34, 38, 53, 64, 66, 69).
Genomic sequence analysis of AM-19226 (an O39 sero-
group, TCP/CT-negative, clinically isolated strain) identi-
fied genes predicted to encode the structural components of
a type three secretion system (T3SS) (25). The genes lie
within an ?55-kb region that displays characteristics of hor-
izontal transmission, and similar sequences have been iden-
tified in other non-O1/non-O139 serogroup strains (16, 25,
64). In other bacteria, the T3SS island typically encodes
three classes of proteins in addition to the structural com-
ponents of the translocation apparatus: effector proteins
(which mediate disease), their chaperones, and transcrip-
tional regulators dedicated to controlling T3SS gene expres-
sion. The V. cholerae T3SS most closely resembles T3SS2 of
V. parahaemolyticus in linear organization and sequence
similarity but appears unique in comparison to the systems
* Corresponding author. Mailing address: Box 672, University of
Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue,
Rochester, NY 14642. Phone: (585) 273-4459. Fax: (585) 473-9573.
† Present address: Institute for Systems Biology, 1441 N. 34th Street,
Seattle, WA 98103.
?Published ahead of print on 12 April 2010.
encoded by Yersinia, Salmonella, and Shigella species (16,
17, 25). Nearly half of the genes within the V. cholerae T3SS
island are predicted to encode hypothetical proteins with
little or no homology to proteins in current databases. None-
theless, experiments using strain AM-19226 demonstrated
that the V. cholerae T3SS is essential for colonization in the
infant mouse model, and one effector protein, VopF, was
shown to function in the reorganization of host cell actin
(75). Although additional effector proteins have not yet
been identified, it is hypothesized that for T3SS-positive V.
cholerae strains, the coordinated functions of multiple ef-
fector proteins promote unique mechanisms of host coloni-
zation and disease manifestation that result in TCP/CT-
Strain AM-19226 encodes two putative transcriptional reg-
ulatory proteins within the T3SS island (25; unpublished ob-
servations). Both proteins show significant sequence similarity
to the ToxR protein, a transmembrane DNA binding protein
encoded by nearly all strains of V. cholerae, including AM-
19226. In epidemic O1 and O139 serogroup strains, the ToxR
protein and the ToxR regulatory network have been studied in
detail and serve as a paradigm for understanding coordinated
virulence gene expression and transcriptional regulation by a
transmembrane protein. Briefly, maximal activation of viru-
lence genes requires that ToxR interact with other proteins,
including ToxS, TcpP, and TcpH, to activate the expression of
the toxT gene. toxT is found within the TCP island and encodes
an AraC-related transcriptional regulator that directly binds
and activates the transcription of the genes encoding TCP and
CT (48). ToxR directly binds DNA through amino acids found
in the amino-terminal winged helix-turn-helix (HTH) domain,
which is necessary for both the positive and negative transcrip-
tional regulation of genes (45, 50, 52, 57, 62). In addition to its
role as a transcriptional activator of virulence genes, ToxR also
functions to regulate the expression of the outer membrane
porins OmpU and OmpT and the expression of metabolic
pathway components (8, 52).
Numerous studies have contributed to our understanding of
how epidemic V. cholerae strains direct the transcription of
virulence factors in response to specific stimuli that the bacte-
ria might encounter during infection (41, 48). pH, mucus, bile,
temperature, anaerobiosis, and osmolarity have been identified
as potentially important signals in the human intestine that
modulate the expression of genes belonging to the ToxR regu-
lon, and several of these signals can be reproduced in vitro to
promote virulence gene expression (22, 44, 49, 58). For exam-
ple, bile and bile salt components have been used to mimic
physiologically relevant in vivo conditions during in vitro stud-
ies aimed at probing the regulatory mechanisms controlling
TCP and CT expression (32, 37, 71). Although the interpreta-
tion of data has been complex, such studies have significantly
contributed to our ability to dissect and understand the regu-
latory networks governing V. cholerae virulence gene expres-
sion. Similarly, the role of bile and bile salts in promoting the
expression of T3SS genes in other enteric pathogens has also
been explored (31).
In many T3SSs, regulation occurs at multiple levels and is
coordinated by several regulatory systems (29, 76). It is not
known how strain AM-19226 regulates T3SS-mediated patho-
genesis, nor do we currently understand if ToxR is involved in
controlling virulence gene expression in TCP/CT-negative,
pathogenic non-O1/non-O139 strains. Since ToxR is a protein
known to regulate the expression of horizontally acquired vir-
ulence genes (e.g., tcp and ctx), its role in effecting T3SS gene
expression clearly warrants investigation. The presence of two
putative transcriptional regulators within the T3SS pathoge-
nicity island, each with amino acid similarity to ToxR, suggests
that T3SS gene expression may instead (or also) be controlled
by the activity of ToxR homologues. Regulation of T3SS gene
expression might therefore be achieved by ToxR-directed
mechanisms (similar to the regulation of TCP/CT gene expres-
sion), by the proteins encoded within the T3SS pathogenicity
island, or by a combination of factors. We therefore sought to
determine the roles of the AM-19226 ToxR protein and the
two T3SS-encoded putative regulatory proteins in the viru-
lence of strain AM-19226. We evaluated the in vitro effect of
crude bile and deoxycholate, a bile acid, on the expression of
the structural genes encoding the T3SS apparatus and deter-
mined whether ToxR and the ToxR-like proteins modulate the
expression of T3SS genes under these conditions. We present
results indicating that two novel proteins related to ToxR have
important roles in directing T3SS-mediated pathogenesis in
strain AM-19226, thus allowing us to begin to develop models
of how virulence is regulated in TCP/CT negative, T3SS-pos-
itive, non-O1/nonO139 V. cholerae strains.
MATERIALS AND METHODS
Strains and growth conditions. The bacterial strains and plasmids used in this
study are shown in Table 1. Escherichia coli and V. cholerae strains were main-
tained at ?80°C in Luria-Bertani (LB) broth containing 25% glycerol. For E. coli
and V. cholerae, ampicillin and streptomycin were each used at 100 ?g/ml.
chloro-3-indolyl-phosphate p-toluidine salt (X-Phos) were added to LB agar at
20 ?g/ml. Sodium deoxycholate (D-6750) and bovine bile (B-3883) were pur-
chased from Sigma. Stock solutions of 10% crude bile and 4% deoxycholate were
prepared in deionized water and centrifuged for 10 min at 16,000 ? g, and the
supernatant was filtered through a 0.45 ?m filter.
Strain and plasmid constructions. Nucleic acid manipulations were performed
using standard molecular biological techniques (70). The primers used are shown
in Table 2. Nonpolar in-frame deletions of toxR, A33_1664 (vttRA), and
A33_1675 (vttRB) were constructed using overlapping PCR (splicing by overlap-
ping extension) and standard allelic-exchange methods (23, 35), leaving se-
quences coding for 7, 13, and 20 amino acids (aa). The number of amino acid
residues in the N-terminal and C-terminal ends of the proteins left after the
in-frame deletions are as follows: 2 and 5 aa for ?toxR, 3 and 10 aa for ?vttRA,
and 3 and 17 aa for ?vttRB. Deletions were confirmed by sequencing, PCR, and
Southern analysis (70).
Alkaline phosphatase analysis. For PhoA fusion analysis, plasmid pKB1 was
constructed by cloning the signal sequenceless phoA gene into the PstI site of
pBSSK? (Invitrogen). The coding regions for AM-19226 ToxR (aa 1 to 293),
A33_1664 (aa 1 to 247), and A33_1675 (aa 1 to 182) were cloned upstream of the
phoA gene to generate C-terminal translational fusions, resulting in plasmids
pKN1, pKN2, and pKN3, respectively. All three fusions code for a glycine-
cysteine-arginine triplet between the C-terminal coding amino acid and PhoA.
Liquid alkaline phosphatase assays were performed as previously described (11).
lacZY transcriptional reporter studies. pAAC3 is a multicopy transcriptional
fusion vector constructed for T3SS gene expression analysis. The promoterless E.
coli lacZY genes (including the native Shine-Dalgarno sequence) and the tran-
scriptional terminator sequence rrnT1T2 were cloned into pBSSK?. Putative
promoter sequences upstream of V. cholerae strain AM-19226 T3SS genes were
amplified by PCR using iProof High-Fidelity DNA polymerase (Bio-Rad) and
cloned into the multiple cloning site of pAAC3. The resulting transcriptional
fusion plasmids were introduced into a genetically responsive lacZ mutant de-
rivative of strain AM-19226, MD996, by electroporation (unpublished data; 75).
pEH3 was constructed as a suicide vector based on pCVD442 to facilitate the
integration of single-copy transcriptional reporter fusions at the lacZ locus of V.
VOL. 78, 2010VttRAAND VttRBREGULATE V. CHOLERAE T3SS GENES2555
TABLE 1. Bacterial strains and plasmids used in this study
AM19226 R?M??lacZ Strr
MD992 ?vttRA(A33_1664) ?vttRB(A33_1675)
MD992 integrated vcsRTCNS2-lacZY fusion
MD992 integrated vspD-lacZY fusion
MD992 integrated vcsVUQ2-lacZY fusion
MD992 integrated vcsJ2-lacZY fusion
MD992 integrated vopF-lacZY fusion
MD992 integrated vttRB(A33_1675)-lacZY fusion
MD992 integrated vttRA(A33_1664)-lacZY fusion
MD992 integrated promoterless lacZY fusion
AAC40 integrated vcsRTCNS2-lacZY fusion
AAC40 integrated vspD-lacZY fusion
AAC40 integrated vcsVUQ2-lacZY fusion
AAC40 integrated vcsJ2-lacZY fusion
AAC40 integrated vttRB(A33_1675)-lacZY fusion
AAC40 integrated vttRA(A33_1664)-lacZY fusion
AAC40 integrated promoterless lacZY
AAC228 integrated vcsRTCNS2-lacZY fusion
AAC228 integrated vspD-lacZY fusion
AAC228 integrated vcsVUQ2-lacZY fusion
AAC228 integrated vcsJ2-lacZY fusion
AAC228 integrated vttRB(A33_1675)-lacZY fusion
AAC228 integrated vttRA(A33_1664)-lacZY fusion
AAC228 integrated promoterless lacZY
AD10 integrated vcsRTCNS2-lacZY fusion
AD10 integrated vspD-lacZY fusion
AD10 integrated vcsVUQ2-lacZY fusion
AD10 integrated vcsJ2-lacZY fusion
AD10 integrated vttRB(A33_1675)-lacZY fusion
AD10 integrated vttRA(A33_1664)-lacZY fusion
AD10 integrated promoterless lacZY
MD1069 integrated vcsRTCNS2-lacZY fusion
MD1069 integrated vspD-lacZY fusion
MD1069 integrated vcsVUQ2-lacZY fusion
MD1069 integrated vcsJ2-lacZY fusion
MD1069 integrated vttRB(A33_1675)-lacZY fusion
MD1069 integrated vttRA(A33_1664)-lacZY fusion
MD1069 integrated promoterless lacZY
F? endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 ?U169(lacZYA-argF) (?80 dlac?M15)
thi thr leu tonA lacY supE recA RP4-2–Tc::(?pir) Kanr
Expression vector; Ampr
Suicide vector with unique multiple cloning site, Ampr
Allelic exchange suicide vector based on pCVD442; facilitates chromosomal
integration of lacZY transcriptional fusions into V. cholerae lacZ; Ampr
vttRB(A33_1675) deletion plasmid; Ampr
vttRA(A33_1664) deletion plasmid; Ampr
toxR deletion plasmid; Ampr
phoA fusion vector based on pBSSK?; Ampr
toxR-phoA fusion; Ampr
vttRA-phoA fusion; Ampr
vttRB-phoA fusion; Ampr
aAmpr, ampicillin resistant; Strr, streptomycin resistant; Kanr, kanamycin resistant; R?, type II restriction endonuclease deletion; M?, methyltransferase positive;
UMCS, unique multiple cloning site introduced into pCVD442 (MD1003).
2556ALAM ET AL.INFECT. IMMUN.
TABLE 2. Primers used in this study
Primer used and gene/ORF amplified
Primer name Sequence
A33_1675-A33_1674936 RT toxR 2332 F
RT 2332 toxR R
A33_1674-vcsR2 2533?VcsR2 XbaI R
RT 2332 vcsR2 NR
vcsR2-vcsT2 764RT vcsR vcsT2 F
RT vcsT vcsR R
vcsT2-A33_1671 516RT vcsT 2329 F
RT 2329 vcsT R
A33_1671-vcsC21,061 RT 2329 vcsC F
RT 2329 vcsC R
vcsC2-vcsN2 1,002RT vcsC vcsN F
RT vcsN vcsC R
vcsN2-A33_16681,012 RT vcsN 2326
RT 2326 vcsN R
A33_1668-A33_1667676RT 2326 ORF32 F
RT 2326 ORF32 R
A33_1667-vcsS2 458RT ORF32 vcsS F
RT ORF32 vcsS R
vcsS2-ORF69 2955? ToxR2.A Del SalI
RT ORF69 vcsS R
ORF69-ORF65317 RT ORF69 ORF65 F
RT ORF69 ORF65 R
A33_1684-A33_1683 1,014RT fw 2342 2341
RT NRv 2341 2342
A33_1683-vcsV2 961 RT Fd 2341 vcsV
RT Rv vcsV 2341
vcsV2-vcsU2 1,128RT Fd vcsV vcsU
RT Rv vcsV vcsU
vcsU2-A33_16801,114RT vcsU2 Fd
RT Rv vcsU 2338
A33_1680-A33_1679 994RT 2338 2337 F
RT 2338 2337 R
A33_1679-A33_1678 1,096 RT 2337 2336 F
RT 2337 2336 R
A33_1678-A33_1677-vcsQ2 990RT 2336 vcsQ2 F
RT vcQ 2335 R
vcsQ2-A33_1675 2803? ToxR2PRMkd R XbaI
ToxR2B R SB
Transcriptional reporter fusions (plasmid and
Upstream region of A33_1674 (vcsRTCNS2) 9693? PM 2332 XbaI
5? PM 2332 PstI
Upstream region of A33_1683 (vcsVUQ2) 3903? PM 2341 XbaI
5? PM 2341 PstI
Continued on following page
VOL. 78, 2010VttRAAND VttRBREGULATE V. CHOLERAE T3SS GENES 2557
cholerae (23). The E. coli lacZY genes and the transcriptional terminator se-
quence rrnT1T2 were cloned into a derivative of pCVD442 that contains unique
multiple cloning sites, including XbaI, SacI, FseI, PmeI, RsrII, PmeI, SphI, and
SacI (M. Dziejman, unpublished construct). Approximately 775-bp DNA frag-
ments representing the 5? and 3? flanking regions of V. cholerae lacZ sequences
were amplified from AM-19226 and cloned into the SalI site immediately up-
stream of the rrnBT1T2 sequence and the SphI and SmaI sites downstream of the
E. coli lacZY genes. The resulting plasmid is pEH3, which has the following
relevant features: a ? protein-dependent origin of replication (oriR6K), a mul-
tiple cloning site between the transcriptional terminator sequences and the E.
coli lacZY reporter fusion to facilitate the cloning of putative promoter regions,
5? and 3? regions of homology to the V. cholerae lacZ locus to facilitate allelic
replacement at the AM-19226 lacZ locus, and the bla and sacB genes for the
selection of primary integrants and recombinants, respectively.
Single-copy lacZ transcriptional fusions to T3SS genes were then constructed
by PCR amplification using the primers specified in Table 2, followed by ligation
of the product into pEH3. The lacZY reporter constructs were integrated into the
chromosome of AM-19226 strains MD992, AAC40 (?A33_1675), AAC228
(?A33_1664), MD1069 (?A33_1675 ?A33_1664), and AD10 (?toxR). Integra-
tion of the promoterless E. coli lacZY reporter (pEH3) into the same strain
backgrounds served as the control in each case. Integration of the reporter
constructs at the V. cholerae lacZ locus was confirmed by PCR and Southern blot
?-Galactosidase assay. Single colonies were inoculated into 5 ml of LB broth
with or without deoxycholate or bile and grown with aeration at 37°C for 16 to
18 h to stationary phase. Logarithmic-phase cultures were grown in LB broth at
37°C until an optical density at 600 nm (OD600) of 0.3 to 0.6 was reached after
a 1:500 dilution of an overnight culture. ?-Galactosidase assays were performed
following the protocol described by Slauch and Silhavy (73). Briefly, bacterial
cultures were centrifuged and pellets were resuspended in Z buffer at pH 7.0
(with ?-mercaptoethanol) and the OD600was determined. One percent sodium
dodecyl sulfate and chloroform were added to the cell suspensions and mixed
well. The reaction was initiated by adding 10 ?g/ml ONPG (o-nitrophenyl-?-D-
galactopyranoside; Sigma), and the OD420was read every 5 min for 60 min at
room temperature using a PowerWave XS spectrophotometer (Bio-Tek). The
results are shown as ?-galactosidase activity, calculated as (units per A600unit ?
milliliters of cell suspension) ? 103, where the units are micromoles of
o-nitrophenol formed per minute.
RNA isolation and reverse transcriptase PCR (RT-PCR). Strain AM-19226
was grown overnight in LB broth with 0.04% deoxycholate. RNA was extracted
using Trizol (Invitrogen) following previously described methods (8, 43). Total
RNA was further purified using the RNeasy Mini kit (Qiagen). Contaminating
Primer used and gene/ORF amplified
Upstream region of A33_1689 (vspD) 4603? VspD R XbaI
5? VspD F PstI
Upstream region of A33_1693(vcsJ2) 5273? VcsJ2 R XbaI
5? VcsJ2 F PstIN
Upstream region of A33_1696 (vopF) 4215? WH2 PM F XbaI
3? WH2 PM R PstI
Upstream region of A33_1675 (vttRB)
3103? ToxR2PRMkd R XbaI
5? ToxR2PMRkd F PstI
Upstream region of A33_1664 (vttRA)
412ToxR2A F XbaI
ToxR2A R PstI
Deletion of toxR (VC0984 and A33_0921)
5? toxRdel OL
3? toxR OL
Deletion of vttRB(A33_1675)
3? ToxR2Rkd SacIGACGGAGCTCTTACGATGAACTGGGTCGAT
5? ToxR2Fkd SalI
Deletion of vttRA(A33_1664)
3? SOE N ToxR2A GAATTGATCTCAAAAATGTATAAACGTGAGC
5? ToxR2.A SOE F
3? ToxR2.A del SacI
5? ToxR2.A Del SalI
aSOE, splicing by overlapping extension.
2558ALAM ET AL.INFECT. IMMUN.
DNA was eliminated by DNase I treatment according to the manufacturer’s
specifications (amplification grade; Invitrogen). The RT-PCR used 1 ?g of RNA
as the template and each primer (listed in Table 2) at 0.20 ?M. Forty cycles were
performed using SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen)
according to the manufacturer’s protocols.
Infant mouse competition assay. Competition assays using 4- to 5-day-old
CD-1 mice were performed as previously described (2, 30). Strains carrying
in-frame deletions in the toxR (AD10), A33_1664/vttRA (AAC228), and
A33_1675/vttRB(AAC40) genes were lacZ?and were competed against the
isogenic parent strain that was ?lacZ. The competitive index (CI) was calculated
based on the input and output ratios of bacteria for each strain, where CI ?
(mutant output/wild-type output)/(mutant input/wild-type input).
In silico analyses. Clone Manager Professional Suite v9 was used for basic
sequence analyses and manipulations. Kyte-Doolittle plots (42) were generated
by Clone Manager. Transmembrane helix predictions were performed using the
TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Transcrip-
tion terminator sequences were predicted by the RibEx online program (http:
//184.108.40.206:8080/cgi-bin/ribex.cgi). The Basic protein BLAST (NCBI) was
used to find protein similarities, and the ClustalW2 program (http://www.ebi.ac
.uk/Tools/clustalw2/index.html) was used to perform multiple-sequence align-
ments. The SCRATCH Protein Predictor (http://www.ics.uci.edu/?baldig
/scratch/index.html) was used for secondary-structure predictions.
The AM-19226 T3SS island encodes two predicted tran-
scriptional regulators with homology to ToxR. Partial genomic
sequencing of strain AM-19226 originally identified genes
within an ?30-kb contig (contig 247) that were predicted to
encode proteins comprising the structural apparatus of a T3SS
(25). Annotation at that time also identified NTO1VC2333, an
open reading frame (ORF) within contig 247 that was pre-
dicted to encode a protein, initially named ToxR2, with 55%
sequence similarity to the transcriptional regulator ToxR (25).
Subsequent sequencing efforts by the National Institute of
Allergy and Infectious Diseases (NIAID)-sponsored J. Craig
Venter Institute Microbial Sequence Center expanded the size
of the T3SS island to ?55 kb and resulted in the identification
of a second ToxR homolog encoded within the T3SS island,
locus tag A33_1664 (gene ID 6826006). In the NIAID anno-
tation, ToxR2 was assigned locus tag A33_1675 (gene ID
6825995). For consistency, the two ToxR-related, T3SS-en-
coded ORFs will be referred to by the A33 locus tags as
annotated in the NCBI database and shown in Fig. 1A. Like
other strains of V. cholerae, strain AM-19226 also encodes a
ToxR protein, locus tag A33_0921, that is 99% identical to that
encoded by the O1 El Tor N16961 strain (gene designation
VC0984) and found in a similar chromosomal context (Fig.
1A). This protein will be referred to as strain AM-19226 ToxR.
A33_1664 is predicted to encode a 249-aa product, while
A33_1675 is predicted to encode a protein of 183 residues. Fig-
ure 1B shows the amino acid sequence alignment of the full-
length AM-19226 and N16961 ToxR proteins with the AM-19226
T3SS-encoded homologues. A33_1664 and A33_1675 show com-
parable levels of amino acid sequence similarity to ToxR (59 and
55%, respectively). Although sequence similarity is found
throughout the length of the proteins, significant amino acid
identity aligns mainly with the amino-terminal DNA binding
domain of ToxR. Consistent with this finding, BLAST analysis
of the A33_1664- and A33_1675-encoded proteins revealed
putative conserved DNA binding domains (trans_reg_C) in the
N-terminal regions of both proteins, indicating that the T3SS-
encoded proteins share the conserved winged HTH DNA
binding motif present in ToxR (Fig. 1B). Secondary-structure
prediction analysis supports the conclusion that A33_1664 and
A33_1675 encode HTH-containing transcriptional regulatory
proteins (data not shown). Interestingly, a BLAST search anal-
ysis performed using the full-length A33_1664-encoded pro-
tein as the query sequence also revealed considerable amino
acid similarity to the TcpP protein of V. cholerae O1 El Tor
strain N16961 (54% similarity with E ? 2e?5), whereas
A33_1675 did not. Both A33_1664- and A33_1675-encoded
proteins have homologues in T3SS-positive V. parahaemolyti-
cus strain RIMD2210633; the A33_1664-encoded protein is
61% identical in amino acid content to VPA1332, and the
A33_1675 gene product is 83% identical to VPA1348.
Kyte-Doolittle and TMHMM analyses predicted that, like
ToxR, each AM-19226 T3SS-encoded ToxR-like protein has a
single transmembrane domain (Fig. 1C). Hydrophobicity plots
for the ToxR proteins of strains N16961 and AM-19226 ToxR
(52) are shown for reference. The topology of the A33_1664
gene product is predicted to be similar to that of ToxR, con-
sisting of an amino-terminal cytoplasmic domain of 129 resi-
dues, a stretch of ?23 hydrophobic residues that are predicted
to span the inner membrane (aa 130 to 152), and a periplasmic
domain of ?97 aa (aa 153 to 249, Fig. 1C and D). Similarly,
A33_1675 is predicted to encode a protein with an ?159-aa
cytoplasmic domain, followed by a 22-aa membrane-spanning
segment (aa 159 to 181). However, the hydrophobic residues of
A33_1675 lie at the C terminus of the protein and are pre-
dicted to represent a membrane-spanning alpha helix (second-
ary-structure prediction, data not shown) that anchors the
cytoplasmic domain. Thus, only 2 aa of A33_1675 are pre-
dicted to be localized in the periplasm. The alignment pre-
sented in Fig. 1B indicates amino acid similarity in the C-
terminal periplasmic region of ToxR to the region of the
A33_1675-encoded protein that is predicted to reside in the
cytoplasm. It is therefore also possible that the true transmem-
brane domain of A33_1675 lies closer to the N terminus than
To confirm the predicted topology of the ToxR homologs,
we constructed alkaline phosphatase translational fusions to
the C-terminal final coding amino acid of the AM-19226 ToxR,
A33_1664, and A33_1675 proteins, resulting in plasmids
pKN1, pKN2, and pKN3 (47, 52). The parental plasmid, pKB1,
which expresses the signal sequenceless phoA gene, was used
as a negative control. pKB1, pKN1, pKN2, and pKN3 were
each singly introduced into V. cholerae strain AM-19226 by
electroporation, and the resulting strains were grown on LB
agar plates containing the chromogenic substrate X-Phos.
Strain AM-19226 carrying the parent plasmid pKB1 produced
white colonies, whereas strains expressing the ToxR and ToxR-
related fusion proteins produced dark blue colonies (data not
shown). The results were confirmed by measuring the alkaline
phosphatase activity in liquid cultures (data not shown; 11).
Figure 1D therefore represents the results of PhoA fusion
experiments that confirm the predicted topology of the AM-
19226 ToxR-like proteins.
The T3SS-encoded ToxR-like proteins and ToxR are re-
quired for full colonization in the infant mouse model. We
used the suckling mouse model to determine whether the
T3SS-associated putative transcriptional regulatory proteins
encoded by A33_1664 and A33_1675 are each required for
AM-19226 to colonize the infant mouse intestine (30). Un-
VOL. 78, 2010 VttRAAND VttRBREGULATE V. CHOLERAE T3SS GENES2559
FIG. 1. (A) The flanking genes for the ancestral toxR gene (hatched arrow) and the region of the T3SS pathogenicity island encoding the
structural components (dark-gray arrows) and the two putative transcriptional regulators (black and checkered arrows) are shown. White arrows
represent genes encoding hypothetical or conserved hypothetical proteins. The dotted arrow represents a gene present in our annotation but not
annotated by the J. Craig Venter Institute. Genes encoding known proteins are shown in light gray. The seven small arrows above the genes
indicate the locations of predicted promoter sequences. (B) Multiple-sequence alignment (ClustalW2 with default settings) of the ToxRN16961,
ToxRAM-19226, A33_1664, and A33_1675 protein sequences. Amino acid residues that constitute the predicted transmembrane domains are in bold
and underlined. The predicted secondary structures of the ToxR domains comprising the winged HTH motif are indicated above the N16961 ToxR
sequence. Residues forming beta sheets are indicated by thin lines, those forming alpha helices are indicated by thick lines, and wing residues are
indicated by the letter W. (C) Hydrophilicity plots of ToxR and the ToxR-related proteins using Kyte-Doolittle analysis. Hydrophilic residues have
a negative score and hydrophobic residues have a positive score on the plot. The numbers at the bottom of each panel refer to amino acid positions
within the four proteins. (D) Domain structure and membrane localization of ToxR paralogs based on TMHMM analysis, hydrophilicity plots, and
phoA fusion analysis. OM, outer membrane; IM, inner membrane.
2560ALAM ET AL.INFECT. IMMUN.
marked in-frame deletions in A33_1664, A33_1675, and toxR
that retained 7, 13, and 20 aa, respectively, were constructed
using standard allelic-exchange methods, resulting in strains
AD10, AAC228, and AAC40. Each deletion strain was coin-
oculated along with the isogenic parent strain, and organisms
were recovered from the small intestine after 18 h of infection.
The results were calculated as CIs and are shown in Fig. 2.
Deletion of A33_1664 significantly reduced colonization by
?100-fold, suggesting that its gene product is required for full
colonization. Deletion of A33_1675 reduces colonization
?1,000-fold compared to the wild-type strain, suggesting that it
is essential for AM-19226 colonization. We therefore con-
cluded that both proteins are required for the full virulence of
In strains that possess TCP and CT, deletion of ToxR results
in a dramatic colonization defect (51). While strain AM-19226
does not encode TCP or CT, an analogous situation exists in
that the pathogenicity of AM-19226 is due to functions asso-
ciated with the laterally acquired T3SS pathogenicity island
(75). Thus, it is conceivable that the AM-19226 ToxR protein
might play a role in regulating the expression of the T3SS
virulence genes. We therefore tested whether a deletion in the
AM-19226 toxR gene had an effect on the ability of the strain
to colonize in the infant mouse model. The results indicate that
the toxR deletion strain displayed a 10-fold colonization defect
compared to the isogenic parent, suggesting that even in the
absence of known downstream virulence factors such as TCP,
CT, and the toxT gene, ToxR may still contribute to the full
virulence of strain AM-19226 (Fig. 2).
Identification of T3SS gene regulatory sequences using mul-
ticopy lacZ reporter fusions. We wanted to evaluate the ex-
pression of genes encoding three different classes of T3SS
proteins: those that comprise the structural apparatus, putative
regulators of the T3SS, and effector proteins. We constructed
pAAC3, which facilitates the insertion of putative promoter
sequences downstream of a transcriptional terminator and up-
stream of the promoterless E. coli lacZY genes. pAAC3 was
used as the basis for all initial reporter fusion constructs. An-
notation and sequence analysis of the T3SS island sequence
suggested that the 10 genes encoding the structural compo-
nents of the apparatus are organized within four operons,
based on the presence of intergenic regions indicated by the
small arrows above genes in Fig. 1A. We postulated that the
intergenic regions contained promoter sequences that con-
trolled the expression of the structural genes. The VcsJ2 cod-
ing sequence lies downstream from and overlaps the predicted
hypothetical protein encoded by A33_1694; the two genes are
likely cotranscribed, and the proteins are likely to be transla-
tionally coupled (Fig. 1A). vspD is predicted to be the first gene
in an operon. vcsVUQ2 lie downstream of ORF A33_1683,
which is predicted to encode a hypothetical protein, with four
ORFs interspersed between vcsU2 and vcsQ2 (Fig. 1A and D).
vcsRTCNS2 are predicted to be cotranscribed downstream of
the hypothetical protein encoded by A33_1674 (Fig. 1A and
D). We therefore predicted that the expression of structural
genes vcsRTCNS2, vcsVUQ2, and vcsJ2 is controlled by regu-
latory sequences found in the intergenic regions upstream of
A33_1674, A33_1683, and A33_1694, respectively. Since vspD
is the first gene of its predicted operon, sequences including
the region immediately upstream of the coding region were
chosen to construct a vspD-lacZ transcriptional reporter fu-
sion. Sequences directly upstream of the putative ToxR-like
transcriptional regulators encoded by A33_1664 and A33_1675
and upstream of the known effector protein VopF coding re-
gion were also chosen for transcriptional reporter analysis.
Each plasmid expressing a lacZ transcriptional fusion was
introduced into strain MD996 (AM-19226 R?M??lacZ), and
the resulting reporter strains were evaluated for ?-galactosi-
dase activity after overnight growth in LB medium at 37°C
(data not shown). We observed detectable levels of expression
for most reporter fusions, suggesting that we had correctly
targeted putative regulatory sequences for the reporter con-
structs. To determine whether the regulatory sequences could
modulate reporter fusion expression in response to different
growth parameters, we grew the strains under several different
in vitro conditions, including temperature, minimal medium
with different carbon sources, and LB broth supplemented with
0.04% deoxycholate. Growth of strains in the presence of
0.04% deoxycholate resulted in maximal reporter fusion ex-
pression under the initial conditions tested (data not shown).
The results suggested that the intergenic sequences included in
the constructs contained transcriptional regulatory regions that
were active and responsive to environmental modulation.
Operon organization of T3SS structural genes. As stated
earlier, sequence data suggested that the expression of the
vcsVUQ2 and vcsRTCNS2 structural genes was likely con-
trolled by sequences upstream of ORFs A33_1683 and
A33_1674 (arrows above genes in Fig. 1A), resulting in poly-
cistronic messages. To confirm that the putative regulatory
regions chosen for lacZ transcriptional reporter fusions were
responsible for controlling the expression of all of the struc-
tural genes within the predicted operons, we performed RT-
FIG. 2. ToxR homologs are essential for full colonization in the
infant mouse model. Competition assays with CD-1 infant mice were
performed using a lacZ mutant derivative of strain AM19226 (MD996)
and a strain with the following gene deleted: ?A33_1664 (strain
AAC228, diamonds), ?A33_1675 (strain AAC40, triangles), or ?toxR
(strain AD10, circles). The results of a single experiment are shown,
where each symbol represents the CI from a single animal (n ? 9, n ?
8, and n ? 8, respectively). The bars indicate the mean CI for each
experiment. Experiments were repeated with similar results.
VOL. 78, 2010VttRAAND VttRBREGULATE V. CHOLERAE T3SS GENES2561
PCR analysis using RNA isolated from cultures grown in the
presence of 0.04% deoxycholate as a template. Figures 3A and
D show the predicted organization of the transcriptional units
within each operon.
An overlapping-amplicon strategy was employed to deter-
mine whether the vcsVUQ2 genes are cotranscribed in a single
operon. Primer pairs were designed to produce products (am-
plicons) that overlapped the coding sequences of adjacent
genes; bars above the gene designations denote the regions
expected to be amplified (primer sequences are listed in Table
2). Lanes labeled “No RT” in Fig. 3B and E show a represen-
tative reaction using only Platinum Taq polymerase and RNA
as the template to test for genomic DNA (gDNA) contamina-
tion. Similar negative results were obtained for each primer
pair (data not shown). We did not observe a product in the
RT-PCR using primers designed to amplify sequences over-
lapping the A33_1684 and A33_1683 coding regions, suggest-
ing that the intergenic region may contain promoter sequences
that control the expression of downstream genes (Fig. 3B, lane
1). Products were generated using primers designed to produce
products that overlap subsequent pairs of genes beginning with
A33_1683 and vcsV2, continuing with vcsV2 and vcsU2, and
FIG. 3. The transcriptional organization of the two main operons encoding the VcsVUQ2 and VcsRTCNS2 structural components are depicted
in panels A and D. Primer pairs were designed to amplify the regions shown by the bars above the genes. The open bars above the genes in panels
A and D indicate that RT-PCR did not produce an amplicon, whereas the solid bars indicate that an amplicon was obtained using RT-PCR. The
numbers above the bars correspond to the gel lanes in panels B, C, E, and F, showing the results of RT-PCR analyses using RNA extracted from
cells grown in LB broth with 0.04% deoxycholate as the template (B and E) and PCR using the same primer pairs with gDNA as the template
(C and F). Lanes marked “No RT” in panels B and E show representative PCRs conducted using RNA as the template, indicating that no product
was observed, consistent with a lack of gDNA contamination.
2562 ALAM ET AL.INFECT. IMMUN.
then gene pairs downstream through vcsQ2. No RT-PCR prod-
uct was found using the primer pair designed to amplify vcsQ2
and A33_1675, suggesting that the vcsQ2 gene lies at the 3? end
of the transcript (Fig. 3B, lane 8). In addition, in silico analysis
(RibEx; http://220.127.116.11:8080/cgi-bin/ribex.cgi) of the in-
tergenic region of the vcsQ2 and A33_1675 genes reveals a
potential factor-independent transcriptional terminator (data
not shown; 1). Figure 3C lanes 1 to 8 show the results of a
reaction using the same primer pairs, Taq polymerase, and
gDNA as a template. The results indicate that each primer pair
can successfully bind a template to produce an amplicon of the
expected size. Together, these data suggest that vcsVUQ2 are
indeed cotranscribed as part of a larger operon of eight genes
from a promoter upstream of A33_1683.
We used a similar strategy to confirm that the expression of
the vcsRTCNS2 genes is controlled by sequences upstream of
A33_1674, resulting in a polycistronic message that included at
least 10 genes. Primers that bind to sequences within A33_1675
and A33_1674 did not produce an amplicon in the RT-PCR
(Fig. 3E, lane 1), whereas those designed to bind within
A33_1674 and vcsR2 did (Fig. 3E, lane2). Primers designed to
produce overlapping products for each successive pair of
genes, including vcsS2 as the most distal gene encoding a
structural subunit, also resulted in detectable amplicons of the
expected sizes (lanes 3 to 9). Figure 3F shows the results of
reactions that used gDNA as a template and Taq polymerase
to produce amplicons of the expected size using the primer
pairs described in Table 2 (as shown in Fig. 3C for vcsVUQ2).
The combined results suggest that sequences upstream of
A33_1674 likely encode the promoter for the operon encoding
the VcsRTCNS2 structural proteins for the type three secre-
Although previous AM-19226 annotation identified A33_
1665 as an ORF beginning ?250 bp downstream of vcsS2 and
encoding a hypothetical protein, we suggest, based on subse-
quent annotation and BLAST analysis, that the ORF begins
?20 bp downstream of vcsS2 and encodes a 124-aa protein that
has ?94% similarity to VPA1334, a T3SS2-encoded hypothet-
ical protein of V. parahaemolyticus. An additional putative
ORF on the complementary strand, which was not originally
identified in the annotation of AM-19226, is shown as a white
arrow with a dotted outline in Fig. 3D. The predicted protein
product has ?79% amino acid similarity to VPA1333, a pre-
dicted protein encoded by V. parahaemolyticus T3SS2. Notably,
RT-PCR results suggest that gene A33_1665 is cotranscribed
with the vcsRTCNS2 genes (Fig. 3E, lane 10) and that tran-
scription extends at least ?140 downstream of the 3? end of
A33_1665 (Fig. 3E, lane 11).
Chromosomal integration of lacZ reporter fusions. The re-
sults of RT-PCR analysis further suggested that the intergenic
sequences chosen for multicopy lacZ reporter fusion studies
were sufficient to promote the expression of all of the genes
encoding the structural apparatus. To more accurately assess
the effect of in vitro growth conditions in modulating T3SS
gene expression and to determine the role of ToxR and the
putative regulatory proteins encoded by A33_1675 and
A33_1664, seven lacZ transcriptional reporter fusions were
each integrated in single copy into the lacZ locus in strain
MD992. The reporter fusions included (i) genes encoding the
structural components, as described for the multicopy vectors
(vcsRTCNS2-lacZ, vcsVUQ2-lacZ, vspD-lacZ, and vcsJ2-lacZ),
(ii) genes encoding the toxR-like putative transcriptional reg-
ulators A33_1675 and A33_1664, and (iii) the gene encoding
VopF, a known effector protein. The small arrows above genes
in Fig. 1A denote putative promoter locations, and the size of
each region chosen for analysis is indicated in Table 2. The
seven strains carrying the reporter fusions and the isogenic
parent strain carrying a promoterless reporter fusion were
grown in LB medium to logarithmic and stationary phases and
then assayed for ?-galactosidase expression. The results are
shown in Fig. 4. In exponentially growing cells (dark gray bars),
detectable levels of expression over background (3- to 4-fold
over promoterless lacZ) were observed for strains carrying all
reporter fusions except vcsRTCNS2-lacZ and vopF-lacZ. Ex-
pression from the vcsRTCNS2-lacZ fusion was approximately
equal to that observed from the promoterless construct,
whereas vopF-lacZ expression was undetectable. Both the
vspD-lacZ and A33_1664-lacZ fusions showed dramatically
higher levels of activity compared to the other reporter fusions
(Fig. 4, note that the two graphs have different y axis scales).
When cells were grown to stationary phase (Fig. 4, white bars),
moderate increases in expression levels compared to that
achieved during exponential growth (generally, 2- to 3-fold)
FIG. 4. The growth phase influences the expression of lacZ tran-
scriptional fusions to T3SS genes. ?-Galactosidase activity levels were
measured in strains containing single-copy transcriptional lacZ fusions
to the structural genes vcsRTCNS2, vspD, vcsJ2, and vcsVUQ2, the
vopF gene (encoding an effector protein), and the putative transcrip-
tional regulators A33_1675 and A33_1664. The promoterless lacZ
fusion was integrated as a control for the basal level of reporter gene
expression. Strains were grown in LB medium at 37°C to exponential
phase (dark gray bars) or to stationary phase (white bars). The data
shown represent the results of one experiment. Experiments were
performed twice using three individual colonies each time and pro-
duced similar results. Note that the two graphs have different y-axis
VOL. 78, 2010VttRAAND VttRBREGULATE V. CHOLERAE T3SS GENES2563
were observed for all reporter fusions except vopF-lacZ. These
results supported the conclusion that active promoter regions
for the structural and putative regulatory genes were targeted
for analysis and suggested that growth phase might influence
T3SS gene expression.
Bile and deoxycholate promote the expression of T3SS
structural genes. Crude bile and purified bile acids (Na-deoxy-
cholate and cholate) represent relevant in vivo signals that V.
cholerae encounters during infection of the small intestine.
Interestingly, bile and deoxycholate have been shown to elicit
opposing effects on the expression of TCP and CT in epidemic-
causing V. cholerae strains (32, 37). Because strains expressing
multicopy reporter fusions to the T3SS structural gene vectors
displayed increased levels of activity when grown in the pres-
ence of deoxycholate (data not shown), we proceeded to de-
termine the effects of both bile and deoxycholate on T3SS gene
expression when the reporter fusions were integrated in single
copy into the chromosome.
Since bile acids have an antibacterial effect, we first exam-
ined the effect of deoxycholate and bile on the growth of
parent strain AM-19226 and isogenic strains with toxR,
A33_1675, and A33_1664 deleted. Strains were grown over-
night in LB medium or LB medium supplemented with 0.04%
Na-deoxycholate or 0.4% bile. After 16 to 18 h of growth in LB
medium in the presence of bile or deoxycholate, the final OD
of cells was typically within a 2- to 3-fold range when the
A33_1675 and A33_1664 deletion strains were compared to
the wild-type strain, and all of the strains appeared to reach a
stationary phase of growth with similar growth kinetics (data
not shown). The concentrations of bile and deoxycholate used
and the data obtained were consistent with those reported by
other investigators (37, 63).
When strains carrying the vcsRTCN2-lacZ, vspD-lacZ, vcsJ2-
lacZ, vcsVUQ2-lacZ, and A33_1675-lacZ reporter fusions were
grown in the presence of deoxycholate, expression increased
?12- to 45-fold over background levels compared to the ex-
pression levels obtained during growth in LB medium alone
(Fig. 5, compare medium gray bars to white bars). The
A33_1664-lacZ reporter fusion showed a more moderate in-
crease (?4-fold), and the expression of vopF-lacZ was unde-
tectable over background levels in deoxycholate. Growth in the
presence of 0.4% bile resulted in an additional 2- to 3-fold
increase in the expression of the structural gene reporters
(?25- to 115-fold higher compared with expression in LB me-
dium). Similarly, A33_1675-lacZ expression was increased ap-
proximately 3.5-fold over levels achieved during growth in de-
oxycholate, whereas the A33_1664-lacZ reporter fusion
showed similar levels of expression in deoxycholate and bile.
Although the vopF-lacZ reporter fusion did not show any ac-
tivity in the presence of deoxycholate, it did exhibit a very low
level of activity when the strain was grown in the presence of
bile (16.6 ? 7.5 U versus 0 U for the promoterless negative
control). Similar trends were observed in multiple experi-
ments. However, it is not clear at this time whether such low
levels of vopF expression accurately represent bile induced
expression above background levels. In general, we found that
increased expression of the T3SS-lacZ transcriptional fusions
correlated with growth in LB broth containing increasing con-
centrations of Na-deoxycholate (0.01 to 0.04%) and bile (0.1 to
0.4%) (data not shown). The data therefore suggest that, com-
pared to growth in LB broth alone, growth in the presence of
either deoxycholate or bile enhanced the expression of T3SS
genes encoding the structural apparatus and the genes encod-
ing the putative ToxR-like transcriptional regulators.
A33_1664 (vttRA) and A33_1675 (vttRB) gene products reg-
ulate T3SS gene expression in the presence of bile. Having
determined in vitro conditions that increased expression of the
T3SS structural and putative regulatory genes, we next deter-
mined the role of ToxR and the T3SS-encoded ToxR-like
proteins in regulating T3SS island gene expression. The lacZ
transcriptional fusions were integrated into the lacZ locus of
four AM-19226-derived strains containing in-frame deletions
of A33_1675 alone, A33_1664 alone, A33_1675 and A33_1664,
and toxR. Figure 6A shows the results of ?-galactosidase assays
conducted with strains grown in LB broth alone. Reporter
fusion activity was measured, and background levels (promot-
erless constructs) were subtracted. Results were compared
among strains with a deletion of ToxR and the ToxR-like
proteins by calculating the percent activity relative to that
achieved in the isogenic parent strain (carrying the wild-type
allele). For each fusion, the activity in the isogenic strain car-
rying wild-type alleles of A33_1664, A33_1675, and toxR was
assigned a value of 100%. We did not observe any difference in
reporter fusion expression levels when fusions were expressed
in the different deletion backgrounds (Fig. 6A).
FIG. 5. Deoxycholate and bile increase the expression of T3SS
promoter-lacZ transcriptional fusions. ?-Galactosidase activity levels
were measured in strains containing single-copy chromosomal tran-
scriptional lacZ fusions to the indicated promoter regions. Strains were
grown at 37°C overnight in LB medium alone (white bars), LB broth
containing 0.04% Na-deoxycholate (dark gray bars), or LB broth con-
taining 0.4% bile (light gray bars). The data shown represent the
results of a single experiment using three individual colonies of each
strain. The experiment was repeated twice with similar results. Note
that the two graphs have different y-axis scales.
2564ALAM ET AL.INFECT. IMMUN.
We then proceeded to assay the reporter fusions in different
strain backgrounds with growth in the presence of 0.4% bile.
Figure 6B shows the results of ?-galactosidase assays calcu-
lated as described for Fig. 6A. Again, for all of the strains, the
level of background activity from the promoterless reporter
construct was subtracted and the level of expression from the
wild-type allele strain was assigned a value of 100%. In the
?A33_1664 strain background, vcsRTCNS2-lacZ, vspD-lacZ,
vcsJ2-lacZ, and vcsVUQ2-lacZ expression levels decreased to
less than 20% of that seen in the isogenic parent strain (Fig. 6,
checkered bars). A similar decrease in activity for structural
gene fusions was observed in the ?A33-1675 strain background
(Fig. 6, black bars). Although expression levels of the vspD2
reporter fusion in bile were typically very high (Fig. 4), levels
were dramatically reduced in the ?A33_1664 and ?A33_1675
deletion strains. Expression of the structural gene reporter
fusions was not further decreased when expression was assayed
in the double-deletion background (Fig. 6, vertical lined bars).
The results suggest that both T3SS-encoded transcriptional
regulatory proteins contribute to T3SS structural gene expres-
sion in the presence of 0.4% bile.
Individual deletions of A33_1664 and A33_1675 also af-
fected the level of expression of reporter fusions to their own
putative promoter regions. Deletion of A33_1675 resulted in
an ?2-fold increase in A33_1675-lacZ reporter fusion activity
compared to the level observed in the wild-type strain (when
cells were grown in the presence of bile). The increase in
expression was reproducible over the course of several exper-
iments and appeared statistically significant (P ? 0.0004, Stu-
dent’s paired t test with a two-tailed distribution), suggesting
that the A33_1675-encoded protein might negatively regulate
its own expression under these conditions. Deletion of
A33_1664 resulted in an ?2-fold increase in its own reporter
fusion expression and an ?2-fold decrease in the expression of
the A33_1675 reporter fusion over the course of multiple ex-
periments. Expression levels were also consistently lower for
the A33_1675 reporter fusion when assayed in the ?A33_1675
?A33_1664 double-deletion background (Fig. 6B, vertically
striped bars), suggesting that A33_1664 may positively contrib-
ute to A33_1675 expression levels.
Collectively, the data presented in Fig. 5 and 6 indicate
that bile and deoxycholate promote the expression of the
structural components of the T3SS apparatus. The expres-
sion of additional genes contained within the T3SS island,
FIG. 6. T3SS pathogenicity island-encoded transcriptional regulators VttRA(encoded by A33_1664) and VttRB(encoded by A33_1675)
regulate T3SS structural gene expression when strains are grown in LB broth containing 0.4% bile but not when they are grown in LB broth alone.
Single-copy transcriptional lacZ fusions in five different genetic backgrounds were assayed after overnight growth at 37°C in LB medium alone
(A) or containing 0.4% bile (B). The ?-galactosidase activity for each fusion was measured in each of the following five backgrounds: wild type
(light gray bars), ?A33_1664 (checkered bars), ?A33_1675 (black bars), ?A33_1664 ?A33_1675 (striped bars), and ?toxR (hatched bars). The data
presented represent the results of three experiments using at least three individual colonies of each strain. All values are background subtracted
(using strains expressing the promoterless construct), and the activity for each fusion in the isogenic parent strain was assigned a value of 100%.
The percent activity for each reporter fusion in each deletion strain was calculated relative to the expression obtained with the isogenic parent
strain, and the standard deviation was calculated based on at least three experiments.
VOL. 78, 2010VttRAAND VttRBREGULATE V. CHOLERAE T3SS GENES2565
such as A33_1664 and A33_1675, are also positively regu-
lated by growth in deoxycholate and bile. Because A33_1664
and A33_1675 (previously annotated as toxR2-B) are each
required for colonization in the infant mouse model (Fig. 2)
and for maximal expression of the structural genes during
growth in bile (Fig. 6B), we propose to rename A33_1664 as
Vibrio type three regulator A (vttRA) and A33_1675 as
Vibrio type three regulator B (vttRB).
Effect of ToxR on T3SS structural gene expression. Since the
strain with a deletion of the toxR gene showed a 10-fold defect
in colonization (Fig. 2), we assayed the reporter fusions in the
?toxR background to determine if the colonization defect
might be mediated by T3SS gene expression (Fig. 6B, hatched
bars). Compared to other detection backgrounds, deletion of
toxR did not dramatically alter the expression level of any
of the reporter fusions to structural genes, although a trend of
2-fold decreased expression (?50% of wild-type expression)
was observed. In the ?toxR strain background, we observed an
?2-fold increase in the expression of the reporter fusion to
A33_1664 (vttRA) compared to the expression level observed
in the wild-type background and a ?2-fold decrease for the
vttRB-lacZ deletion (60% of the activity observed in the iso-
genic parent strain, Fig. 6B). In both cases, the standard devi-
ation from multiple experiments suggests that this trend must
be further evaluated before arriving at any conclusion as to the
role of ToxR in vttRAand vttRBexpression.
In many organisms, the T3SS genes are typically found clus-
tered within a large pathogenicity island. The linear organiza-
tion and operon structures differ among bacteria, but certain
similarities, as well as phylogenetic analysis based on protein
homologies and gene positions, suggest that the systems can be
grouped into clades (17). Although the V. cholerae T3SS re-
sides on an ?55-kb pathogenicity island, the gene organization
does not resemble that found in any of the established clades.
Instead, the V. cholerae T3SS appears mosaic in nature and
most closely resembles T3SS2 of V. parahaemolyticus isolates
(16, 25, 46). In V. cholerae strain AM-19226, the highly con-
served protein structural subunits are encoded within four
operons, with the structural genes interspersed with ORFs that
are predicted to encode hypothetical proteins and, based on
our analysis, are likely coexpressed along with the structural
genes. The mosaic nature of the Vibrio T3SS suggests that this
system may have been derived from multiple T3SS systems,
resulting in a unique island whose function likely provides an
advantage specific to Vibrio spp. for host infection, survival in
the environmental reservoir, or perhaps both. Additional
T3SS-containing Vibrio genomes have been sequenced, and it
is becoming clear that diversity exists among the different
genes carried by the T3SSs, even within strains of the same
species (16, 55). Nonetheless, the operon organization of the
structural genes appears to be conserved among different
Vibrio strains and is consistent with our data demonstrating the
coordinated regulation of the structural genes by VttRAand
VttRBin response to growth in medium containing bile and
deoxycholate (discussed below).
The ToxR protein has long served as the foundation for
understanding V. cholerae virulence gene regulation. Data
from numerous laboratories have shown that the expression of
the horizontally acquired virulence factors for colonization
(TCP) and toxin production (CT) is mediated by a complex
circuitry involving not only the transmembrane ToxR protein
but multiple membrane-associated and cytoplasmic transcrip-
tional regulators. Some genes encoding products that are part
of the ToxR regulon are horizontally acquired along with the
virulence factors (e.g., toxT), whereas others, such as toxR, are
considered “ancestral” or core genes that are found in all
strains. The V. cholerae non-O1/non-O139 T3SS is encoded on
a horizontally acquired pathogenicity island that carries two
genes encoding proteins with significant amino acid similarity
to the ToxR protein. These observations prompted our inves-
tigation of whether T3SS gene expression might be regulated
by the ancestral ToxR and/or the T3SS-encoded ToxR-related
The results of colonization studies using the infant mouse
model demonstrated that the T3SS-encoded VttRAand
VttRBtranscriptional regulators are essential for full viru-
lence. This is consistent with the finding that T3SS island
sequences encode dedicated transcriptional regulators in
other bacteria (36). For example, the Y. enterocolitica T3SS
gene cluster encodes VirF, which belongs to the AraC family
of transcriptional regulators and controls yop expression
(17). Similarly, ExsA, an AraC-like transcriptional activator,
is located in the Pseudomonas aeruginosa T3SS gene cluster.
ExsA activates the transcription of genes encoding the se-
creted effector proteins and the T3SS structural apparatus
(79). Both AraC-like transcriptional regulators and two-
component regulatory systems are commonly responsible
for regulating the expression of T3SS genes (29). In that
regard, it is important to note that the V. cholerae T3SS
encodes regulatory proteins most similar to the ToxR family
of transmembrane transcriptional regulators. Since related
proteins are encoded by V. parahaemolyticus T3SS2, we
speculate that the activity of ToxR-related proteins influ-
ences T3SS gene expression in this bacterium as well.
In other organisms, global regulatory proteins (e.g., HIS, Fis,
and quorum-sensing components) also contribute to T3SS
gene regulation. The increase in reporter fusion expression
observed for strains grown to stationary phase in LB broth
compared to logarithmic phase was not due to ToxR, VttRA,
or VttRB, suggesting that growth phase-dependent regulation
may function in controlling T3SS expression in V. cholerae
(data not shown). The effect could be mediated either through
the requirement for alternative sigma factors such as RpoS/?S
(40) or for density-dependent signals such as the LuxO-HapR/
LuxR quorum-sensing system, as seen in V. harveyi (33) and P.
aeruginosa. Alternatively, other factors might be necessary to
ensure a basal level of expression or to relieve the repression of
T3SS gene expression in the absence of inducing conditions.
For example, H-NS is involved in the repression of T3SS genes
in Yersinia, Shigella, enterpathogenic E. coli and enterohemor-
rhagic E. coli (29). It is therefore reasonable to speculate that
additional contributors to V. cholerae T3SS regulation may
have characteristics similar to those of factors found in other
organisms. We favor this hypothesis in light of the recent
report by Shakhnovich et al. that identified Hfq as an impor-
tant factor regulating T3SS virulence gene expression in patho-
genic E. coli and demonstrated that V. cholerae AM-19226
2566 ALAM ET AL.INFECT. IMMUN.
vopF transcription was detected in a ?Hfq background but not
in the wild-type strain (72). Further studies are needed to
identify additional regulatory candidates that control Vibrio
T3SS expression, either in concert with or independently of
Deletion of the AM-19226 ancestral toxR gene produced
a colonization defect in the infant mouse model, although it
was not as severe as that resulting from the deletion of
VttRAor VttRB(10-fold versus 100- to 1,000-fold). The
results of transcriptional fusion studies suggest that ToxR is
required for the maximal expression of structural genes un-
der that condition, although it does not affect gene expres-
sion to the same extent as VttRAand VttRB(discussed
below). It is well established that ToxR regulates the ex-
pression of porin genes and components of metabolic path-
ways, and it is therefore plausible that the regulation of
non-T3SS genes by ToxR is important for AM-19226 fitness
in the mouse intestine (8, 18, 45, 62). Alternatively, ToxR
may regulate T3SS-related genes that are important for full
virulence but are as yet unidentified (e.g., effector proteins,
chaperones, or additional regulatory factors).
We used lacZ transcriptional fusion analyses to identify an in
vitro condition that stimulated T3SS gene expression so that we
could then assess whether the ToxR, VttRA, and VttRBpro-
teins contributed to virulence by modulating the expression of
T3SS genes. Although host cell contact typically serves as an in
vivo signal to induce T3SS gene expression, it is presumed that
other in vivo signals (e.g., temperature, divalent cation concen-
tration, pH) can modulate T3SS gene expression (29). For
many enteric pathogens, bile and deoxycholate are important
host intraintestinal signaling molecules that serve to regulate
the expression of virulence factors during infection. For exam-
ple, bile can repress SPI1 T3SS-mediated invasion of Salmo-
nella spp., and in V. parahaemolyticus, bile acids enhance the
production of the thermostable direct hemolysin, which is an
essential virulence factor (56, 59–61). Regulation can also oc-
cur at the protein level, and bile salts have been shown to act
as environmental signals for the stable recruitment of IpaB
onto the Shigella needle tip complex (74). Because previous
studies reported that bile and the bile acid deoxycholate reg-
ulate virulence gene expression in epidemic V. cholerae strains
(32, 37, 62, 63, 71), we chose similar growth conditions to test
the induction and regulation of T3SS genes. We found that
deoxycholate stimulated the expression of T3SS structural and
regulatory genes, consistent with the reports of deoxycholate
stimulating virulence gene expression in epidemic O1 and
O139 strains. In V. cholerae O1 serogroup classical-biotype
strains O395 and 569B, bile has been shown to dramatically
reduce the expression of the ctxAB and tcpA genes (32). The
repression by crude bile was shown to be mediated by H-NS
and is independent of the ToxR regulon (13). However, Hung
et al. showed that the purified bile acid deoxycholate or cholate
induced CT and TCP expression through ToxR (37). In con-
trast, our studies show that, like deoxycholate, bile promotes
the expression of T3SS structural genes and the genes encod-
ing the VttRAand VttRBregulatory proteins. As shown in Fig.
6B, maximal bile-dependent expression of the structural genes
required VttRA, VttRB, and ToxR. Preliminary studies sug-
gested that deoxycholate-induced expression was dependent on
VttRAand VttRBas well (data not shown). It is not clear why, in
contrast to regulation in epidemic strains, both crude bile and
purified bile acids can act as stimulatory signals for virulence gene
expression in AM-19226. Perhaps it is not surprising given that
the T3SS encodes an inherently different mechanism of patho-
genesis compared to TCP/CT-mediated colonization and disease.
The in vivo signals perceived temporally during infection and at
specific locations within the intestine may also play a role (71).
Clearly, the roles of VttRAand VttRBin coordinating gene ex-
pression in response to environmental stimuli and the identifica-
tion of additional proteins that have a role in the T3SS regulatory
network require additional investigation; it seems likely that fur-
ther studies will identify both conserved features and mechanistic
differences used by diverse V. cholerae strains to control virulence
We were surprised to find comparatively high levels of ex-
pression of the vspD-lacZ and vttRA-lacZ (A33_1664) reporter
fusions. High levels of vspD expression might be related to the
role of VspD as the protein that comprises the multisubunit
translocator component of the T3SS. The elevated level of
expression of the vttRA-lacZ fusion is more difficult to explain,
since transcriptional regulators are typically expressed at rela-
tively low levels and the vttRAdeletion strain was less impaired
for colonization than the vttRBdeletion strain. Since multiple
signals are typically sensed by bacteria in the host, it is possible
that a combination of stimuli result in a more moderate level
of expression in vivo. For both the vspD-lacZ and vttRA-lacZ
constructs, it is formally possible that the intergenic regions
chosen for transcriptional fusion analysis lack sequences that
bind repressor proteins when in the native chromosomal con-
text. It is interesting to speculate that increased vttRAexpres-
sion in the ToxR deletion strain is consistent with a role for
ToxR as a repressor of T3SS gene expression in specific cases,
but any firm conclusions necessitate further investigation. Fu-
ture studies that more precisely define the promoter regions
and identify additional regulators should help to clarify this
Our studies did not identify conditions that promoted vopF
expression to levels comparable to those observed for other
reporter fusions, although VopF is clearly expressed and trans-
located in vitro when AM-19226 is cocultured with HEp-2 cells
(75). vopF expression might respond to in vitro signals that
differ from those to which the structural or regulatory protein
genes respond, or perhaps vopF expression and translocation
are tightly linked with host cell contact. Alternatively, low
levels of vopF expression might be sufficient to produce the
levels of protein necessary for pathogenesis. The results of
Shakhnovich et al. (mentioned above) suggest that vopF ex-
pression is, at least in part, negatively regulated by Hfq (72).
The identification of additional effector proteins, the analysis
of their expression patterns, and studies conducted with strains
having deletions of multiple regulators are expected to provide
insights into the mechanisms contributing to effector protein
Our data indicate that VttRAand VttRBare both necessary
for maximal structural gene expression in the presence of bile.
One possible explanation is that VttRAand VttRBmust inter-
act with each other to bind target sequences and promote
transcription. Interaction could occur either as heterodimers
or as homodimeric complexes that function cooperatively to
regulate T3SS gene expression. Alternatively, the two proteins
VOL. 78, 2010VttRAAND VttRBREGULATE V. CHOLERAE T3SS GENES2567
may not interact with each other and might instead bind dif-
ferent regions of promoter sequences. Another possibility in-
volves a transcriptional regulatory hierarchy whereby VttRA
and VttRBexhibit an epistatic interaction with each other or
with another transcriptional regulator that might affect T3SS
gene expression. For example, a situation analogous to the
interaction of ToxR and TcpP may exist, where the resulting
protein interactions result in the activation of toxT expression
in TCP/CT-positive strains. The C-terminal periplasmic do-
mains of VttRAand ToxR share less sequence similarity than
the N-terminal regions that contain the HTH DNA binding
domains, perhaps indicating that the VttRAperiplasmic do-
main differs functionally or structurally from its ToxR coun-
terpart. In this context, it is interesting to again note that
VttRBhas no or a very small periplasmic domain and appears
unusual in that respect among ToxR-like proteins.
That bile is perceived as a signaling molecule for the expres-
sion of virulence factors is complicated in light of its antimi-
crobial nature. Bile has been shown to modulate the expression
of the V. cholerae outer membrane porins OmpU and OmpT in
a ToxR-dependent manner (9, 62). Recent microarray analysis
indicates that bile regulates the expression of more than 100
genes, and three RND efflux systems are reported to contrib-
ute to bile resistance in V. cholerae (9, 10, 12). Cerda-Maira et
al. have reported that the BreAB (VexCD) RND efflux pump
is upregulated specifically by bile and its expression is regu-
lated by the BreR protein, a bile-responsive autoregulatory
transcriptional repressor (12). The authors also proposed that
BreR requires bile acids as inducer molecules to dissociate
from the breAB or breR promoter under conditions of “high
bile” similar to the 0.4% bile concentration used in our and
others’ experiments. BreR represses its own transcription in
the presence of “low bile,” and deoxycholate alone was re-
ported to provide the most robust induction of a bre-lacZ
reporter fusion. Strain AM-19226 does contain a gene pre-
dicted to encode BreR, although we do not know its effect on
virulence gene expression. The AM-19226 protein responsible
for bile sensing is unknown, and although it is tempting to
speculate that the periplasmic domain of VttRAmay have a
role in this function, it is prudent to note that previous studies
of ToxR suggest that the ToxR periplasmic domain functions
in protein-protein interactions rather than environmental sens-
ing and signaling (24, 41, 48).
We do not know whether the T3SS has a role in the aquatic
existence of V. cholerae or whether its role is restricted to
virulence in the human host. It is formally possible that T3SS
activity could influence the relationship of V. cholerae with
chitinaceous organisms in the marine environment. In this
regard, determining whether the VttRAand VttRBproteins
regulate the transcription of genes that lie outside the T3SS
island will expand our understanding of whether T3SS-en-
coded regulator activity is restricted to the T3SS pathogenicity
island or whether they can impact global gene expression to
affect other parameters of the V. cholerae lifestyle.
We thank Scott Butler and Marty Pavelka for critically reading the
manuscript, the members of the Dziejman lab for helpful discussions,
and Adam Derr, Peter Hong, Edward Katich, and Katelin Noble for
generation of preliminary data and assistance with plasmid and strain
constructions. We are especially grateful to John Mekalanos for shar-
ing resources and for supportive discussions.
This work was supported by grant AI073785 from NIH/NIAID
ADDENDUM IN PROOF
Kodama et al. (T. Kodama, K. Gotoh, H. Hiyoshi, M.
Morita, K. Izutsu, Y. Akeda, K. S. Park, V. V. Cantarelli, R.
Dryselius, T. Iida, and T. Honda, PLoS One 5:e8678, 2010)
have recently demonstrated that the Vibrio parahaemolyticus
homologues of VttRAand VttRBregulate genes within the
type III secretion system (T3SS2) (the V. parahaemolyticus
pathogenicity island region) and are essential for T3SS2-me-
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