JOURNAL OF BACTERIOLOGY, Oct. 1998, p. 5256–5259 Vol. 180, No.

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0021-9193/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Identification of Multiple s54-Dependent Transcriptional

Activators in Vibrio cholerae
KARL E. KLOSE,1* VERONICA NOVIK,2 AND JOHN J. MEKALANOS2,3
Department of Microbiology, University of Texas Health Science Center, San Antonio, Texas 78284-7758,1
and Department of Microbiology and Molecular Genetics2 and Shipley Institute of Medicine,3
Harvard Medical School, Boston, Massachusetts 02115
Received 12 June 1998/Accepted 29 July 1998

In the pathogenic bacterium Vibrio cholerae, the alternate sigma factor s54 is required for expression of
multiple sets of genes, including an unidentified gene(s) necessary for enhanced colonization within the host.
To identify s54-dependent transcriptional activators involved in colonization, PCR was performed with V.
cholerae chromosomal DNA and degenerate primers, revealing six novel and distinct coding sequences with
homology to s54-dependent activators. One sequence had high homology to the luxO gene of V. harveyi, which
in that organism is involved in quorum sensing. Phenotypes of V. cholerae strains containing mutations in each
of the six putative s54-dependent activator genes identified one as a probable ntrC homologue. None of the
mutant strains exhibited a defect in the ability to colonize infant mice, suggesting the presence of additional
s54-dependent activators not identified by this technique.

Cholera is a life-threatening diarrheal disease caused by the
gram-negative bacterium Vibrio cholerae. V. cholerae enters
humans orally through ingestion of contaminated food or wa-
ter, swims toward and penetrates the mucus gel lining of the
small intestine, and eventually adheres to the apical surface of
the intestinal epithelial cells. V. cholerae bacteria respond to
the specific environment encountered within the host intestine
and express a number of virulence factors which allow the
organisms to colonize the surface of the epithelial cells and
cause disease; under normal in vitro growth conditions, viru-
lence factors are not expressed (3). Several major virulence
factors have been identified, most notably, cholera toxin (CT)
and toxin coregulated pilus (TCP) (9, 16). However, it has
become clear that other, unidentified factors are produced
which aid in colonization and induce disease. Identification of
such factors remains problematic, given that they are probably
expressed only within a specific host environment. Although in
vitro conditions have been developed which elicit V. cholerae
CT and TCP production, these conditions are clearly not those
encountered within the host (for example, CT and TCP are
expressed optimally at 30°C in the Classical biotype). Thus, the
identification of genes required for V. cholerae pathogenesis
and the environmental signals that regulate their expression
remains an active area of research.
RNA polymerase containing the alternate sigma factor s54
54
(s -holoenzyme) transcribes genes with diverse physiological
roles in different bacteria, including pilin genes in pathogenic
Neisseria and Pseudomonas spp., which are important for col-
onization by these organisms (8). s54 is likewise required for
some aspect of V. cholerae colonization. A V. cholerae strain
containing a mutation in the gene encoding s54 (rpoN) has a
significant defect in the ability to colonize, but this defect is
distinct from two other phenotypes associated with the rpoN
strain, namely, nonmotility and low levels of glutamine syn-
thetase expression (7). The rpoN strain also produces normal
amounts of TCP under laboratory inducing conditions, so the
* Corresponding author. Mailing address: Department of Microbi-
ology, University of Texas Health Science Center, 7703 Floyd Curl Dr.,
San Antonio, TX 78284-7758. Phone: (210) 567-3990. Fax: (210) 567-
6612. E-mail: klose@uthscsa.edu.
nature of the s54-dependent gene(s) which enhances coloniza-
tion remain unknown. s54-Holoenzyme has a requirement for
an activator protein to initiate transcription; s54-dependent
transcriptional activators generally bind to DNA enhancer el-
ements located within the promoter region and activate tran-
scription by direct contact with s54-holoenzyme (8, 14).
We have previously identified two s54-dependent transcrip-
tional activators, FlrA and FlrC, which are required for V.
cholerae flagellar synthesis (7). However, strains containing
mutations in either flrA or flrC do not demonstrate the severe
colonization defect observed in a V. cholerae rpoN strain, thus
implying that an additional s54-dependent activator(s) is in-
volved in colonization. We utilized PCR with degenerate oli-
gonucleotides to identify six additional s54-dependent tran-
scriptional activators of V. cholerae. We have tentatively
assigned roles in quorum sensing and nitrogen assimilation to
two of these newly discovered s54-dependent transcriptional
activators, based on sequence and phenotype, but none ap-
pears to be involved in colonization. Our results suggest the
presence of yet more unidentified s54-dependent transcrip-
tional activators in V. cholerae and demonstrate the involve-
ment of s54 in the expression of multiple sets of genes in this
pathogen.
Identification of multiple V. cholerae s54-dependent tran-
scriptional activators. The V. cholerae rpoN mutant strain is
defective in the ability to colonize a host and is, additionally,
nonmotile (nonflagellated) and expresses low levels of glu-
tamine synthetase (7); these phenotypes are distinct from the
colonization defect. We thus predicted the presence of more
s54-dependent transcriptional activators in addition to the two
flagellar activators FlrA and FlrC. All s54-dependent activators
are modular proteins, and they contain a highly conserved
catalytic domain required for activation of transcription by
s54-holoenzyme (17). Kaufman and Nixon (5) demonstrated
that s54-dependent transcriptional activators could be isolated
by PCR utilizing degenerate primers designed to recognize this
conserved catalytic domain. We utilized their technique with
identical primers recognizing the conserved amino acids (W/F)
PGNV and ELFGH(V/A/D/E/G) and chromosomal DNA
from V. cholerae Classical biotype strain O395 (11); the prim-
ers also incorporated restriction sites for EcoRI and BamHI.

5256
VOL. 180, 1998
FIG. 1. Homology of s54act gene products with the catalytic domains of
s54-dependent transcriptional activators. Deduced amino acid sequences of
PCR-derived fragments containing putative s54-dependent transcriptional acti-
vators from V. cholerae were aligned by GCG Pileup with the amino acid se-
quences of the s54-dependent activators FlrA (amino acids 154 to 302) and FlrC
(amino acids 146 to 295) from V. cholerae and NtrC (amino acids 157 to 306)
from S. typhimurium. Amino acids found in at least five activators are in capital
letters, and amino acids common to all nine activators are designated by aster-
isks. Sequences recognized by degenerate primers used in PCR are designated by
arrows.
The PCR with Taq DNA polymerase consisted of 30 cycles of
92°C for 45 s, 42°C for 1 min, and 72°C for 1.5 min. The
resulting fragments were first digested with EcoRI and BamHI
and then ligated into pTZ19U (10) that had been similarly
digested.
Restriction analysis of the resultant recombinant clones re-
vealed seven distinct PCR-derived fragment inserts. These
seven fragments were sequenced directly from the plasmid by
utilizing an ABI 373AStretch sequencer, and the deduced
amino acid sequences are shown in Fig. 1. One fragment was
identical to the sequence of flrA, which encodes a V. cholerae
s54-dependent transcriptional activator of flagellar genes pre-
viously described (7), thus validating this technique for the
identification of such activators in V. cholerae. The other six
sequences were novel, and the predicted amino acid sequences
share high homology with s54-dependent transcriptional acti-
vators from different bacteria. Interestingly, flrC, which en-
codes the only other V. cholerae s54-dependent transcriptional
activator previously described (7), was not isolated by this
technique. This may be due to two amino acid deviations from
the consensus ELFGH sequence in FlrC (Fig. 1), resulting in
the failure of the degenerate oligonucleotide primers to am-
plify the coding sequence, raising the possibility that perhaps
yet more s54-dependent activators that deviate from the con-
sensus remain to be identified in this organism.
The partial sequences of the putative V. cholerae s54-depen-
dent activators were named s54act1 to s54act6. The predicted
protein product of s54act1 shares the highest homology with
the dicarboxylic acid transport regulatory protein DctD from
Rhizobium meliloti (62% identity), while the s54act2 gene prod-
uct is most homologous to the nitrogen regulatory protein
NtrC from Salmonella typhimurium (77% identity). The puta-
tive protein encoded by s54act3 has very high homology (90%
identity) to the LuxO protein of the closely related symbiotic
bacterium V. harveyi. LuxO is a repressor of luminescence, and
NOTES 5257
its activity is modulated by signals perceived through quorum
sensing (2). Curiously, Bassler and colleagues failed to ac-
knowledge the possibility that LuxO exerts its effects by acti-
vation of s54-dependent transcription, despite noting the ho-
mology of LuxO with the s54-dependent activator NtrC.
Bassler et al. have recently shown that V. cholerae produces an
autoinducer substance that can be recognized by the V. harveyi
quorum sensing system (1), and quorum sensing has been
shown to modulate the expression of virulence factors in
Pseudomonas aeruginosa (13). V. cholerae is not biolumines-
cent, so we predict that s54act3 encodes a LuxO homologue
modulated by quorum sensing signals, similar to V. harveyi
LuxO, which may modulate the expression of virulence factors.
The predicted protein encoded by s54act4 shares the highest
homology with a hypothetical s54-dependent transcriptional
activator from Escherichia coli (YgaA; 64% identity), while
that encoded by s54act5 is most homologous to NtrC from
Proteus vulgaris (43% identity). Finally, the putative s54act6
gene product is most homologous to PspF from E. coli, the
s54-dependent transcriptional activator of the phage shock
protein operon (70% identity). Given the modular nature of
s54-dependent activators, the function of the particular V.
cholerae activator cannot necessarily be deduced by homology
with the catalytic domain of another s54-dependent activator,
with the possible exception of s54act3 because of the extremely
high degree of homology with luxO and the close relationship
between these two Vibrio spp.
s54act4 probably encodes an NtrC homologue. To determine
the function of the s54act1 to s54act6 gene products, V. chol-
erae strains containing mutations in each gene were con-
structed. The internal fragments amplified by PCR and ligated
into pTZ19U as described above were first digested with
BamHI, made blunt ended with the Klenow fragment of DNA
polymerase, digested with EcoRI, and then ligated into
pGP704 (12) that had been digested with EcoRI and EcoRV.
Since pGP704 requires the pir gene product for replication,
mobilization of the recombinant plasmids containing internal
s54act gene fragments into V. cholerae (which lacks pir) results
in a chromosomal insertion into the s54act genes caused by
integration of the plasmid through homologous recombina-
tion. These plasmids were mobilized into V. cholerae wild-type
strain O395 (11) from E. coli SM10lpir (12), as previously
described (6), to form strains KKV216 (s54act1::pGP704),
KKV217 (s54act2::pGP704), KKV218 (s54act3::pGP704),
KKV219 (s54act4::pGP704), KKV221 (s54act5::pGP704), and
KKV220 (s54act6::pGP704).
The V. cholerae rpoN strain expresses low levels of glutamine
synthetase, a Gln phenotype which results in slow growth on
glutamine-limited nutrient broth medium (Fig. 2) (7). Wild-
type growth can be restored by transforming the rpoN strain
with pKEK71, which expresses S. typhimurium glutamine syn-
thetase (glnA) from a s54-independent promoter. The V. chol-
erae s54act4 mutant displays a Gln phenotype similar to that of
the rpoN strain when grown on nutrient broth, and wild-type
growth can likewise be restored when it is transformed with
pKEK71 (Fig. 2). None of the other s54act mutant strains
exhibited such a Gln phenotype. These results are consistent
with the idea that s54act4 encodes a homologue of NtrC, the
s54-dependent nitrogen regulatory protein which activates
glnA transcription in many different bacteria (14).
All of the V. cholerae s54act1 to s54act6 mutant strains are
able to grow on minimal glucose ammonia medium (although
the s54act4 strain grows slowly due to lack of glutamine), and
all displayed wild-type motility in a soft agar swarm assay (data
not shown), indicating that none of these regulators activates
the expression of genes necessary for prototrophy or motility.
5258 NOTES
FIG. 2. The s54act4 mutant displays a growth defect on glutamine-deficient
medium that is complemented by expression of glnA. Strains were grown for 24 h
on nutrient broth agar. The strains shown are KKV55 (DrpoN [7]) and KKV219
(s54act4::pGP704), either without or with plasmid pKEK71 (“pglnA” [7]), which
expresses S. typhimurium glnA from a s54-independent promoter.
The V. cholerae rpoN strain is unable to grow on minimal
medium containing succinate as the sole carbon source (Fig.
3), a phenotype associated with a lack of expression of dicar-
boxylic acid transport genes, which are transcribed by s54-
holoenzyme in several bacteria (4). However, all of the V.
cholerae s54act mutant strains grow similarly to a wild-type
strain on minimal succinate medium, indicating that none of
these novel activators encodes a homologue of DctD, the s54-
dependent activator of dicarboxylic acid transport genes. V.
cholerae strains containing mutations in either flagellar s54-
dependent activator gene flrA or flrC are also able to grow by
utilizing succinate as a carbon source (data not shown); we thus
predict the presence of at least one additional unidentified V.
cholerae s54-dependent activator, namely, a DctD homologue.
None of the newly identified s54-dependent activators is
required for colonization. The ability of V. cholerae to colonize
the intestines of infant mice is correlated with its ability to
colonize the human intestine (15), and thus, the infant mouse
model is a widely used model of V. cholerae virulence. The V.
cholerae s54act mutant strains were tested for the ability to
colonize infant mice by utilizing a competition assay previously
FIG. 3. s54act mutants are able to utilize succinate as a carbon source, unlike
an rpoN mutant. Strains were grown for 48 h on minimal medium containing 10
mM ammonium, 2 mM glutamine, and 0.4% succinate. The strains shown are
O395 (wild type [11]), KKV55 (DrpoN [7]), KKV216 (s54act1), KKV217
(s54act2), KKV218 (s54act3), KKV219 (s54act4), KKV221 (s54act5), and
KKV220 (s54act6).
J. BACTERIOL.
TABLE 1. Infant mouse colonization assaya results
Strain Relevant genotype Competitive indexb
0395 Wild type 1.35c
KKV56 DrpoN 0.030c
KKV216 s54act1 0.888
KKV217 s54act2 1.809
KKV218 s54act3 1.904
KKV219 s54act4 1.013
KKV221 s54act5 0.801
KKV220 s54act6 0.711
KKV59 DflrA 0.141c
a
The assay was performed as previously described (7). The values shown are
based on a group of at least seven mice.
b
The competitive index is the ratio of output mutant to wild-type bacteria
recovered from the intestine divided by the ratio of input mutant to wild-type
bacteria inoculated into mice. Thus, if a mutant strain has no colonization defect,
we expect a competitive index of close to 1. The P values for the rpoN and flrA
strains were ,0.05 by Student’s t test, indicating significant differences in colo-
nizing ability, but none of the other strains showed any statistically significant
differences from a wild-type strain in the ability to colonize.
c
The value has been reported previously (7).
described (7). Briefly, a mutant strain is coinoculated orally
into infant mice along with an isogenic wild-type strain, and the
amounts of mutant and wild-type strains which successfully
colonize are determined after 24 h from intestinal homoge-
nates. Any colonization defect of the mutant is reflected in a
lower ratio of mutant to wild-type strains recovered from the
intestine.
None of the V. cholerae s54act strains demonstrated reduced
colonization in infant mice (Table 1). We have previously
shown that mutations in the s54-dependent flagellar activators
FlrA and FlrC result in approximately 10-fold defects in colo-
nization. Because FlrA is required for transcription of flrC, the
defect in both flagellar regulatory mutants is probably due to a
lack of FlrC. However, an rpoN strain exhibits an approxi-
mately 100-fold defect in colonization (7). Since none of the
newly identified s54-dependent activators appears to be re-
quired for efficient colonization, an additional, unidentified
activator(s) may be present in V. cholerae which transcribes a
gene(s) involved in colonization. Alternately, the absence of
expression of multiple sets of s54-dependent genes mediated
by more than one activator may have an additive effect and
diminish colonization efficiency. If the putative LuxO homo-
logue encoded by s54act4 modulates colonization genes, it
must act as a repressor in V. cholerae also, since inactivation
does not prevent colonization. Not surprisingly, all of the
s54act mutant strains expressed similar wild-type levels of the
virulence factors CT and TCP under laboratory inducing con-
ditions (data not shown); the same was shown previously for
the rpoN strain (7). Thus, the nature of the s54-dependent
genes required for colonization remains unknown.
We have identified eight putative and proven s54-dependent
transcriptional activators in V. cholerae and inferred the pres-
ence of at least one, and possibly two, more which has yet to be
identified. Our results establish the involvement of s54 in the
expression of multiple sets of genes in V. cholerae, including
those for flagellar synthesis, glutamine synthetase expression,
and dicarboxylic acid transport; some genes involved in colo-
nization; and possibly some quorum sensing-regulated genes;
as well as other, unidentified genes regulated by the activators
with no known function.
Nucleotide sequence accession numbers. The following se-
quences have been deposited in GenBank and assigned the
corresponding accession numbers: s54act1, AF069055; s54act2,
VOL. 180, 1998
AF069056; s54act3, AF069387; s54act4, AF069388; s54act5,
AF069389; s54act6, AF069390.
We thank Dan Steiger for DNA sequence support and Darren
Schuhmacher for assistance with figures.
This study was supported by an institutional new faculty award of the
Howard Hughes Medical Institute to K.E.K. and NIH grant AI-18045
to J.J.M.
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