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Journal of Bacteriology, October 2000, p. 5342-5350, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Regulation of Vibrio cholerae Genes
Required for Acid Tolerance by a Member of the "ToxR-Like" Family
of Transcriptional Regulators
D. Scott
Merrell and
Andrew
Camilli*
Department of Molecular Biology and
Microbiology, Tufts University School of Medicine, Boston,
Massachusetts 02111
Received 2 March 2000/Accepted 6 July 2000
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ABSTRACT |
The ability of the intestinal pathogen Vibrio cholerae
to undergo an adaptive stress response, known as the acid tolerance response (ATR), was previously shown to enhance virulence. An essential
component of the ATR is CadA-mediated lysine decarboxylation. CadA is
encoded by the acid- and infection-induced gene cadA. Herein, cadA is shown to be the second gene in an operon
with cadB, encoding a lysine/cadaverine antiporter.
cadC, which is 5' of cadB, encodes an
acid-responsive, positive transcriptional regulator of
cadBA. Unlike in Escherichia coli, V. cholerae cadB and cadA are also transcribed
monocistronically. Of note, bicistronic cadBA is
transcribed at low constitutive levels in an acid- and CadC-independent
manner. CadC represents a new member of the "ToxR-like" family of
transcriptional regulators in V. cholerae and, in addition, exhibits extensive amino acid and functional similarity to E. coli CadC. The amino-terminal, putative DNA binding domains of ToxR and CadC are highly conserved, as are the putative promoter elements recognized by these transcription factors.
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INTRODUCTION |
Vibrio cholerae is the
causative agent of the endemic and epidemic diarrheal disease cholera.
Since the natural environmental reservoir for this intestinal pathogen
is aquatic, it stands to reason that ingestion by a human and
subsequent colonization of the relatively sterile small intestine
involve the expression of genes that are crucial for survival and
adaptation in this new environment. Several research strategies
designed to identify pathogen genes that are upregulated during
infection have been used to show that adaptation/stress systems are
often induced upon entry of a pathogen into its host environment
(5, 13, 21, 40, 42). Although much emphasis has been placed
on the identification and characterization of V. cholerae
virulence factors, such as those within the ToxR/ToxT regulon
(37), very little is known about the survival and adaptation
systems employed by this gram-negative bacterium during infection.
We recently used recombinase-based in vivo expression technology to
identify V. cholerae genes that are transcriptionally induced within two separate animal models of cholera. One such gene was
cadA, which encodes a lysine decarboxylase; cadA
was subsequently shown to be essential for V. cholerae's
ability to undergo an adaptive stress response known as the acid
tolerance response (ATR) (25). This stress response has been
well characterized in the two closely related enteric pathogens,
Escherichia coli and Salmonella enterica serovar
Typhimurium, and has been shown to be necessary for pathogenicity of
the latter (4, 23, 24, 29). We have found that V. cholerae cells that are acid adapted are more virulent than cells
grown at a neutral pH. This finding suggests that the V. cholerae ATR may play an important role in the fitness of this
pathogen, with respect to both infectivity of a single host and rapid
epidemic spread within populations (25).
Here we extend our characterization of the V. cholerae cadA
locus and show that, as in E. coli, it is the downstream
gene in and acid-inducible operon, cadBA. However, in
contrast to what is observed in E. coli, the V. cholerae cadA also possesses an independent promoter. In addition,
we have identified a positive transcriptional regulator of the
cadBA operon, CadC, which shows significant homology to both
ToxR and the E. coli CadC protein. Mutational analyses
revealed that CadC is essential for acid induction of cadBA
but is not needed for basal-level transcription of cadBA. The basal-level expression of cadBA was insufficient for a
functional ATR, as cadC mutants were unable to mount either
an inorganic or organic ATR.
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MATERIALS AND METHODS |
Sequence analysis.
DNA sequences were analyzed for open
reading frames (ORFs) by using DNA Strider version 1.2 (C. Marck).
E. coli CadB, CadC, and Lcd amino acid sequences were used
to search for putative homologs in the V. cholerae genome
sequence (The Center for Genomic Research [TIGR]) using the BLAST
algorithm (1). Multiple alignments of conserved protein
domains and hydropathy profile predictions were performed using
MacVector 6.0.1 (Oxford Molecular). cadC, -B, and
-A are located on the V. cholerae large
chromosome (replicon 1) beginning at positions 306411, 308670, and
310117, respectively (TIGR database).
Strain and plasmid construction.
All strains, plasmids, and
primers used in this study are listed in Tables
1 and 2.
Mutagenesis of cadB and cadC was by insertional
inactivation of a suicide plasmid, pGP704. The mutagenic plasmids were
constructed as follows. A 253-bp internal fragment of cadB
was amplified with Pfu polymerase (Stratagene) by PCR using
primers BF1 and BR1. SfiI adapters were ligated onto the PCR
product, and the resulting DNA fragment was ligated into the SfiI-digested pAC212 backbone as previously described
(25) to generate pDSM373. A 288-bp internal fragment of
cadC was amplified with Taq polymerase using
primers CadCF1 and CadCR1 and was subsequently cloned into pCR-Script
Amp SK(+) (Stratagene) according to the manufacturer's directions,
generating pDSM582. The cadC fragment was liberated from
pDSM582 by SacI/SalI double digestion and cloned into similarly digested pGP704 (26) to generate pDSM588. A
recombinant PCR method, gene splicing by overlap extension
(15) using primers BA
F1, BAR1, BAF2, and BAR2,
was used to generate strain DSM-V783, which contains a deletion of the
entire cadBA coding sequence.
All plasmids used for construction of insertion mutations were
mobilized into
V. cholerae from
E. coli
SM10
pir as previously
described (
25), and all
integration mutations were subsequently
verified by Southern blot
analysis (data not shown). The loss
of the ability to decarboxylate
lysine was shown by utilization
of lysine decarboxylation indicator
broth (Difco) as previously
described (
25).
Growth conditions.
All strains were maintained at 
80°C
in Luria-Bertani (LB) broth containing 30% glycerol. All strains were
grown at 37°C in LB broth. The pH of the medium was adjusted with
HCl. Ampicillin and streptomycin were used at concentrations of 100 µg ml
1. RNA was harvested from strains grown in the
following manner. Overnight cultures of each test strain were grown in
LB broth containing ampicillin and then diluted 1:150 into 30 ml of
fresh medium plus ampicillin. The diluted cultures were grown with
aeration until they reached an optical density (600 nm) of
approximately 0.16 to 0.20. At this point, cells were pelleted at
5,000 × g for 5 min at 24°C, and the supernatants
were removed by aspiration. Cells were resuspended in 1 ml of LB broth
(pH 7.0), and then 10 and 90% of the cells were placed into two
microcentrifuge tubes. The cells were pelleted at 12,000 × g for 1 min at 24°C, and the supernatants were removed by
aspiration. The 10% cell pellet was resuspended in 1 ml of LB broth
(pH 7.0), and the 90% cell pellet was resuspended in 1 ml of LB broth
(pH 5.7); then both were transferred to culture tubes and grown at
37°C with aeration for 1 h. After 1 h, all of the pH
7.0-grown cells and half of the pH 5.7-grown cells were pelleted and
then flash frozen in a 80°C isopropanol bath. The remainder of each
pH 5.7 culture was resuspended in LB (pH 4.5) and incubated at 37°C
for 15 min. These cells were then pelleted and flash frozen as above.
Strains which were exposed to organic acids were grown exactly as above
except that pH 5.7 LB and pH 4.5 LB were supplemented with 0.075× and
0.1× organic acid cocktail, respectively (1× cocktail was 87 mM
acetic acid, 25 mM butyric acid, and 37 mM propionic acid). Cell
pellets were then used to collect total RNA.
RPAs and primer extension.
RNase protection assays (RPAs)
were conducted on total RNA isolated from AC-V168 and DSM-V591 as
previously described (25). A 318-bp probe 1 and 732-bp probe
2 fragment were generated by PCR using Taq polymerase and
primer pairs AF1-AR1 and CadBOF-CadAOR, respectively. The amplification
products were ligated to pGemT (Promega), proper orientation was
confirmed, and riboprobes were synthesized using a Maxiscript kit
(Ambion) and 50 µCi of [32P]UTP (NEN) as previously
described (25). In each case, riboprobes transcribed from
the pGemT SP6 or T7 promoters contain pGemT-specific sequence that
results in riboprobes that are slightly larger than the original probe
1 or probe 2 V. cholerae-specific PCR fragments. Therefore,
hybridization of the probe with its target mRNA followed by digestion
with RNase results in a smaller protected fragment as the
nonhybridizing pGemT-specific transcript is cleaved. RPAs were
conducted with an RPAII kit using 1 to 3 µg of RNA as described by
the manufacturer (Ambion). The products of RNase protection were
separated on 5% denaturing polyacrylamide gels and exposed to phosphor
screens (Kodak). Quantification and peak analysis of bands was
conducted using a PhosphorImager and the ImageQuant program (Molecular Dynamics).
Primer extension to map the 5' ends of
cadA,
cadB, and
cadC mRNA transcripts produced after
acid challenge was conducted using
primers CadAPE (and CadAP2), CadBPE,
and CadCPE, respectively.
Approximately 10 pmol of each primer was end
labeled and used
for primer extensions with a primer extension
system-avian myeloblastosis
virus reverse transcriptase kit as
described by the manufacturer
(Promega). pDSM567, which contains the
entire
cadBA promoter region
and coding sequences, and
pDSM673, which contains the
cadC promoter
region, were
generated by PCR using primer pairs Cad8F1-Cad8R1
and
CadCCF-CadCRI, respectively. Amplification products were ligated
into pGemT, and the plasmids were used as templates for sequencing
reactions using the appropriate end-labeled primer. Sequencing
reactions were conducted by denaturation of template DNA as described
elsewhere (
16), followed by utilization of the denatured
template
in manual sequencing reactions using a Sequenase DNA
sequencing
kit (version 2.0) as described by the manufacturer
(Amersham).
Products of primer extensions and sequencing reactions were resolved in
7% denaturing polyacrylamide gels, dried, and exposed
to phosphor
screens. Products were analyzed using a
PhosphorImager.
ATR and competition assays.
ATR and competition assays were
conducted as previously described (25). The output ratio
from each in vivo and in vitro competition was corrected for any
deviations in the inoculum ratio from a value of 1:1. The competitive
index was calculated by dividing the in vivo output ratio by the in
vitro output ratio.
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RESULTS |
Identification of CadB and CadC homologs.
In an earlier study,
cadA was identified as an infection-induced gene of V. cholerae and shown to encode an inducible lysine decarboxylase
(25). Searches against the V. cholerae genome sequence revealed the presence of V. cholerae DNA sequences
5' of cadA on the large chromosome coding for putative
polypeptides with 67 and 27% identity to E. coli CadB and
CadC, respectively. Due to the high degree of identity of the putative
CadB and the similar location of its gene immediately upstream of
cadA, we designated this gene cadB.
In
E. coli, CadB acts as an antiporter that is responsible
for the transport of cadaverine, the by-product of lysine
decarboxylation,
from the bacterial cell, concomitant with import of
lysine (
24,
30). The
V. cholerae cadB ORF
consists of 1,338 bp and encodes
a predicted protein of 446 amino acids
(46.8 kDa). It lies upstream
of, and in a putative bicistronic operon
with, the previously
identified
cadA (Fig.
1). To verify the requirement of the
cadBA operon for lysine decarboxylation and cadaverine
export, a plasmid
insertion mutation within
cadB was
constructed; the resulting
mutant was unable to decarboxylate lysine in
a lysine decarboxylase
indicator medium. Although this result does not
distinguish between
loss of CadB function and a polar effect of the
mutation on
cadA,
given that
cadB represents the
only lysine/cadaverine antiporter
homolog in the
V. cholerae
genome, and given data below that substantiate
bicistronic
transcription of
cadBA, it is likely that both effects
occur.
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FIG. 1.
Schematic depiction of the V. cholerae cadCBA
locus. Arrows above the designated ORFs depict transcriptional
orientation and represent promoters based on 5' end mapping of mRNA
species by primer extension. The locations of primers used for primer
extension analysis are depicted by small arrows within the ORFs.
Riboprobes used for RPAs and corresponding to the V. cholerae-specific component of the probe are represented by
hatched boxes labeled probe 1 and probe 2. Relative sizes of
cadBA, cadB, and cadA transcripts are
represented by black lines beneath the ORFs. The locations of putative
transcriptional terminators sequences (t) are also shown.
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CadC has been shown to act as a positive transcriptional regulator of
the
cadBA operon in
E. coli (
24,
41).
The putative
V. cholerae cadC ORF consists of 1,560 bp and
encodes a predicted
protein of 519 amino acids (58.6 kDa). CadC lies
upstream of,
and in the same transcriptional orientation as,
cadB but is separated
from the
cadB open reading
frame by region of 699 bp (Fig.
1).
The
V. cholerae CadC
also shares a high degree of identity with
the N-terminal portion of
V. cholerae ToxR (Fig.
2).
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FIG. 2.
Multiple sequence alignment of amino-terminal portions
of E. coli (E. c.) CadC and V. cholerae (V. c.) CadC and ToxR. Identical amino acids
are highlighted by a gray background; conservative changes are outlined
in black. A consensus sequence is depicted below the multiple
alignment.
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ToxR is a transcriptional activator localized within the inner membrane
that is required for proper expression of virulence
factors such as
cholera toxin and the toxin coregulated pilus
(reviewed in reference
37). The N-terminal domain of ToxR is
cytoplasmic
and encodes an OmpR-like DNA binding domain that is
necessary for both
positive and negative transcriptional regulation
of ToxR-regulated
genes. In addition, ToxR possesses a transmembrane
segment and
periplasmic domain that are believed to be involved
in receiving and
transmitting proper signals to elicit ToxR-mediated
regulation
(
26,
27,
31). Based on amino acid sequence similarity
at the
N-terminal end of
V. cholerae ToxR and CadC and a conserved
hydropathy profile throughout (data not shown), CadC is predicted
to be
a transmembrane transcriptional activator with topological
and
localization features similar to those of
ToxR.
Transcription of cadA is regulated by CadC.
Since
CadC has previously been shown to be a positive transcriptional
regulator of the cadBA operon in E. coli
(24, 41), we sought to determine if CadC was similarly
required for cadA expression in V. cholerae. V. cholerae strain DSM-V591, which contains an insertion mutation in
cadC, was unable to decarboxylate lysine upon inoculation
into lysine decarboxylase indicator medium whereas the wild-type
parental strain did, suggesting that cadC was necessary for
proper expression of cadBA.
To determine whether the inability of the
cadC strain to
decarboxylate lysine was due to an inability to properly regulate
transcription of
cadA, RPAs were conducted with probe 1, a
riboprobe
specific for
cadA transcripts (Fig.
1). Total RNA
was collected
from isogenic strains AC-V168 and DSM-V591 after growth
in a rich
medium at pH 7.0, 5.7, or 4.5. As previously reported
(
25),
a large induction of
cadA transcription
occurs in wild-type
V. cholerae cells upon exposure to pH
4.5 (Fig.
3A). In contrast,
the
cadC strain showed no induction of
cadA
transcription upon
exposure to low pH. Of note, a basal level of
cadA transcript
is seen which appears both pH and CadC
independent (Fig.
3A and
B).
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FIG. 3.
RPA for cadA transcription in wild-type (WT)
and cadC mutant strains. Total RNA was prepared from
bacteria grown in either low-pH media (A) or low-pH media supplemented
with organic acids (B). The lanes corresponding to undigested probe
represent the size of probe 1 used in the assays. Negative controls
lacking bacterial RNA () show no protected bands. The protected
cadA transcript is indicated. Relative molecular sizes are
indicated on the left.
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To determine if CadC was also necessary for the previously reported
induction of
cadA transcription upon exposure to low pH
plus
organic acids (
25), similar RPAs were conducted using total
RNA collected from AC-V168 and DSM-V591 grown in the presence
of
organic acids. As expected, AC-V168 showed an increase in
cadA transcription when grown in medium at pH 5.7 and 4.5 that had
been supplemented with organic acids (Fig.
3B and reference
25).
In contrast, no increase in
cadA
transcript was evident in samples
prepared from DSM-V591, indicating
that CadC is required for organic
acid-induced transcription of
cadA at pH 5.7.
Transcription of the cadBA operon.
Since
cadA was shown to play an essential role in the ATR of
V. cholerae (25) but not of E. coli
(34), we sought to develop a better understanding of the
differences in regulation of cadA expression in these two
organisms. Previous studies of E. coli have shown that
cadA exists as part of a bicistronic operon with cadB. Transcription of this operon occurs from a promoter
that has been designated pCad (41). pCad has been shown to
be stimulated in a CadC-dependent manner by low pH, oxygen limitation,
and high lysine concentrations (2, 23, 29, 35, 39, 41). To determine if cadB and cadA were cotranscribed in
V. cholerae, RPA analysis was performed using probe 2, a
riboprobe designed to span the entire cadB-cadA intergenic
region and portions of cadB and cadA (Fig. 1).
Three possibilities for potential transcripts were considered: (i)
bicistronic cadB and cadA transcripts, (ii) specific cadB and cadA transcripts, and (ii) both
bicistronic and single gene-specific transcripts. Based on probe 2, we
expected that bicistronic cadBA would result in protection
of a 732-bp fragment. A monocistronic cadB transcript would
result in protection of a fragment that spanned the 3' end of the
cadB ORF (532 bp) as well as downstream sequences, depending
on the site(s) of transcriptional termination. A monocistronic
cadA transcript would result in a protected fragment that
encompassed the 5' end of the cadA ORF (101 bp) as well as
upstream sequences, depending on the site of transcriptional initiation.
Total RNA was prepared from AC-V168 grown at pH 7.0, 5.7, and 4.5, and
RPAs using probe 2 revealed protected fragments that
correspond to
sizes expected for a bicistronic transcript. Therefore,
cadB
and
cadA are transcribed as a bicistronic operon in
V. cholerae (Fig.
4; depicted in Fig.
1). Upon shift to pH 4.5, a condition
known to increase transcription
of
cadA, an increase in the amount
of bicistronic
cadBA message was seen. In addition, two protected
fragments
that correspond to the sizes expected of transcripts
terminated at the
end of the
cadB ORF appeared. A minor band that
would
correspond to the expected size of a
cadA-specific
transcript
also appeared (Fig.
4).
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FIG. 4.
RPA for cadBA transcription in wild-type,
(WT) cadC mutant, and cadBA strains. Total RNA
was prepared from strains grown at the pH values indicated. The lanes
corresponding to undigested probe represents the size of synthesized
probe 2 used in the assays. Bands corresponding to undigested
full-length probe and cadBA, cadB, and
cadA transcripts are indicated. Molecular weight markers are
shown with relative sizes. The undigested probe and molecular weight
markers are shown from a shorter exposure, which was necessary to
prevent overexposure in comparison to protected fragments.
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Since additional fragments of unexpected sizes were protected in each
lane, we wished to confirm the identities of the indicated
transcripts.
An isogenic
V. cholerae strain that contained a deletion
of
the entire
cadBA coding sequence was constructed and used in
similar RPAs. As shown in Fig.
4, deletion of the
cadBA
coding
sequence resulted in loss of bicistronic
cadBA and of
monocistronic
cadB and
cadA bands, thus
confirming that the indicated bands
are specific for
cad transcripts.
In Fig.
4, the protected fragments corresponding to the
cadB-specific transcript, which would result from
termination or processing
downstream of the
cadB ORF, was
shown to be visible only upon
shift to pH 4.5. Analysis of the
nucleotide sequence immediately
downstream of
cadB revealed
two large inverted repeats, designated
as IR 1 and IR 2, which could
potentially code for factor-independent
transcriptional terminators
(see Fig.
6). IR 1 is a perfect inverted
repeat whose predicted stem
and loop structure is followed by
three uracil nucleotides. IR 2 is an
imperfect repeat; the spacing
of the run of uracil nucleotides is four
positions distal to the
base of the predicted hairpin structure.
Termination at IR 1 would
result in a 562-bp
cadB
transcript, while termination at IR 2
would result in a 621-bp
cadB transcript. These sizes are in good
agreement with the
sizes for
cadB transcripts indicated in Fig.
4. The presence
of only three uracil nucleotides at the base of
IR 1 and the spacing of
uracil nucleotides distal to the stem
of IR 2 would be consistent with
low efficiency termination at
these sites (
8).
Since we had shown that CadC was required for low-pH-induced
transcription of
cadA (Fig.
3), we investigated whether
regulation
by CadC occurred at the bicistronic
cadBA
promoter, the internal
cadA promoter, or both. Total RNA was
collected from DSM-V591,
and RPA analysis using probe 2 was carried
out. No significant
increase in
cadBA (or
cadB or
cadA) transcript was seen upon shift
of DSM-V591 to lower
pHs (Fig.
4). Furthermore, basal-level transcription
of
cadBA is CadC independent (Fig.
4). These results indicate
that CadC regulation of both
cadA and
cadB
expression occurs at
the
cadBA and
cadA promoters
(Fig.
4).
Identification of the transcription start sites of
cadC, cadB, and cadA.
Primer
extension analysis was carried out to map the 5' termini of the
cadB, cadA, and cadC transcripts to
begin to define the promoter elements within this region. Total RNA was
extracted and used for primer extension as described in Materials and
Methods. For cadB, two primer extension products separated
by three nucleotides were detected using RNA collected after acid
challenge (Fig. 5A). This suggests a
transcriptional start site(s) that is located 96 to 99 bp upstream of
the translational start codon. Putative promoter elements were deduced
according to the apparent transcriptional start site(s). The predicted
35 element (TATTTT) bears little resemblance to the E. coli 
70 consensus sequence (TTGACA). In addition,
the 10 region (TACTCT) varies from the consensus (TATAAT) by three
bases (Fig. 5B). Interestingly this 10 region shows greater
similarity to the S consensus sequence (TATACT) in that
it varies by only two nucleotides. A mutation in rpoS that
encodes S, though, has previously been shown not to
affect cadA transcription under the tested conditions
(25). The predicted weakness of these 10 and 35 elements
may account for the low level of basal transcription seen in the
absence of the positive regulator CadC.
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FIG. 5.
(A) Primer extension analysis of cadB
transcripts. The two primer extension products obtained with primer
CadBPE are depicted in lane 1. Lane 2 represents a negative control
where no RNA was added. The sequencing ladder obtained with the same
primer is shown to the left. The +1 sites are indicated by arrows, and
a likely 10 box is shown. The length of exposure of the sequencing
ladder was reduced to prevent overexposure in comparison to primer
extension products. (B) Nucleotide sequence of the cadB
promoter region. Direct (DR) and inverted (IR) repeats are indicated by
arrows. Transcription start sites are indicated by asterisks, and a
likely 10 and a 35 site are underlined. The probable ribosomal
binding site (RBS) is shaded, and the translational start is
indicated.
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Analysis of the region upstream of the
cadB transcription
start sites revealed regions rich in A and T nucleotides. Two direct
repeats of TGATAT
5NG are located starting at position
180, and
two smaller direct repeats of T
7NG overlap the
35 region (Fig.
5B). These repeats represent potential CadC binding
sites and
show striking similarity to A/T-rich regions that ToxR has
been
shown to bind (
7,
14,
20,
27). In particular, the
TGATAT
5NG
repeat is similar to the consensus repeat
sequence TG(a/T)
3TTTNN
bound by ToxR within the
ompT promoter (
20).
Primer extension of
cadA transcripts was conducted using two
separate primers, and in each case a single primer extension
product
was detected (Fig.
6A and data not
shown). This probable
transcriptional start site is located 73 bp
upstream of the translational
start codon. The predicted
35
element (TTGACC) differs from the
E. coli
70 consensus sequence by only one nucleotide, but the
10 region
(CAGCTT) bears little similarity to the consensus (Fig.
6B). Comparison
of these regions to consensus sequences utilized by
other sigma
factors revealed no other significant sigma factor motifs.
Two
inverted repeats of 8 and 11 bp are found upstream of the
cadA translational start site and, as mentioned above,
potentially
represent the site(s) of inefficient termination of
transcripts
originating from the
cadB promoter.
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FIG. 6.
(A) Primer extension analysis of cadA
transcript. The primer extension product obtained with primer CadAPE is
depicted in lane 2. Lane 1 represents a negative control where no RNA
was added. The sequencing ladder obtained with the same primer is shown
to the right. The +1 site is indicated by an arrow, and a likely 10
box is shown. The length of exposure of the sequencing ladder was
reduced to prevent overexposure in comparison to primer extension
products. (B) Nucleotide sequence of the cadA promoter
region. The transcription start site is indicated by an asterisk, and a
likely 10 and a 35 site are underlined. IR 1 and IR 2 are indicated
by arrows. The probable ribosomal binding site (RBS) is shaded, and the
translational start is indicated.
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A single primer extension product was detected for
cadC
(Fig.
7A). This product suggests a
transcriptional start site that
is located 75 bp upstream of the
translational start codon. A
poorly conserved
35 element was
predicted (TAGGTG), but a consensus
10 was evident (Fig.
7B).
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FIG. 7.
(A) Primer extension analysis of cadC
transcript. The primer extension products obtained with primer CadCPE
are depicted in lanes 2 and 3. Lane 3 represents threefold less RNA
than lane 2. Lane 1 represents a negative control where no RNA was
added. The sequencing ladder obtained with the same primer is shown to
the right. The +1 site is indicated by an arrow, and a likely 10 box
is shown. The length of exposure of the sequencing ladder was reduced
to prevent overexposure in comparison to primer extension products. (B)
Nucleotide sequence of the cadC promoter region. The
transcriptional start is indicated by an asterisk, and a likely 10
and a 35 site are underlined. The probable ribosomal binding site
(RBS) is shaded, and the translational start is indicated.
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CadC does not regulate other genes required for acid tolerance or
genes essential for virulence.
Since CadC acts as a
transcriptional regulator of the cadBA operon and shares a
high degree of similarity to ToxR, which directly or indirectly
regulates as many as 17 genes, we sought to determine whether CadC
regulates additional pathways, other than lysine decarboxylation, that
are required for acid tolerance. This possibility was tested by
assessment of the ability of DSM-V591 to undergo an attenuated ATR. We
reasoned that if CadC regulated additional pathways required for ATR,
the ATR defect exhibited by this cadC strain should be more
severe than the ATR defect exhibited by a cadA strain. ATR
assays using AC-V168 (wild type), DSM-V591 (cadC), and
DSM-V376 (cadA) were performed as previously described (25). All three strains, when unadapted, were quickly killed when exposed to pH 4.5 (data not shown). Acid-adapted AC-V168 was able
to mount a productive ATR and showed complete survival out to 60 min
(Fig. 8). However, both DSM-V591 and
DSM-V376 exhibited attenuated ATRs, and the rates of acid killing for
both of these adapted strains were virtually identical (Fig. 8). Note
that the rates of death of adapted DSM-V376 and DSM-V591 were not as
severe as that for the unadapted wild-type strain, suggesting that some acid adaptation is occurring in these mutant strains. Similar results
were obtained in organic ATR assays (data not shown). The sum of these
experiments supports the hypothesis that CadC does not regulate
additional genes (besides cadBA) that are required for the
ATR of V. cholerae.
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|
FIG. 8.
ATR assays of wild-type (WT; AC-V168), cadA
(DSM-V376), and cadC (DSM-V591) V. cholerae
strains. Strains were acid adapted or unadapted as described in
Materials and Methods, and percent survival was calculated as a
function of time after resuspension of bacteria in acid challenge
medium, pH 4.5.
|
|
The ability of DSM-V591 (
cadC) to colonize the infant mouse
intestine was tested in a competition assay. DSM-V591 and the
virulent,
isogenic strain AC-V66 were mixed to attain an input
ratio of
approximately 1:1, and the mixture was used in competition
assays as
previously described (
25). DSM-V591 exhibited no detectable
defect in colonization, giving a competitive index of 0.96, which
is
essentially identical to the competitive index reported for
a
cadA mutant (
25). These results suggest that in
this model
system, CadC is not required for regulation of factors
essential
for
V. cholerae intestinal
colonization.
| |
DISCUSSION |
The cadA gene of V. cholerae was previously
identified as an infection-induced gene in two different animal models
of V. cholerae (25). In vitro transcription
analysis revealed that the gene was induced by low pH, high lysine
concentration, and oxygen limitation. It was shown that cadA
plays an essential role in the ATR of V. cholerae.
Additionally, it was found that V. cholerae cells that were
acid adapted prior to intragastric inoculation into animals greatly
outcompeted unadapted cells, suggesting interesting implications for
ATR in the epidemic spread of V. cholerae. In the present study, we examined the gene arrangement, transcription, and regulation of the cadA locus in more detail. The regulation of
cadA has not been investigated in S. enterica
serovar Typhimurium, the only other organism known to require lysine
decarboxylation by cadA for ATR (30).
We found that V. cholerae contains cadB and
cadC homologs, which are linked to cadA and
arranged similarly as in E. coli. CadB in E. coli
functions as a lysine/cadaverine antiporter, and its structural gene,
cadB, is transcribed in a bicistronic operon with
cadA. The cadBA operon is positively regulated by
CadC (2, 23, 29, 35, 39, 41). RPA analysis revealed that
cadB and cadA are also transcribed as a
bicistronic operon in V. cholerae. However, unlike in
E. coli, cadB- and cadA-specific
transcripts are also present in V. cholerae.
Neely and Olson (29) previously determined the kinetics of
cadBA expression in E. coli and found that a
cadBA transcript is not detectable at neutral pH but becomes
apparent upon shift to pH 5.8. Similarly, in V. cholerae,
cadBA transcription was greatly increased upon shift from
neutral to acidic pH, although pH 4.5 was necessary to see induction
(or pH 4.5 and 5.7 plus organic acids [data not shown]). In addition,
cadA monocistronic transcript appears upon shift to low pH.
Together, these results suggest that our previous demonstration of
low-pH induction of cadA transcription (25)
occurs at the level of increased bicistronic cadBA and
monocistronic cadA mRNA. pH-regulated induction of
cadBA in E. coli is completely dependent on CadC
in that a cadC null mutant does not express cadBA
under any conditions tested (28). Unlike the case for
E. coli, though, V. cholerae shows basal-level expression of cadBA transcript at neutral pH. In addition,
this basal-level transcription is CadC independent, as mutations in cadC resulted in no decrease in transcription at any pH tested.
The location of cadC adjacent to cadBA on the
V. cholerae large chromosome, and the similarity of the
predicted CadC primary sequence to that of E. coli CadC,
suggested that CadC might act as a transcriptional regulator required
for low-pH induction of cadBA. Indeed, a greater than
fivefold increase in cadBA mRNA was detected upon exposure
of wild-type cells to low pH, and this increase was completely ablated
in a cadC mutant. Again, however, basal-level transcription
of cadBA still occurs in a cadC mutant, suggesting that functional lysine decarboxylase may be produced at a
low level.
What might be the role of low constitutive levels of CadA and CadB in
V. cholerae? Amino acid decarboxylases contribute to either
biosynthetic or biodegradative pathways and have been shown to play
important roles in polyamine synthesis or pH homeostasis, respectively
(23, 24, 30). Biosynthetic amino acid decarboxylases are
generally expressed constitutively, while biodegradative amino acid
decarboxylases require low-pH induction for expression (3, 23, 24,
35, 38). E. coli is known to possess both biosynthetic and biodegradative lysine decarboxylases, Lcd and CadA, respectively. Lcd is responsible for the constitutive, low-level biosynthesis of
cadaverine from lysine (17, 19). However, after acid
induction, the bulk of lysine decarboxylase activity is due to the
induction of cadA (23, 29). Extensive searches
conducted against the V. cholerae genome sequence reveal no
other potential lysine decarboxylase-encoding genes, suggesting that
CadA is the sole enzyme responsible for the decarboxylation of lysine
by V. cholerae (D. S. Merrell and A. Camilli,
unpublished data). We hypothesize that the CadC-independent, basal-level expression of cadBA results in low levels of
lysine decarboxylase necessary for biosynthetic decarboxylation, but at
a level that is undetectable in our broth indicator medium assay. Upon
exposure of V. cholerae to low pH, oxygen limitation, and
high lysine concentrations, high levels of CadA and CadB are expressed
in a CadC-dependent manner to assist with maintenance of pH homeostasis.
The N-terminal domain of CadC shares a high degree of identity with the
N-terminal DNA binding domain of ToxR. ToxR is responsible for
transcriptional activation of the ToxR/ToxT virulence gene regulon.
Upon receiving unknown signals in the host intestine, ToxR stimulates
transcription of toxT and several other genes; ToxT
subsequently acts as a major transcription factor for a large set of
virulence genes (37). V. cholerae also contains
TcpP, an additional inner membrane protein that shares homology with ToxR in both sequence and function (6, 12). Recent studies have shown that both TcpP and ToxR are required for maximal expression of ToxT (12). toxR and tcpP are
cotranscribed with toxS and tcpH, respectively;
ToxS and TcpH are inner membrane proteins required for maximal activity
of ToxR and TcpP, respectively (9, 12, 32). The V. cholerae and E. coli CadC proteins, though similar to
ToxR in their N-terminal domains and cellular locations, appear not to
require a ToxS-like component for activity (20). This
suggests that the V. cholerae CadC protein is independently capable of receiving and transmitting signals. However, the possibility remains that an unidentified protein is capable of associating with
CadC and facilitating its function.
It has long been known that the ability to respond and adapt to low-pH
environments is an important aspect of the life cycle of many
pathogenic organisms that colonize within the gastrointestinal tract.
For instance, mutant strains of S. enterica serovar
Typhimurium, Listeria monocytogenes, and Helicobacter
pylori that are compromised in their ability to survive acid
exposure have been shown to exhibit attenuated virulence (4, 10,
22, 36). Interestingly, the CadC homolog of S. enterica serovar Typhimurium was previously identified using in
vivo expression technology as an infection-induced gene expressed
within both the small intestines and spleens of BALB/c mice
(13). The fact that two genes within the cad
locus have been shown to be upregulated in two pathogens (V. cholerae and S. enterica serovar Typhimurium) upon
entry into the host suggests that homologs of these genes may play
crucial roles in the survival and adaptation of other pathogenic
organisms as well.
Studies of the mechanism of transcriptional regulation by ToxR have
shown that it binds to promoter elements exhibiting high degrees of A/T
richness (7, 14, 20, 27). Analysis of the region upstream of
the cadB promoter revealed the presence of direct repeats
that are strikingly similar to ToxR-recognized elements in the
ompT, ompU, ctxAB, and toxT
promoters (20). Despite this sequence similarity, we found
that a toxR null mutant shows normal levels of
cadA expression (25), suggesting that ToxR does
not regulate transcription of the cad locus under the conditions tested. Instead, these findings suggest that ToxR and CadC
recognize very similar A/T-rich sequences but with a high degree of fidelity.
| |
ACKNOWLEDGMENTS |
This research was supported by NIH training grant AI 07422 (D.S.M.), NIH grants AI 40262 and Pew Scholars Award P0168SC (A.C.), and the Center for Gastroenterology Research on Absorptive and Secretory Processes, NEMC (P30 DK34928).
We thank E. Olson for helpful discussion, and we thank E. Joyce and E. Lloyd Angelichio for critical discussion and support. In addition, we
thank A. Sonenshein, J. Mecsas, and M. Waldor for critical readings of
the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Tufts University
School of Medicine, Department of Molecular Biology and Microbiology, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2144. Fax: (617)
636-0337. E-mail: andrew.camilli{at}tufts.edu.
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Top
Abstract
Introduction
