Regulatory elements involved in the expression of competence genes in naturally transformable Vibrio cholerae
© Lo Scrudato et al.; licensee BioMed Central. 2014
Received: 3 September 2014
Accepted: 16 December 2014
Published: 24 December 2014
The human pathogen Vibrio cholerae normally enters the developmental program of natural competence for transformation after colonizing chitinous surfaces. Natural competence is regulated by at least three pathways in this organism: chitin sensing/degradation, quorum sensing and carbon catabolite repression (CCR). The cyclic adenosine monophosphate (cAMP) receptor protein CRP, which is the global regulator of CCR, binds to regulatory DNA elements called CRP sites when in complex with cAMP. Previous studies in Haemophilus influenzae suggested that the CRP protein binds competence-specific CRP-S sites under competence-inducing conditions, most likely in concert with the master regulator of transformation Sxy/TfoX.
In this study, we investigated the regulation of the competence genes qstR and comEA as an example of the complex process that controls competence gene activation in V. cholerae. We identified previously unrecognized putative CRP-S sites upstream of both genes. Deletion of these motifs significantly impaired natural transformability. Moreover, site-directed mutagenesis of these sites resulted in altered gene expression. This altered gene expression also correlated directly with protein levels, bacterial capacity for DNA uptake, and natural transformability.
Based on the data provided in this study we suggest that the identified sites are important for the expression of the competence genes qstR and comEA and therefore for natural transformability of V. cholerae even though the motifs might not reflect bona fide CRP-S sites.
Vibrio cholerae is a Gram-negative bacterium that often lives in aquatic environments in association with the chitinous exoskeleton of zooplankton ,. Chitin, a polymer of β-1,4-linked N-acetylglucosamine, is one of the most abundant biopolymers in nature . In addition to its role as a nutrient source, chitin also induces natural competence for transformation in V. cholerae and other Vibrio species (reviewed by ).
In addition to TfoX expression, pathways that regulate quorum sensing (QS) and carbon catabolite repression (CCR) are also necessary to induce the competence regulon of V. cholerae, (Figure 1). QS is a process of bacterial communication and is based on the production and secretion of small molecules called autoinducers (reviewed by ). V. cholerae produces and secretes at least two different autoinducers: the intra-species cholera autoinducer 1 (CAI-1) and the universal autoinducer 2 (AI-2) -. At high cell density, the concentration of autoinducers is sufficient to lead to the production of HapR, the master regulator of QS that is known to regulate virulence repression ,,, biofilm formation  and natural competence for transformation ,,-,,- (Figure 1). In the absence of HapR, the extracellular DNA is degraded by the action of the nuclease Dns, preventing DNA uptake ,,. HapR regulates natural transformation by direct repression of dns and concomitantly with TfoX-mediated induction, directly drives the expression of qstR, which encodes the newly identified transcription factor QstR  (Figure 1). Notably, the contribution of QstR to natural transformation was recently confirmed by Dalia et al. using a genome-wide transposon sequencing (Tn-seq) screen .
The third pathway involved in the regulation of natural competence for transformation is CCR ,. This term indicates the mechanism by which, in the presence of a preferred carbon source such as glucose, the expression of genes necessary for the metabolism of other carbon sources is repressed . The major players in CCR are the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), adenylate cyclase (CyaA), the metabolite 3′,5′-cyclic adenosine monophosphate (cAMP) and the CRP protein. Unsaturated PTS transporters enhance the synthesis of cAMP by CyaA. High levels of cAMP trigger the formation of an active complex of CRP and cAMP, which binds the promoters of the target genes (e.g., those genes encoding proteins that are involved in the transport and utilization of alternative carbon sources). Conversely, when the PTS is saturated cAMP is not produced and the CRP-cAMP complex cannot form. Central metabolism and transport of the carbon sources are not the exclusive targets of CCR; cAMP and the CRP protein, as well as the PTS components (independent of cAMP), also control biofilm formation in V. cholerae-. With respect to natural competence for transformation, the presence of PTS sugars significantly decreases the transformability of V. cholerae; moreover, knockout strains for crp or cyaA are non-transformable .
The role and function of the CRP protein have been primarily studied in E. coli (reviewed by ,). CRP, formerly known as catabolite activator protein (CAP), forms a dimer of two identical subunits. Each CRP subunit contains an N-terminal cAMP binding domain, a flexible hinge region and a C-terminal helix-turn-helix DNA binding motif. CRP recognizes and binds 22 bp-long symmetrical sequences called CRP sites. Under physiological conditions, CRP is likely present either as a free apo-CRP dimer (in the absence of cAMP) or as a dimer with each subunit bound to a molecule of cAMP. V. cholerae CRP and E. coli CRP (EcCRP) share 95% identity in amino acid sequence . As with EcCRP, V. cholerae CRP displayed a biphasic dependence on cAMP levels in vitro. Moreover V. cholerae CRP is able to activate the transcription of E. coli gal promoters ,. These findings strongly suggest that the CRP protein functions similarly in these two bacterial species.
In H. influenzae, the expression of the competence genes requires the CRP-cAMP complex , along with the master regulator of transformation Sxy  (TfoX in V. cholerae). The competence regulon of H. influenzae consists of genes characterized by the presence of competence regulatory elements (CRE) ,. Due to their Sxy dependency, these specific competence-related CRP binding sites were later renamed CRP-S sites to distinguish them from the canonical Sxy-independent CRP-N sites . Indeed, Cameron and Redfield suggested that in H. influenzae, and most likely in other competent Gram-negative bacteria, the induction of competence genes is under the control of CRP and Sxy/TfoX acting in concert at the CRP-S site . Based on previously published expression data from V. cholerae, Cameron and Redfield also predicted a 22-bp CRP-S and CRP-N consensus motif for the Vibrionaceae family in silico, which was nnntTTnAAnTnnnTCGAAnnn for CRP-S and tnntGTGAnnnnnnTCACanan for CRP-N (the most common bases are indicated in upper case, the less likely bases in lower case; 'n' refers to any base even though minor preference might be valid for some of these positions; for details see ).
In this study, we tested the role of QstR as an activator of comEA. We demonstrated that overexpression of qstR was sufficient to increase the abundance of the comEA transcripts, although not to the same level observed under competence-inducing conditions. We therefore hypothesized that TfoX and CRP-cAMP were also involved in driving the expression of comEA and qstR (Figure 1). In agreement with this hypothesis, we identified putative CRP-S sites in the promoter regions of qstR and comEA, which were not part of the in silico predicted Vibrionaceae CRP-S sites described above . We investigated the importance of these motifs using site-directed mutagenesis, followed by the analysis of the respective mutants. Our results suggest that these sites are important for the transcriptional regulation of the respective competence genes but might not represent bona fide CRP-S sites.
Bacterial strains and plasmids
Strains and plasmids used in this study
Strains or plasmids
V. cholerae strains
Wild-type, O1 El Tor Inaba, RifR
A1552 strain with aph cassette in lacZ gene; RifR, KanR
A1552 containing mini-Tn7-araC-P BAD -tfoX; RifR, GentR
A1552ΔhapR containing mini-Tn7-araC-P BAD -tfoX; RifR, GentR
A1552ΔVC1917 (=A1552VC1917 in (Ref)), RifR
A1552ΔcomEA containing mini-Tn7-araC-P BAD -tfoX; RifR, GentR
A1552ΔqstR containing mini-Tn7-araC-P BAD -tfoX; RifR, GentR
CRP-S site upstream of comEA deleted in strain A1552-TntfoX using the TransFLP method; RifR, GentR
CRP-S site upstream of comEA inverted in strain A1552-TntfoX using the TransFLP method; RifR, GentR
CRP-S site upstream of comEA changed for a CRP-N site (see scheme in Figure 4) in strain A1552-TntfoX using the TransFLP method; RifR, GentR
CRP-S site upstream of comEA changed for the in silico predicted CRP-N site preceding the frdA gene in strain A1552-TntfoX (see scheme in Figure 4) using the TransFLP method; RifR, GentR
CRP-S site upstream of comEA changed in the 3′ conserved region (see scheme in Figure 4) in strain A1552-TntfoX using the TransFLP method; RifR, GentR
WT_qstR (FRT control)
HapR-binding site determined in vitro deleted from strain A1552-TntfoX using the TransFLP method; RifR, GentR
CRP-S site upstream of qstR deleted in strain A1552-TntfoX (see scheme in Figure 4) using the TransFLP method; RifR, GentR
CRP-S site upstream of qstR changed for the in silico predicted CRP-N site preceding the frdA gene (see scheme in Figure 4) in strain A1552-TntfoX using the TransFLP method; RifR, GentR
CRP-S site upstream of qstR changed in the 3′ conserved region (see scheme in Figure 4) in strain A1552-TntfoX using the TransFLP method; RifR, GentR
pBR322-derived expression vector; araBAD promoter (PBAD); AmpR
qstR gene cloned into pBAD/Myc-HisA; arabinose inducible; AmpR
ori R6K, helper plasmid with Tn7 transposition function; AmpR
pGP704 with mini-Tn7
pGP704 with mini-Tn7 carrying araC and P BAD -driven tfoX; AmpR
pBR322 derivative deleted for Tet promoter and part of tet R gene; new MCS included; AmpR
pBR322 derivative deleted for Tet promoter and part of tet R gene; new MCS included; AmpR
comEA gene preceded by 900 bp of upstream region cloned into pBR-Tet_MCSII; AmpR
comEA gene preceded by 700 bp of upstream region; plasmid generated by inverse PCR of pBR-[own]comEA; AmpR
comEA gene preceded by 500 bp of upstream region; plasmid generated by inverse PCR of pBR-[own]comEA; AmpR
comEA gene preceded by 300 bp of upstream region; plasmid generated by inverse PCR of pBR-[own]comEA; AmpR
comEA gene preceded by 134 bp of upstream region cloned into Not I site of pBR-Tet_MCSII; AmpR
comEA gene preceded by 100 bp of upstream region; plasmid generated by inverse PCR of pBR-[own]comEA; AmpR
comEA gene preceded by 40 bp of upstream region; plasmid generated by inverse PCR of pBR-[own]comEA; AmpR
Media and growth conditions
V. cholerae and E. coli strains were grown at either 30°C or at 37°C. Overnight cultures were grown in LB medium under aerobic conditions. Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar plates were used to counterselect E. coli strains after triparental mating with V. cholerae strains. The TCBS agar plates were prepared following the manufacturer’s instructions (Fluka). For plasmid maintenance or selection of transformants/transconjugants, antibiotics were added to the growth media at concentrations of 50 or 100 μg ml−1 for ampicillin, 75 μg ml−1 for kanamycin, and 50 μg ml−1 for gentamicin.
Construction of V. cholerae mutant strains
Chromosomally-encoded site-directed mutants were generated using the previously described TransFLP method ,,. The extended FRT scar was located downstream of the native comEA gene and upstream of the native qstR gene. In the latter case, a control strain was designed that was not modified in a site-directed manner but solely contained the integrated FRT scar upstream qstR (strain WT_qstR (FRT control)). This strain behaved as the WT with respect to the expression pattern, natural transformation, DNA uptake, and general growth behavior (described below).
Construction of plasmids
Primers used in this study
(in 5′ to 3′ direction)
For inverse PCR leading to plasmids:
Inverse PCR to generate
Inverse PCR to generate
Inverse PCR to generate
PCR to generate
Inverse PCR to generate
Inverse PCR to generate
qRT-PCR for gyrA transcription 
qRT-PCR for comEA transcription 
qRT-PCR for comEC transcription 
qRT-PCR for qstR transcription 
qRT-PCR for hapR transcription 
qRT-PCR for hapA transcription 
5′RACE: synthesis of first strand cDNA of comEA
5′RACE: PCR amplification of Poly(A) cDNA comEA
oligo dT-Anchor primer (Roche)
5′RACE: PCR fragment of Poly(A) cDNA comEA cloned into plasmid pBR-Tet_MCSI
Natural transformation assay (chitin-dependent and chitin-independent)
Natural transformation assays were performed as previously described, growing V. cholerae strains on chitin flakes with a medium change on day two , or in LB medium supplemented with 0.02% arabinose to express an inducible chromosomal copy of tfoX (preceeded by a PBAD promoter; ). The same growth conditions were used for in trans over-expression of the plasmid-encoded qstR gene. Notably, V. cholerae does not contain any obvious homolog of the low-affinity high-capacity arabinose transporter AraE, which is involved in the all-or-none induction of genes preceeded by the arabinose-inducible promoter PBAD in E. coli. Statistical analyses of transformation data were carried out on log-transformed data  using a two-tailed Student’s t-test.
Whole-cell duplex PCR assay to test for DNA uptake
DNA uptake was verified using a whole-cell duplex PCR assay as previously described ,. Briefly, the respective V. cholerae strains were induced for competence as described above before genomic DNA of E. coli strain BL21(DE3) was added at a final concentration of 2 μg/ml. After a 2 h incubation step the cells were harvested and DNase I-treated. Any excess nuclease was removed by washing of the cells with PBS buffer. ~3×106 bacteria served as template in a whole-cell duplex PCR. The two primer pairs were specific for the donor DNA derived from E. coli BL21(DE3) and for gDNA of the V. cholerae acceptor strain (at a 10-fold lower concentration), respectively ,.
SDS-PAGE and Western blotting
Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and then subjected to western blotting as previously described . The primary antibody against ComEA (GP 1248; see below) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, Switzerland) were diluted at 1:5,000 and 1:20,000, respectively. Luminescent signals were produced and detected by Western Lightning-ECL (PerkinElmer) and chemiluminescence-detecting film (Amersham Hyperfilm ECL, GE Healthcare).
Generation of the antibodies against ComEA
Rabbit anti-ComEA antibodies were raised against synthetic peptides and produced by Eurogentec (Belgium). The antibody was tested in Western blot analysis against the comEA knockout strain to exclude potential cross-reactions with proteins migrating towards the same position as the target protein.
Quantitative reverse transcription PCR (qRT-PCR)
V. cholerae strains were grown in LB medium supplemented with 0.02% arabinose to induce the qstR gene or natural competence. RNA preparation, DNase treatment, reverse transcription, and qPCR were performed as previously described ,,.
5′ Rapid amplification of cDNA ends (5′RACE)
V. cholerae wild-type strain A1552-TntfoX was induced to competence as described above. Cell harvesting and RNA preparation were performed as previously published . The 5′/3′ RACE Kit 2nd Generation (Roche) was used to identify the transcription start of the comEA gene. All steps were performed according to the manufacturer’s protocol unless stated otherwise. Total RNA (2 μg) and the gene-specific primer comEA_284_rev were used to synthesize the first strand cDNA of comEA. The cDNA was then purified using the High Pure PCR Product Purification Kit (Roche, Switzerland). After addition of the Poly(A) tail to the 3′ end, the first strand cDNA was amplified by PCR with the gene-specific primer comEA_217_rev and oligo dT-Anchor primer. The PCR products were visualized on an agarose gel and purified. The double-stranded cDNA of comEA was further amplified by PCR with the primers F-EcoRI_Anchor_P and R-BamHI_comEA_217. The PCR products were cloned into the EcoR I/BamH I sites of the plasmid pBR-Tet_MCSI . To determine the transcription start point of comEA, fifteen of those plasmids were sequenced. 12 out of those 15 sequences pointed to the C at position −24 bp upstream the comEA start codon as transcriptional start site.
Regulation of comEA by QstR and TfoX/CRP-cAMP
Next, we investigated the production of the ComEA protein in order to evaluate whether the transcript levels would also reflect the protein levels. Apart from the strains used to measure the comEA transcripts, we also included wild-type strains harboring an inducible copy of tfoX on the chromosome as previously described  and grew the strains under both competence-inducing and non-inducing conditions. Total protein extracts were prepared from bacterial strains that had reached the high cell density state. As shown in Figure 2B, the ComEA protein was readily detectable in the competence-induced strain (lane 2) and absent in the same strain that was grown without competence induction (lane 1). Moreover, we detected low levels of ComEA in the wild-type strain when qstR was overexpressed but competence (e.g., tfoX) was not induced (lane 4), which was not the case for the vector control (lane 3). This result strengthened the evidence that QstR per se is able to drive the expression of comEA in the absence of competence induction. However, the protein was more abundant in the wild-type strain under competence-inducing conditions, suggesting that full comEA expression requires more than the QstR protein alone, which is consistent with the proposed dual regulation of comEA by TfoX / CRP-cAMP complex and QstR (Figure 1).
Narrowing down the promoter region driving the expression of comEA
Prediction of putative promoter elements within the comEA upstream region
Investigation of the putative CRP-S site upstream of comEA
The CRP-S site upstream of qstR
In bacteria, gene transcription begins only after 1) binding of the RNA polymerase (RNAP) holoenzyme to the promoter region and 2) formation of an open complex of the DNA. Promoters that are non-constitutively active (e.g., those of the competence genes) require one or several activating protein(s), such as the CRP protein, that directly interact with the RNAP and assist the holoenzyme in the steps preceding transcriptional initiation . Using comEA and qstR as important genes of the competence regulon of V. cholerae, this study contributes to the understanding of the regulatory network driving natural competence. For comEA we showed that the region up to 134 bp upstream of the start codon is sufficient to drive comEA expression in this organism. But what initiates transcription of comEA? Trying to answer this question, we followed a common assumption, namely that the expression of the competence genes of V. cholerae is TfoX- and CRP-cAMP-dependent and that competence genes are preceeded by so-called CRP-S sites as previously suggested for H. influenzae,. And indeed, we demonstrated that the deletion of a newly identified motif with striking resemblance to the in silico predicted CRP-S sites  had a negative effect on natural transformability. Moreover, we also identified and investigated a putative CRP-S site upstream qstR, which likewise was required for qstR expression and natural transformability. Interestingly, our site-directed mutagenesis approach resulted in unexpected but interesting phenotypes. CRP-mediated activation at the CRP-S site is expected to be different from that at the canonical CRP binding site (CRP-N site; Sxy/TfoX-independent). We therefore assumed that exchanging the putative CRP-S site for a CRP-N motif would enhance competence gene expression. Notably, and in contrast to this assumption, the expression of comEA and qstR was significantly reduced when preceeded by a CRP-N site (Figures 5 and 6). Moreover, opposite results were observed for the strains carrying the CRP-0 motif variants. Despite the fact that this mutation affects the most highly conserved bases of the in silico predicted CRP-S consensus (those in the 3′ part of the motif), the comEA/qstR genes were expressed at higher levels, which correlated well with increased levels of ComEA protein, enhanced DNA uptake, and higher transformation frequencies (Figures 5 and 6). We therefore speculate that the CRP-0 mutation could either be a better binding site for the RNAP or favor the escape of the RNAP complex from the promoter in order to begin transcription. However, based on these unexpected phenotypes we concluded that the identified motifs indeed play a role in driving the expression of qstR and comEA but that they do not qualify as bona fide CRP-S sites.
Upon visual inspection we did not identify other motifs resembling the CRP-S site within the putative promoter region of comEA. A sequence (TGCGA-N6-AAGCA) centered at −115.5 from the transcription start point and located between −147 bp and −132 bp upstream the comEA start codon, was recently discussed (though never experimentally addressed) by Antonova et al. The authors suggested that this sequence serves as a potential CRE element (competence regulatory element ; the former name for CRP-S sites) . Although we cannot exclude that this sequence is indeed a CRP binding motif, this site is not essential for the transcription of comEA, as it was not part of the construct that in trans complemented the respective knockout strain (e.g. comEA preceded by 134 bp of its upstream region; Figure 3). Furthermore, the localization of this putative CRP-S site within the open reading frame of the adjacent gene (VC1918) also leads to questions regarding its functionality with respect to the regulation of comEA.
Given the absence of any obvious alternative CRP-S site within the 134 bp upstream of comEA we raised the possibility that TfoX and CRP-cAMP only indirectly regulate comEA via the intermediate transcriptional regulator QstR. Indeed, we have previously shown that QstR is necessary for the expression of comEA and comEC but not of the DNA-uptake pilus-encoding genes ,. However, upon artificial induction of qstR in trans only low levels of comEA transcript were measured in accordance with the production of low levels of the ComEA protein (Figure 2). Two hypothesis are therefore possible: either the proteins TfoX and/or CRP-cAMP are somehow involved in the production of the previously suggested co-factor of QstR , or a direct regulation of comEA by TfoX/CRP-cAMP still occurs but involves a CRP-S binding site that significantly differs form the consensus that was in silico predicted for the Vibrionaceae. Further studies involving chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) will be required to ultimately establish the in vivo binding sites of CRP-cAMP, QstR, and potentially also of TfoX in naturally competent V. cholerae cells.
We are grateful for technical assistance by Tiziana Scrignari and to all of the members of the Blokesch laboratory for discussion of the project. We also acknowledge useful comments from the participants of the Cold Spring Harbor Meeting on Bacteria, Archaea & Phages in August 2012 where we first presented our work on the putative CRP-S site upstream of comEA. This work was supported by the Swiss National Science Foundation (SNSF) grants 31003A_127029 and 31003A_143356.
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