A transcriptional regulator linking quorum sensing and chitin induction to render Vibrio cholerae naturally transformable

Abstract

The human pathogen Vibrio cholerae is an aquatic bacterium associated with zooplankton and their chitinous exoskeletons. On chitinous surfaces, V. cholerae initiates a developmental programme, known as natural competence, to mediate transformation, which is a mode of horizontal gene transfer. Competence facilitates the uptake of free DNA and recombination into the bacterial genome. Recent studies have indicated that chitin surfaces are required, but not sufficient to induce competence. Two additional regulatory pathways, i.e. catabolite repression and quorum sensing (QS), are components of the regulatory network that controls natural competence in V. cholerae. In this study, we investigated the link between chitin induction and QS. We show that the major regulators of these two pathways, TfoX and HapR, are both involved in the activation of a gene encoding a transcriptional regulator of the LuxR-type family, which we named QS and TfoX-dependent regulator (QstR). We demonstrate that HapR binds the promoter of qstR in a site-specific manner, indicating a role for HapR as an activator of qstR. In addition, epistasis experiments indicate that QstR compensates for the absence of HapR. We also provide evidence that QstR is required for the proper expression of a small but essential subset of competence genes and propose a new regulatory model in which QstR links chitin-induced TfoX activity with QS.

Figure 1.

Schematic representation of the regulatory circuitry of natural competence and transformation of V. cholerae. Upon growth on chitin surfaces (or chitin-independent artifical induction), the expression of tfoX, encoding for the main regulator of transformation TfoX, occurs. Concomitantly with cAMP binding to CRP, TfoX most likely induces the expression of the competence genes, which include the genes encoding the assembly machinery and structural components of a type IV pilus (pil genes) in V. cholerae. TfoX also positively regulates chitin metabolism genes, such as those encoding chitinases (chiA-1 and chiA-2 depicted as chiA in the scheme). In this study, we provided evidence for the existence of an intermediate transcription factor downstream of TfoX, QstR, which is required for the expression of a small subset of competence genes (comEA and comEC). We showed that the expression of these genes, which are also dependent on the QS circuitry, is mediated through QstR, which itself is dependent on the main regulator of QS, HapR. QstR thus links the TfoX- and QS-dependent signalling in V. cholerae. At this point, an additional regulation of comEA/comEC by TfoX/CRP-cAMP cannot be excluded and is indicated by the grey dashed arrow. HapR is primarily produced in the presence of high levels of the CAI-1, (whereas AI-2 only plays a minor role in the production of HapR) (8), reflecting the high cell density (HCD) of the population (11). Earlier studies have demonstrated that HapR binds to the promoter sequences of the two competence-unrelated genes (aphA and hapA) (12,13), which we used as controls in this study. Here, we identified putative HapR binding sites upstream of qstR and dns (black boxes) based on the in vitro binding of HapR to these promoter regions and previous in silico predictions (grey boxes) (13).

Figure 2.

Artificial expression of comEA increases natural transformation in hapR negative strains. V. cholerae strains were tested for natural transformability through the artificial expressing of the transformation regulatory gene, tfoX, using 0.02% arabinose as inducer. Plasmid-encoded and P_BAD-driven genes were simultaneously induced. The tested strains were either A1552-TntfoX (WT-TntfoX, lane 1) or a hapR minus variant (ΔhapR-TntfoX, lanes 2–4) all harbouring various plasmids. These plasmids were either the empty vector as control (lanes 1 and 2), plasmid pBAD-hapR (lane 3) or plasmid pBAD-comEA (lane 4). The natural transformation frequencies are indicated on the y-axis. The experiments were independently repeated three times, and the error bars reflect standard deviations. <d.l.: below detection limit (average d.l. of strain ΔhapR-TntfoX was 2.9 × 10⁻⁹, as indicated with a dashed grey line). Statistically significant differences were determined using Student’s t-test. *P < 0.05; for strain ΔhapR-TntfoX, the value of the detection limit was used for statistical analysis.

Figure 3.

HapR does not bind to the comEA, comEC and pilA promoters in vitro. EMSA using the comEA (P_comEA), comEC (P_comEC) (panel A) and the pilA (P_pilA) (panel B) upstream regions as a probe did not show any bandshift. The aphA promoter was used as a positive control (P_aphA; *indicates the longer fragment used in panel B as described in the text). A total of 40 ng (panel A)/80 ng (panel B) of DNA fragments were incubated without (−) or with increasing amounts of HapR-N-Strep protein, as schematized in the figure. L: DNA ladder (representative bp are indicated on the left). Solid arrow: unbound DNA probe. Dashed arrow: bound/shifted DNA.

Figure 4.

The HapR protein binds to the dns upstream region. (Panel A) Binding of HapR to the upstream region of dns results in a shifted DNA fragment. Lanes 1–4: EMSA of the dns promoter fragment covering the region −203 to +48 bp with respect to the “ATG” start codon (corresponding to fragment C shown in panel B). The increasing amounts of HapR-N-Strep protein are depicted on the right of the image. (Panel B) Schematic representation of the DNA region surrounding the dns start codon. The tested DNA fragments (A. to K.) spanning the respective region are depicted below the scheme. The dashed line in fragments I. to K. represents unrelated and plasmid-derived DNA. All fragments were tested for HapR-N-Strep-mediated in vitro binding using EMSA, and the EMSA results are indicated in the left column. The large grey arrow depicts the dns gene (not to scale). (Panel C) HapR binding site(s) were associated with two 50 bp regions located within the dns promoter. DNA fragments (40 ng) of ∼200 bp length containing short parts of dns upstream region (−100 to −50 bp for fragment I; −50 to −1 bp for fragment J) surrounded by unrelated and plasmid-derived DNA were subjected to EMSA using increasing amounts of HapR-N-Strep, as indicated in panel A. The negative control (fragment K) did not contain any P_dns-derived DNA sequence. L: DNA ladder. Solid arrow: unbound DNA probe. Dashed arrow: bound/shifted DNA.

Figure 5.

QstR is required for the induction of comEA and comEC. qRT-PCR data showing the expression of the indicated genes relative to gyrA in the wild-type background strain A1552-TntfoX and its hapR or qstR knockout derivatives (ΔhapR-TntfoX and ΔqstR-TntfoX). All three strains were grown under competence non-inducing (TntfoX −) and competence-inducing (TntfoX+) conditions. The highlighted results (shaded boxes) are first discussed in the text and indicate that the expression of qstR is TfoX- and HapR-dependent. The data represent the averages of three biological replicates. The error bars indicate standard deviations. Statistically significant differences were determined using Student’s t-tests. *P < 0.05, **P < 0.01, n.s. = not significant.

Figure 6.

The HapR protein binds to the promoter region of qstR. (Panel A) HapR’s ability to bind to the upstream region of qstR was tested by EMSA. A DNA fragment covering the region −248 to −47 bp upstream the start codon of qstR (corresponding to fragment C shown in panel B) was used as a probe for the in vitro binding of HapR. The aphA promoter was used as a positive control. The concentration of HapR protein used in each lane is indicated above the images and schematized on the right of the figure. (Panel B) Scheme representing the DNA region, which surrounds the qstR start codon. DNA fragments (A. to I.) spanning regions upstream and within qstR are depicted below the scheme. A dashed line indicates the unrelated and plasmid-derived DNA of fragments H. and I. All fragments (A. to I.) were tested using EMSA, and the results are indicated in the left column. The large grey arrow depicts the qstR gene (not to scale). (Panel C) A HapR binding site was located within a 50 bp stretch upstream the qstR gene and site-directed mutagenesis abolished HapR’s ability to bind the qstR promoter. A total of 40 ng of DNA fragments (∼200 bp in length) containing short parts of the qstR upstream region (−150 to −120 bp for fragment H; −150 to −102 bp for fragment I; and a mutated version thereof as indicated in panel D) surrounded by plasmid-derived and therefore qstR-unrelated DNA were subjected to EMSA. Only fragment I, containing the longer qstR upstream region, bound to HapR in vitro, resulting in a bandshift. The amounts of HapR-N-Strep used were as indicated in panel A. L: DNA ladder. Solid arrow: unbound DNA probe. Dashed arrow: bound/shifted DNA. Panel D: A HapR binding motif exists within the qstR promoter region. Simplified scheme of the in silico predicted HapR binding motif 2 [with slight modification from (13); e.g. four gaps (−) were introduced in the consensus sequence to allow proper alignment with the qstR promoter sequence]. A similar sequence located upstream of qstR (−130 to −111 bp from the start codon) is illustrated in the middle row. This DNA sequence has been modified through site-directed mutagenesis (boxed residues), resulting in a mutated P_qstR sequence as depicted in the lower row. The shadings indicate highly conserved (black background), medium conserved (dark grey background) and low conserved (light grey background) bp. Non-conserved bp are not shaded. ‘W' stands for A/T.