CytR Is a Global Positive Regulator of Competence, Type VI Secretion, and Chitinases in <i>Vibrio cholerae</i>

<div><p>The facultative pathogen <i>Vibrio cholerae</i> transitions between its human host and aquatic reservoirs where it colonizes chitinous surfaces. Growth on chitin induces expression of chitin utilization genes, genes involved in DNA uptake by natural transformation, and a type VI secretion system that allows contact-dependent killing of neighboring bacteria. We have previously shown that the transcription factor CytR, thought to primarily regulate the pyrimidine nucleoside scavenging response, is required for natural competence in <i>V</i>. <i>cholerae</i>. Through high-throughput RNA sequencing (RNA-seq), we show that CytR positively regulates the majority of competence genes, the three type VI secretion operons, and the four known or predicted chitinases. We used transcriptional reporters and phenotypic analysis to determine the individual contributions of quorum sensing, which is controlled by the transcription factors HapR and QstR; chitin utilization that is mediated by TfoX; and pyrimidine starvation that is orchestrated by CytR, toward each of these processes. We find that in <i>V</i>. <i>cholerae</i>, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.</p></div

Model for association of the relaxase, helicase, HelP, and Ssb with ICE<i>Bs1</i> DNA.

<p>Following excision from the chromosome (not shown), the double-stranded circular ICE<i>Bs1</i> DNA is nicked at <i>oriT</i> by the relaxase NicK (filled circle). By analogy to related relaxases, NicK likely becomes covalently attached to the 5′ end of the nicked DNA on the strand that is transferred during conjugation. HelP (open circles) and the host-encoded helicase PcrA (gray packman) associate with the nicked ICE<i>Bs1</i> at <i>oriT</i>. HelP facilitates processive unwinding of ICE<i>Bs1</i> by PcrA and subsequent association of the host-encoded Ssb (open triangles). Unwinding of ICE<i>Bs1</i> by PcrA and HelP is required for replication and conjugation of ICEBs1. HelP is depicted as binding to both single strands of ICE<i>Bs1</i> DNA adjacent to PcrA, although it is possible that HelP associates only with one of the two single strands.</p

Strains and plasmids.

<p>Strains and plasmids.</p

Expression of Type VI secretion system genes and T6SS-mediated killing are positively regulated by CytR, TfoX, HapR, and QstR.

<p><i>V</i>. <i>cholerae</i> C6706 with indicated alleles of <i>tfoX</i>, <i>cytR</i>, <i>hapR</i>, and <i>qstR</i> (+, native;-, deletion; *, constitutively expressed) were analyzed for expression of bioluminescence from a plasmid-encoded <i>lux</i> transcriptional reporter fusion to the promoter of first gene of a T6SS auxiliary cluster, <i>vca0017</i> (Panel A). Bioluminescence is defined as relative light production per OD₆₀₀ (RLU). All strains are deleted for <i>luxO</i> and are therefore constitutive for HapR expression (*) when the <i>hapR</i> gene is present. Data shown are mean values ± standard deviation for triplicates from one representative experiment of three performed. ‡ indicates a p-value < 0.01, † indicates a p-value <0.05. N.S. denotes not significant, calculated using a two-tailed Student’s t-test. Bars 2–5 are compared to bar 1 and bars 7–9 are compared to bar 6. Panel B: Chloramphenicol resistant <i>E</i>. <i>coli</i> prey were incubated with the indicated <i>V</i>. <i>cholerae</i> predator strains at a ratio of 1:10 on membrane filters to monitor contact-dependent killing. Total surviving prey cfus are represented in each case.</p

Competence genes are differentially regulated by TfoX, CytR, HapR and QstR.

<p><i>V</i>. <i>cholerae</i> C6706 derivatives with native alleles of <i>tfoX</i>, <i>cytR</i> and <i>qstR</i> (not constitutively expressed, denoted by +), alleles of <i>tfoX</i> or <i>qstR</i> made constitutive by replacing the chromosomal native promoter with a <i>ptac</i> promoter (indicated by *), or containing in-frame deletions of <i>tfoX</i>, <i>cytR</i>, <i>hapR</i> and <i>qstR</i> (-), were analyzed for expression of bioluminescence from plasmid-encoded <i>lux</i> transcriptional reporter fusions. Expression profiles are shown for the transcriptional regulator <i>qstR</i> (Panel A) and for a member of each regulatory class: class I, <i>comEA</i> (Panel B) class II, <i>pilM</i> (Panel C) class III, <i>pilF</i>, (Panel D), and class IV, <i>pilT</i> (Panel E). All strains are deleted for <i>luxO</i> and are therefore constitutive for HapR expression (*) when the <i>hapR</i> gene is present. Bioluminescence is represented as relative light production per OD₆₀₀ (RLU) and data shown are mean values ± standard deviation from three biological replicates of one representative experiment of three. Data are shown as mean values ± standard deviation. ‡ indicates a p-value < 0.01, † indicates a p-value <0.05. N.S. denotes not significant, calculated using a two-tailed Student’s t-test. In Panels A to E, bars 2–5 are compared to bar 1; in Panels A and B, bars 7–9 are compared to bar 6. Panel F: A TfoX* CytR⁺ HapR* QstR* strain is transformable in LB in the absence of chitin induction, but an isogenic strain carrying a <i>qstR</i> deletion was poorly transformable. The <i>hapR</i> deletion strain was partially restored for transformation by constitutive expression of QstR (*), but strains deleted for <i>cytR</i> or <i>tfoX</i> were not restored for competence by the QstR* allele. The limit of detection is 1 x 10⁻⁸ cfu. mL^-1 (d.l.).</p

UvrD permits ICE<i>Bs1</i> replication in a <i>pcrA</i>-defective strain.

<p>Induction of ICE<i>Bs1</i> gene expression was carried out by overproduction of RapI from <i>amyE</i>::{Pxyl-<i>rapI spc</i>} for two hours followed by mating as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003198#s4" target="_blank">Materials and Methods</a>. Data presented are averages from two experiments ± standard deviation. Wild type and all derivates of ICE<i>Bs1</i> contained Δ(<i>rapI</i>-<i>phrI</i>)::<i>kan</i>. All strains were also <i>thrC</i> mutants, with either <i>thrC</i>::<i>cat</i> or <i>E. coli uvrD</i> cloned and integrated into <i>thrC</i> (<i>thrC</i>::Pspank-<i>uvrD mls</i>).</p

Organization of genes for HelP, ConQ (coupling protein) and NicK homologues in ICE<i>Bs1</i> and Tn<i>916</i>.

<p>Schematic diagram showing the organization of genes encoding HelP, ConQ and NicK in ICE<i>Bs1</i> and their homologues Orf23, Orf22, Orf21, and Orf20 in Tn<i>916</i>. Most (>40) of the 72 ICEs in the ICEberg database that have <i>helP</i> homologues have this consecutive (<i>helP</i>)-<i>helP</i>-<i>conQ</i>-<i>nicK</i> configuration. Some ICEs have the same gene order but have an additional one or two genes located between the <i>helP</i> and <i>conQ</i> and/or between <i>conQ</i> and <i>nicK</i>. Not shown is a different gene organization found in ICEs related to ICE<i>6013</i> in which the <i>conQ</i> is separate from <i>helP</i> and <i>nicK</i>.</p

Comparison of HelP to Orf22 and Orf23 of Tn<i>916</i>.

<p>HelP (middle sequence), Orf22 of Tn<i>916</i> (top sequence) and Orf23 of Tn<i>916</i> (bottom sequence) were aligned using Clustal X. Markings above and below the HelP sequence indicate which amino acids are identical (asterisk), highly similar (colon) or weakly similar (period) to Tn<i>916</i> Orf22 (marks above) and Tn<i>916</i> Orf23 (marks below). Markings for the pairwise comparison of Orf22 to Orf23 are shown below the Tn<i>916</i> Orf23 sequence. Needleman-Wunsch alignment scores indicate that HelP is more similar to Orf22 and Orf23 (N-W scores = 142 and 104, respectively) than Orf22 is to Orf23 (N-W score = 31). ICE<i>Bs1</i> HelP is 126 amino acids, Tn<i>916</i> Orf22 is 128 amino acids, and Tn<i>916</i> Orf23 is 104 amino acids.</p

Phylogenic tree of HelP homologues.

<p>Using the ICEberg database and search tools (HMMER3 and WU-BLAST2) (<a href="http://db-mml.sjtu.edu.cn/ICEberg/" target="_blank">http://db-mml.sjtu.edu.cn/ICEberg/</a>), we identified 128 HelP homologues in 72 ICEs. The homologues were analyzed using CLUSTAL X and grouped into clades if they were within a distance of <0.22. The diagram is of an unrooted tree with the number of homologues in each clade shown in parentheses. Branch lengths indicate relative phylogenic distances. The width and length of branch tips indicate the number of homologues and the relative phylogenic distance between homologues in the clade, respectively. Of the seven clades, six are named for a representative ICE and its HelP homologue and one is named “Orf23-like” to reflect its close relationship to the Tn<i>916</i> Orf23 clade. Twelve homologues in the ICE<i>6013</i> Orf14 clade have the identical 106 amino acid sequence. The two homologues in the ICE<i>Bs1</i> HelP clade only differ by 5 of 126 amino acids. The two homologues in the Orf23-like clade only differ by 7 of 108 amino acids. Sixteen ICEs encode a single homologue of HelP. These sixteen homologues are from three clades - ICE<i>Bs1</i> HelP (2), ICE<i>6013</i> Orf14 (12) and Tn<i>916</i> Orf23 (2). The remaining 56 ICEs encode pairs of HelP homologues. The members of each pair of homologues fall into separate clades as exemplified by Tn<i>916</i> Orf22 (128 aa) and Tn<i>916</i> Orf23 (104 aa), and ICE<i>Sa2</i> SAPIG1865 (141 aa) and ICE<i>Sa2</i> SAPIG1866 (107 aa).</p

HelP, PcrA, and Ssb-GFP association with ICE<i>Bs1</i> DNA.

<p>ICE<i>Bs1</i> gene expression was induced by overproduction of RapI from <i>amyE</i>::{Pspank(hy)-<i>rapI spc</i>} for one hour followed by DNA-protein crosslinking with formaldehyde. Association of HelP (A, D), PcrA (B, E), and Ssb-GFP (C, F) with DNA sequences near <i>oriT</i> (A–C) and <i>conE</i> (D–F) were measured by immunoprecipitation of lysates with polyclonal anti-HelP, anti-PcrA and anti-GFP antibodies respectively followed by qPCR (ChIP-PCR), relative to <i>ydbT</i>, adjacent to ICE<i>Bs1</i>. When Ssb-GFP association was measured, strains contained the <i>lacA</i>::{PrpsF-<i>ssb-GFPm tet</i>} allele expressing GFP-tagged Ssb <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003198#pgen.1003198-Lee1" target="_blank">[6]</a>. Strains included: wild type (JMA168 for HelP and PcrA; MMB834 for Ssb-GFP), white bars; <i>ΔnicK</i> (CAL306), horizontally striped bars; <i>nicKY195F</i> (JT340), dotted bars; Δ<i>helP</i> (JT155 for HelP and PcrA; JT252 for Ssb-GFP), light grey bars; and Δ<i>helP</i>, complemented with <i>helP+</i> (JT335 for HelP and PcrA; JT398 for Ssb-GFP), dark grey bars. The c-Myc tag in the <i>nicKY195F</i> allele did not affect HelP association with <i>conE</i> which was unchanged in the NicK-Myc (JT308) control strain (421±96) compared to wild type (551±100) in three independent experiments. Error bars represent standard deviation.</p