Genetics of type VI secretion and natural transformation in Vibrio cholerae

The facultative waterborne pathogen Vibrio cholerae transitions between its human host and the environment where it colonizes chitinous surfaces in aquatic settings. Growth on chitin coordinates the induction of sets of genes for 1) chitin utilization; 2) a type VI secretion system that allows contact-dependent killing of neighboring bacteria; and 3) DNA uptake by natural transformation, which is a mechanism for horizontal gene transfer. This thesis describes the regulatory network controlling these behaviors in V. cholerae and the consequences of their coordinate regulation. Results from high-throughput RNA sequencing (RNA-seq) show that transcription factor CytR is one of four positive regulators comprising the chitin-induced regulatory network. A combination of genetic and phenotypic assays reveal the four regulators TfoX, HapR, QstR and CytR control each behavior in a distinct manner in a commonly used clinical reference strain of V. cholerae. Whole genome sequencing and bioinformatics analyses of a set of strains isolated from diverse sources reveal novel type VI secretion system components present in environmental, but not clinical isolates. Finally, I show that chitin-induced natural transformation can facilitate horizontal gene transfer of distinct type VI secretion system genes between strains. Horizontally acquired effector-immunity proteins are functional in the new genetic background and can be employed in antibacterial antagonism against parental cells and simultaneously protect against attacks by the donor cells. This thesis sheds light on diverse behavioral adaptations that allow this important human pathogen to spread and persist in the environment.Ph.D

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

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

Expression of <i>V</i>. <i>cholerae</i> chitinases requires TfoX and CytR, but not HapR or QstR.

<p>Panel A: <i>V</i>. <i>cholerae</i> strains 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 the chitinase <i>chiA1</i>. 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 defined as relative light production per OD₆₀₀ (RLU). ‡ 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. Panel B and C: Chitin agar plate assays. <i>V</i>. <i>cholerae</i> strains with indicated alleles of <i>tfoX</i>, <i>cytR</i>, <i>hapR</i>, and <i>qstR</i> were assayed for chitinase activity which results in a zone of clearing on LB plates containing 2% colloidal chitin (panel B). Strains constitutive for TfoX (*) and isogenic strains deleted for <i>cytR</i>, <i>tfoX</i> and the CytR-dependent chitinases <i>chiA1</i>, <i>chiA2</i>, <i>vc0769</i>, <i>vca0700</i>, a <i>chiA1 chiA2</i> double mutant and a strain deleted for all four chitinases were assayed for the contribution of individual chitinase genes to chitinase activity (panel C).</p

CytR and TfoX co-regulate natural competence, chitinase expression and the type VI secretion system.

<p>Panel A: <i>V</i>. <i>cholerae</i> C6706 is capable of natural transformation in LB medium lacking chitin if <i>tfoX</i> is constitutively expressed (TfoX*, bar 1) but not if <i>tfoX</i> is under control of its native promoter (TfoX⁺, bars 3 and 4). No transformants were detected in the absence of CytR (CytR^-, bars 2 and 4). Transformation frequency is expressed as the number of kanamycin resistant cfu mL^-1 divided by total cfu mL^-1. The limit of detection (d.l.) is 1 x 10⁻⁸. Data are shown as mean ± standard deviation from three independent biological replicates. Panel B: Heat map of genes differentially regulated by CytR in the absence (TfoX⁺, column 1) or presence (TfoX*, column 2) of TfoX induction, and genes differentially regulated by TfoX in the absence (CytR^-, column 3) or presence (CytR⁺, column 4) of a functional <i>cytR</i> gene. The majority of known competence genes are positively regulated by both TfoX and CytR and can be classified into four distinct regulatory classes (see text for details). CytR and TfoX positively regulate the three known T6SS gene clusters as well as four chitinase genes. CytR negatively regulates nucleoside uptake and catabolism genes in a TfoX-independent manner.</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