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CytR Is a Global Positive Regulator of Competence, Type VI Secretion, and Chitinases in Vibrio cholerae.

Watve SS, Thomas J, Hammer BK - PLoS ONE (2015)

Bottom Line: 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 V. cholerae, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACT
The facultative pathogen Vibrio cholerae 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 V. cholerae. 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 V. cholerae, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.

No MeSH data available.


Related in: MedlinePlus

Competence genes are differentially regulated by TfoX, CytR, HapR and QstR.V. cholerae C6706 derivatives with native alleles of tfoX, cytR and qstR (not constitutively expressed, denoted by +), alleles of tfoX or qstR made constitutive by replacing the chromosomal native promoter with a ptac promoter (indicated by *), or containing in-frame deletions of tfoX, cytR, hapR and qstR (-), were analyzed for expression of bioluminescence from plasmid-encoded lux transcriptional reporter fusions. Expression profiles are shown for the transcriptional regulator qstR (Panel A) and for a member of each regulatory class: class I, comEA (Panel B) class II, pilM (Panel C) class III, pilF, (Panel D), and class IV, pilT (Panel E). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 qstR deletion was poorly transformable. The hapR deletion strain was partially restored for transformation by constitutive expression of QstR (*), but strains deleted for cytR or tfoX were not restored for competence by the QstR* allele. The limit of detection is 1 x 10−8 cfu. mL-1 (d.l.).
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pone.0138834.g002: Competence genes are differentially regulated by TfoX, CytR, HapR and QstR.V. cholerae C6706 derivatives with native alleles of tfoX, cytR and qstR (not constitutively expressed, denoted by +), alleles of tfoX or qstR made constitutive by replacing the chromosomal native promoter with a ptac promoter (indicated by *), or containing in-frame deletions of tfoX, cytR, hapR and qstR (-), were analyzed for expression of bioluminescence from plasmid-encoded lux transcriptional reporter fusions. Expression profiles are shown for the transcriptional regulator qstR (Panel A) and for a member of each regulatory class: class I, comEA (Panel B) class II, pilM (Panel C) class III, pilF, (Panel D), and class IV, pilT (Panel E). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 qstR deletion was poorly transformable. The hapR deletion strain was partially restored for transformation by constitutive expression of QstR (*), but strains deleted for cytR or tfoX were not restored for competence by the QstR* allele. The limit of detection is 1 x 10−8 cfu. mL-1 (d.l.).

Mentions: RNA-seq analyses revealed that both TfoX and CytR positively control the majority of competence genes, (Fig 1), including qstR, the transcription factor shown to be directly up-regulated at high cell density by HapR [15]. To confirm and expand upon our RNA-seq observations (Fig 1), we constructed luciferase-based transcriptional fusions to the promoters of the following genes (or operons): qstR, pilM, pilE, vc0858, pilF, dprA, comF, comEC, and pilT. To uncouple native qstR expression from HapR, TfoX, and CytR control, a constitutively expressed qstR* allele was also constructed in a manner analogous to the tfoX* allele (see Materials and Methods). Expression of each reporter and of the previously published comEA-lux and pilA-lux reporters [12, 16] was measured in a V. cholerae ΔluxO tfoX* (TfoX* CytR+ HapR* QstR+) strain and in isogenic strains also carrying deletions in cytR, tfoX, hapR, or qstR (Fig 2). We find that in agreement with our RNA-seq analysis, competence gene expression falls into four distinct regulatory classes discussed in detail below.


CytR Is a Global Positive Regulator of Competence, Type VI Secretion, and Chitinases in Vibrio cholerae.

Watve SS, Thomas J, Hammer BK - PLoS ONE (2015)

Competence genes are differentially regulated by TfoX, CytR, HapR and QstR.V. cholerae C6706 derivatives with native alleles of tfoX, cytR and qstR (not constitutively expressed, denoted by +), alleles of tfoX or qstR made constitutive by replacing the chromosomal native promoter with a ptac promoter (indicated by *), or containing in-frame deletions of tfoX, cytR, hapR and qstR (-), were analyzed for expression of bioluminescence from plasmid-encoded lux transcriptional reporter fusions. Expression profiles are shown for the transcriptional regulator qstR (Panel A) and for a member of each regulatory class: class I, comEA (Panel B) class II, pilM (Panel C) class III, pilF, (Panel D), and class IV, pilT (Panel E). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 qstR deletion was poorly transformable. The hapR deletion strain was partially restored for transformation by constitutive expression of QstR (*), but strains deleted for cytR or tfoX were not restored for competence by the QstR* allele. The limit of detection is 1 x 10−8 cfu. mL-1 (d.l.).
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Related In: Results  -  Collection

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pone.0138834.g002: Competence genes are differentially regulated by TfoX, CytR, HapR and QstR.V. cholerae C6706 derivatives with native alleles of tfoX, cytR and qstR (not constitutively expressed, denoted by +), alleles of tfoX or qstR made constitutive by replacing the chromosomal native promoter with a ptac promoter (indicated by *), or containing in-frame deletions of tfoX, cytR, hapR and qstR (-), were analyzed for expression of bioluminescence from plasmid-encoded lux transcriptional reporter fusions. Expression profiles are shown for the transcriptional regulator qstR (Panel A) and for a member of each regulatory class: class I, comEA (Panel B) class II, pilM (Panel C) class III, pilF, (Panel D), and class IV, pilT (Panel E). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 qstR deletion was poorly transformable. The hapR deletion strain was partially restored for transformation by constitutive expression of QstR (*), but strains deleted for cytR or tfoX were not restored for competence by the QstR* allele. The limit of detection is 1 x 10−8 cfu. mL-1 (d.l.).
Mentions: RNA-seq analyses revealed that both TfoX and CytR positively control the majority of competence genes, (Fig 1), including qstR, the transcription factor shown to be directly up-regulated at high cell density by HapR [15]. To confirm and expand upon our RNA-seq observations (Fig 1), we constructed luciferase-based transcriptional fusions to the promoters of the following genes (or operons): qstR, pilM, pilE, vc0858, pilF, dprA, comF, comEC, and pilT. To uncouple native qstR expression from HapR, TfoX, and CytR control, a constitutively expressed qstR* allele was also constructed in a manner analogous to the tfoX* allele (see Materials and Methods). Expression of each reporter and of the previously published comEA-lux and pilA-lux reporters [12, 16] was measured in a V. cholerae ΔluxO tfoX* (TfoX* CytR+ HapR* QstR+) strain and in isogenic strains also carrying deletions in cytR, tfoX, hapR, or qstR (Fig 2). We find that in agreement with our RNA-seq analysis, competence gene expression falls into four distinct regulatory classes discussed in detail below.

Bottom Line: 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 V. cholerae, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACT
The facultative pathogen Vibrio cholerae 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 V. cholerae. 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 V. cholerae, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.

No MeSH data available.


Related in: MedlinePlus