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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

Expression of V. cholerae chitinases requires TfoX and CytR, but not HapR or QstR.Panel A: V. cholerae strains with indicated alleles of tfoX, cytR, hapR and qstR (+, native;-, deletion; *, constitutively expressed), were analyzed for expression of bioluminescence from a plasmid-encoded lux transcriptional reporter fusion to the promoter of the chitinase chiA1. All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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. V. cholerae strains with indicated alleles of tfoX, cytR, hapR, and qstR 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 cytR, tfoX and the CytR-dependent chitinases chiA1, chiA2, vc0769, vca0700, a chiA1 chiA2 double mutant and a strain deleted for all four chitinases were assayed for the contribution of individual chitinase genes to chitinase activity (panel C).
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pone.0138834.g004: Expression of V. cholerae chitinases requires TfoX and CytR, but not HapR or QstR.Panel A: V. cholerae strains with indicated alleles of tfoX, cytR, hapR and qstR (+, native;-, deletion; *, constitutively expressed), were analyzed for expression of bioluminescence from a plasmid-encoded lux transcriptional reporter fusion to the promoter of the chitinase chiA1. All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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. V. cholerae strains with indicated alleles of tfoX, cytR, hapR, and qstR 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 cytR, tfoX and the CytR-dependent chitinases chiA1, chiA2, vc0769, vca0700, a chiA1 chiA2 double mutant and a strain deleted for all four chitinases were assayed for the contribution of individual chitinase genes to chitinase activity (panel C).

Mentions: V. cholerae has five genes encoding predicted chitinases that may participate in degradation of chitinous material, such as crab and shrimp shells and zooplankton molts [3, 33]. We showed previously that CytR positively regulates expression of a chiA1-lux reporter. TfoX was also identified as a critical activator of several predicted or validated chitinase genes (chiA-1, chiA-2, vc0769, and vca0700), but not the fifth predicted chitinase vc1073 [3, 4]. Consistent with these findings, robust up-regulation of each of these four chitinases (but not vc1073) was observed in the CytR+ TfoX* strain, compared to corresponding strains lacking CytR and TfoX (Fig 1B). To investigate the effect of CytR and TfoX on expression of chitinase genes, we constructed transcriptional luciferase fusions to the promoters of each chitinase and measured expression of these reporters (and of the previously constructed chiA1-lux reporter) in a ΔluxO tfoX* qstR* (TfoX* CytR+ HapR* QstR*) strain and in isogenic strains carrying deletions in cytR, tfoX, hapR or qstR. Maximal expression of each of the four chitinases occurred in the TfoX* CytR+ HapR* QstR* strain. Deletions in cytR or tfoX greatly impaired expression of each reporter, but deletion of qstR or hapR did not significantly impact chitinase expression (Fig 4A and S4 Fig), suggesting that chitinase expression, like Class II competence gene expression, is controlled by TfoX and CytR, but not HapR or QstR.


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)

Expression of V. cholerae chitinases requires TfoX and CytR, but not HapR or QstR.Panel A: V. cholerae strains with indicated alleles of tfoX, cytR, hapR and qstR (+, native;-, deletion; *, constitutively expressed), were analyzed for expression of bioluminescence from a plasmid-encoded lux transcriptional reporter fusion to the promoter of the chitinase chiA1. All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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. V. cholerae strains with indicated alleles of tfoX, cytR, hapR, and qstR 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 cytR, tfoX and the CytR-dependent chitinases chiA1, chiA2, vc0769, vca0700, a chiA1 chiA2 double mutant and a strain deleted for all four chitinases were assayed for the contribution of individual chitinase genes to chitinase activity (panel C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581735&req=5

pone.0138834.g004: Expression of V. cholerae chitinases requires TfoX and CytR, but not HapR or QstR.Panel A: V. cholerae strains with indicated alleles of tfoX, cytR, hapR and qstR (+, native;-, deletion; *, constitutively expressed), were analyzed for expression of bioluminescence from a plasmid-encoded lux transcriptional reporter fusion to the promoter of the chitinase chiA1. All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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. V. cholerae strains with indicated alleles of tfoX, cytR, hapR, and qstR 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 cytR, tfoX and the CytR-dependent chitinases chiA1, chiA2, vc0769, vca0700, a chiA1 chiA2 double mutant and a strain deleted for all four chitinases were assayed for the contribution of individual chitinase genes to chitinase activity (panel C).
Mentions: V. cholerae has five genes encoding predicted chitinases that may participate in degradation of chitinous material, such as crab and shrimp shells and zooplankton molts [3, 33]. We showed previously that CytR positively regulates expression of a chiA1-lux reporter. TfoX was also identified as a critical activator of several predicted or validated chitinase genes (chiA-1, chiA-2, vc0769, and vca0700), but not the fifth predicted chitinase vc1073 [3, 4]. Consistent with these findings, robust up-regulation of each of these four chitinases (but not vc1073) was observed in the CytR+ TfoX* strain, compared to corresponding strains lacking CytR and TfoX (Fig 1B). To investigate the effect of CytR and TfoX on expression of chitinase genes, we constructed transcriptional luciferase fusions to the promoters of each chitinase and measured expression of these reporters (and of the previously constructed chiA1-lux reporter) in a ΔluxO tfoX* qstR* (TfoX* CytR+ HapR* QstR*) strain and in isogenic strains carrying deletions in cytR, tfoX, hapR or qstR. Maximal expression of each of the four chitinases occurred in the TfoX* CytR+ HapR* QstR* strain. Deletions in cytR or tfoX greatly impaired expression of each reporter, but deletion of qstR or hapR did not significantly impact chitinase expression (Fig 4A and S4 Fig), suggesting that chitinase expression, like Class II competence gene expression, is controlled by TfoX and CytR, but not HapR or QstR.

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