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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 Type VI secretion system genes and T6SS-mediated killing are positively regulated by CytR, TfoX, HapR, and QstR.V. cholerae C6706 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 first gene of a T6SS auxiliary cluster, vca0017 (Panel A). Bioluminescence is defined as relative light production per OD600 (RLU). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 E. coli prey were incubated with the indicated V. cholerae 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.
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pone.0138834.g003: Expression of Type VI secretion system genes and T6SS-mediated killing are positively regulated by CytR, TfoX, HapR, and QstR.V. cholerae C6706 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 first gene of a T6SS auxiliary cluster, vca0017 (Panel A). Bioluminescence is defined as relative light production per OD600 (RLU). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 E. coli prey were incubated with the indicated V. cholerae 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.

Mentions: Each T6SS reporter was introduced into the ΔluxO tfoX* strain (TfoX* CytR+ HapR* QstR+) carrying the native qstR allele, an isogenic qstR* strain that expresses QstR constitutively, and derivatives singly deleted for tfoX, cytR, hapR, and qstR. Robust expression from the vca0017-lux T6SS promoter fusion was observed in the TfoX* CytR+ HapR* QstR+ strain that expresses QstR from its native promoter (Fig 3A). Expression levels were modestly reduced in the corresponding TfoX- and CytR- strains, supporting the interpretation that CytR, like TfoX, plays a significant role in T6SS gene expression [8]. Expression was lowest in the isogenic HapR- strain, consistent with reports of at least three distinct levels of quorum sensing dependent regulation of T6SS genes: direct binding by quorum regulatory small RNAs [29] and HapR [30], and through QstR [8]. The QstR- strain unable to auto-activate was reduced relative to the parental QstR+ strain. Surprisingly, expression of the constitutive qstR* allele bypassed deletion of each of the other four regulators (Fig 3A, black bars). A similar pattern of expression with minor differences was also obtained for luciferase fusions to the other two T6SS promoters (S3 Fig). Thus, heterologous qstR expression, uncoupled from its native regulatory role, is sufficient for T6SS gene expression.


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 Type VI secretion system genes and T6SS-mediated killing are positively regulated by CytR, TfoX, HapR, and QstR.V. cholerae C6706 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 first gene of a T6SS auxiliary cluster, vca0017 (Panel A). Bioluminescence is defined as relative light production per OD600 (RLU). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 E. coli prey were incubated with the indicated V. cholerae 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581735&req=5

pone.0138834.g003: Expression of Type VI secretion system genes and T6SS-mediated killing are positively regulated by CytR, TfoX, HapR, and QstR.V. cholerae C6706 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 first gene of a T6SS auxiliary cluster, vca0017 (Panel A). Bioluminescence is defined as relative light production per OD600 (RLU). All strains are deleted for luxO and are therefore constitutive for HapR expression (*) when the hapR 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 E. coli prey were incubated with the indicated V. cholerae 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.
Mentions: Each T6SS reporter was introduced into the ΔluxO tfoX* strain (TfoX* CytR+ HapR* QstR+) carrying the native qstR allele, an isogenic qstR* strain that expresses QstR constitutively, and derivatives singly deleted for tfoX, cytR, hapR, and qstR. Robust expression from the vca0017-lux T6SS promoter fusion was observed in the TfoX* CytR+ HapR* QstR+ strain that expresses QstR from its native promoter (Fig 3A). Expression levels were modestly reduced in the corresponding TfoX- and CytR- strains, supporting the interpretation that CytR, like TfoX, plays a significant role in T6SS gene expression [8]. Expression was lowest in the isogenic HapR- strain, consistent with reports of at least three distinct levels of quorum sensing dependent regulation of T6SS genes: direct binding by quorum regulatory small RNAs [29] and HapR [30], and through QstR [8]. The QstR- strain unable to auto-activate was reduced relative to the parental QstR+ strain. Surprisingly, expression of the constitutive qstR* allele bypassed deletion of each of the other four regulators (Fig 3A, black bars). A similar pattern of expression with minor differences was also obtained for luciferase fusions to the other two T6SS promoters (S3 Fig). Thus, heterologous qstR expression, uncoupled from its native regulatory role, is sufficient for T6SS gene expression.

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