Small non-coding RNAs (sRNAs) are being increasingly recognized as critical regulators of a wide variety of processes in bacteria. To investigate the contribution of unknown sRNAs to virulence gene regulation in Vibrio cholerae, we undertook a screen to identify previously uncharacterized sRNAs under the control of the major virulence gene activator in V. cholerae, ToxT. Using a combination of direct sRNA cloning and sequencing together with a genome-wide ToxT in vitro binding assay, we identified 18 putative ToxT-regulated sRNAs. Two of these ToxT regulated sRNAs were located within the Vibrio Pathogenicity Island-1 (VPI-1), the genetic element that encodes ToxT and the Toxin Co-regulated Pilus (TCP). We verified regulation of these sRNAs by ToxT and showed that deletion of one of them, now designated tarB, caused a variable colonization phenotype when competed against the parental strain in an infant mouse model of V. cholerae infection. Infections progressing for 18 hours or less showed the ΔtarB strain was out-competed by the wild type strain, while those carried out longer, showed Δ tarB out-competing the wild type. Additionally, if inoculated from a resource poor environment the ΔtarB strain also showed decreased colonization relative to wild type. Using a bioinformatic approach, we identified that tarB-mediated regulation of the gene tcpF was primarily responsible for the tarB mutant's in vivo colonization phenotype. Further investigation of genes regulated by tarB using genome-wide transcriptional profiling of a tarB over-expressing strain revealed that tarB also directly regulates genes involved in iron and amino acid uptake. We determined that tarB has a repressive effect on many genes within the VPI-1, but has an activating effect on tcpP/tcpH, encoding regulators upstream of ToxT. Taken together, the data suggest that tarB plays an important role in regulating virulence and metabolic genes early after V. cholerae infection, but that this repressive effect on virulence genes later in infection may lead to reduced replication in vivo.
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|Commitee:||Hu, Linden, Isberg, Ralph, Malamy, Michael|
|School:||Sackler School of Graduate Biomedical Sciences (Tufts University)|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 75/10(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology|
|Keywords:||Illuimina sequencing, Small RNA, TarB, Vibrio cholerae, tcpF|
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