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Decoding The Regulatory Mechanisms Of Two Quorum Sensing Proteins, Cepr And Cepr2, Of Burkholderia Cenocepacia

Author
Ryan, Gina
Abstract
Burkholderia cenocepacia is an opportunistic pathogen of humans that encodes two LuxR-type acylhomoserine (AHL) synthases and three LuxR-type AHL receptors. Of these, cepI and cepR are tightly linked and form a cognate synthase/receptor pair, as do cciI and cciR. In contrast, CepR2 is unlinked from the other four genes, and lacks a genetically linked cognate AHL synthase gene. In the first study, a CepR-binding site (cep box) was systematically altered to identify nucleotides essential for CepR activity in vivo and CepR binding in vitro. The consensus cep box determined from these experiments was used to screen the genome and identify CepR-regulated genes containing this site. Four new regulated promoters were found to be induced by OHL and required the cep box for induction and CepR binding. In the second study, the regulatory mechanism of CepR2 at two divergently transcribed genes predicted to direct the synthesis of secondary metabolites was investigated. These cepR2-linked genes were induced by OHL and required CepR2, indicating CepR2 acts as a repressor at these promoters and is antagonized by OHL. A lacZ reporter fused to the divergent promoters was used to confirm these hypotheses. Promoter resections and DNase I footprinting assays revealed a single cepR2 binding site located in the intergenic region upstream of both promoters and was required for CepR2-dependent regulation. An AraC homolog, CepS, encoded adjacent to cepR2, was found to be essential for expression of both promoters, regardless of the CepR2 status or OHL concentration. CepS therefore acts downstream of CepR2 and CepR2 appears to function as a CepS antiactivator. iii
Date Issued
2012-08-20Subject
quorum sensing; CepR2; repression; transcriptional antiactivation; CepR
Committee Chair
Winans, Stephen C
Committee Member
Helmann, John D; Stanhope, Michael J
Degree Discipline
Microbiology
Degree Name
Ph.D. of Microbiology
Degree Level
Doctor of Philosophy
Type
dissertation or thesis