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Title: Saturation mutagenesis of a CepR binding site as a means to identify new quorum-regulated promoters in Burkholderia cenocepacia

Author
item WEI, YUPING - Cornell University
item FLORES-MIRELES, ANA - Cornell University
item COSTA, ESTHER - Cornell University
item RYAN, GINA - Cornell University
item Schneider, David
item WINANS, STEPHEN - Cornell University

Submitted to: Molecular Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2010
Publication Date: 12/23/2010
Citation: Wei, Y., Flores-Mireles, A.L., Costa, E.D., Ryan, G.T., Schneider, D.J., Winans, S.C. 2010. Saturation mutagenesis of a CepR binding site as a means to identify new quorum-regulated promoters in Burkholderia cenocepacia. Molecular Microbiology. 79(3):616-632.

Interpretive Summary: Some single-cell organisms such as bacteria can coordinate their behavior by exchanging certain chemical signals through a process called "quorum sensing." The collective behavior made possible through quorum sensing is known to play a key role in response to environmental stresses in many species of bacteria. In addition, it is also often involved in the regulation of virulence-related genes in both plant and animal pathogens. This paper focuses on essential molecular mechanisms of quorum sensing in Burkholderia cenocepacia, a species of bacteria that can cause disease in both plants and animals. These mechanisms are characterized using a combination of classical laboratory genetics and a novel computational method.

Technical Abstract: Burkholderia cenocepacia, an opportunistic pathogen of humans, encodes the CepI and CepR proteins, which resemble the LuxI and LuxR quorum sensing proteins of Vibrio fischeri. CepI directs the synthesis of octanoylhomoserine lactone (OHL), while CepR is an OHL dependent transcription factor. In previous studies, we identified two direct target promoters of CepR. In the current study, we systematically dissected one of these promoters to identify nucleotides that are essential for CepR activity in vivo. We constructed 34 self complementary oligonucleotides containing altered CepR binding sites (cep boxes), and measured CepR affinity for each. These experiments allowed us to identify a consensus CepR binding site. A search of the genome for similar sequences led to the identification of many such sequences adjacent to probable promoters. To determine the subset of these promoters that are directly regulated by CepR, fifteen putative promoters were subcloned with and without intact cep boxes and fused to a reporter gene. This allowed us to identify seven new regulated promoters that were induced by OHL and that needed cep boxes for induction. CepR binding was confirmed for six of the seven promoters by electrophoretic mobility shift assays.