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J. Bacteriol. doi:10.1128/JB.01779-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Analysis of LuxR Regulon Gene Expression during Quorum Sensing in Vibrio fischeri

Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061; Department of Microbiology, University of Hawaii, Honolulu, HI 96822; Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109

^* To whom correspondence should be addressed. Email: ams{at}vt.edu.

	Abstract

The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. 2D-SDS PAGE analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and acyl-homoserine lactone (AHL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and AHL or instead was due to indirect control, via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and AHL-dependence in recombinant Escherichia coli. The promoters for qsrP, acfA and ribB were found to be directly activated via LuxR-AHL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near -40, all three appear to have a Class-II type promoter structure. In order to more fully characterize the LuxR-regulon in V. fischeri MJ-100, real time reverse transcription PCR was used to study the temporal expression of qsrP, acfA and ribB across the exponential and stationary phases of growth, and electrophoretic mobility shift assays (EMSA) were used to compare the binding affinity of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing a LuxR regulon in V. fischeri MJ-100, the genes of which are coordinately expressed during mid-exponential growth.

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