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Journal of Bacteriology, June 2007, p. 4127-4134, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01779-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Nan Qin,¹ Sean M. Callahan,² Paul V. Dunlap,³ and Ann M. Stevens^1*

Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061,¹ Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822,² Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109³

Received 22 November 2006/ Accepted 19 March 2007

The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL 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 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. 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 position –40, all three promoters 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 during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities 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 that there is a LuxR regulon in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth.

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* Corresponding author. Mailing address: Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061. Phone: (540) 231-9378. Fax: (540) 231-9307. E-mail: ams{at}vt.edu

Published ahead of print on 30 March 2007.

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Journal of Bacteriology, June 2007, p. 4127-4134, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01779-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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