The Vibrio fischeri quorum-sensing signal N-3-oxohexanoyl-L-homoserine lactone (3OC6-HSL) activates expression of the seven-gene luminescence operon. We used microarrays to unveil 18 additional 3OC6-HSLcontrolled genes, 3 of which had been identified by other means previously. We show most of these genes are regulated by the 3OC6-HSL-responsive transcriptional regulator LuxR directly. This demonstrates that V. fischeri quorum sensing regulates a substantial number of genes other than those involved in light production.Quorum sensing allows a species to measure its population density and control gene expression in a population densitydependent manner. Bacterial quorum sensing involves cell-cell communication mediated by extracellular signal compounds. Various species of proteobacteria use acyl-homoserine lactones (acyl-HSLs) as quorum-sensing signals (2, 12-15, 36, 42). Acyl-HSL quorum sensing was first described in the marine luminescent bacterium Vibrio fischeri, where it controls transcription of the luminescence (lux) operon (7, 10, 11). The LuxI acyl-HSL synthase and the LuxR transcriptional activator constitute the quorum-sensing system, which controls the lux operon directly. The LuxI-generated signal is N-3-oxohexanoyl-L-HSL (3OC6-HSL), and LuxR activates the lux operon in response to this signal. The LuxR-3OC6-HSL complex binds to a 20-bp inverted repeat centered at Ϫ42.5 from the transcriptional start site of the lux operon (for reviews, see references 14, 36, and 41).Vibrio fischeri occurs at low population densities in seawater and at high densities in specific light organ symbioses with certain fish and squid (3,21,29). Quorum sensing allows V. fischeri to discern its existence in the symbiosis and activate transcription of the lux operon (4). Many proteobacteria have acyl-HSL signaling systems, which serve as global regulators of extracellular factor production and are often important for successful interactions with plant or animal hosts (26,36). Existing evidence shows that besides the lux operon, LuxR and 3OC6-HSL activate five other genes (5). However, the search for quorum-controlled genes in V. fischeri has been limited to proteomic analyses. We recently reported the complete genome sequence of a strain of V. fischeri isolated from a squid light organ (30). Thus, it is now possible to use DNA microarrays to identify LuxR-regulated V. fischeri genes on a global scale. We have designed and constructed a microarray and used it to perform an analysis of 3OC6-HSL-regulated genes in V. fischeri. We furthered this analysis by examining the activity of relevant promoters in response to 3OC6-HSL and LuxR in recombinant Escherichia coli and by analyzing the binding of LuxR to promoter DNA fragments in vitro.We used E. coli DH12S (Invitrogen, Carlsbad, CA) and V. fischeri ES114 (3). The genome sequence of V. fischeri ES114 is publicly available at http://www.ergo-light.com (30). We used Luria-Bertani broth (31) without added sodium chloride to grow E. coli, at 30°C, and seawater-tryptone broth (3) with s...

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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