Two-Component Response Regulators ofVibrio fischeri: Identification, Mutagenesis, and Characterization

Abstract

Two-component signal transduction systems are utilized by prokaryotic and eukaryotic cells to sense and respond to environmental stimuli, both to maintain homeostasis and to rapidly adapt to changing conditions. Studies have begun to emerge that utilize a large-scale mutagenesis approach to analyzing these systems in prokaryotic organisms. Due to the recent availability of its genome sequence, such a global approach is now possible for the marine bioluminescent bacterium Vibrio fischeri, which exists either in a free-living state or as a mutualistic symbiont within a host organism such as the Hawaiian squid species Euprymna scolopes. In this work, we identified 40 putative two-component response regulators encoded within the V. fischeri genome. Based on the type of effector domain present, we classified six as NarL type, 13 as OmpR type, and six as NtrC type; the remaining 15 lacked a predicted DNA-binding domain. We subsequently mutated 35 of these genes via a vector integration approach and analyzed the resulting mutants for roles in bioluminescence, motility, and competitive colonization of squid. Through these assays, we identified three novel regulators of V. fischeri luminescence and seven regulators that altered motility. Furthermore, we found 11 regulators with a previously undescribed effect on competitive colonization of the host squid. Interestingly, five of the newly characterized regulators each affected two or more of the phenotypes examined, strongly suggesting interconnectivity among systems. This work represents the first large-scale mutagenesis of a class of genes in V. fischeri using a genomic approach and emphasizes the importance of two-component signal transduction in bacterium-host interactions.The symbiosis between the Hawaiian squid Euprymna scolopes and the bacterium Vibrio fischeri serves as a model for symbiotic bacterium-host interactions. Previous studies have revealed a number of bacterial factors required for host colonization (24, 56), including motility (25, 49, 57) and luminescence (78). Many of these factors were identified by generating and testing specific hypotheses developed from an understanding of the general colonization process. To date, however, large-scale studies of colonization factors have been hampered by a deficit of genetic tools needed for bacterial mutant construction. Recently, however, the genome sequence of V. fischeri has been published (65). In addition, the availability of a useful suicide vector (17) has greatly facilitated mutant construction (73, 90). Thus, it is now possible to approach the investigation of V. fischeri biology from a genomic perspective.

H-NOX–mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri

“…The hnoX-insertion mutant (YLW1) was constructed by plasmid integration (29), and the ΔhnoX in-frame deletion mutant (YLW37) was constructed by allelic exchange (30) (Table S1). …”

RscS Functions Upstream of SypG To Control the syp Locus and Biofilm Formation in Vibrio fischeri

“…However, many of these 12 RRs have established homologs and functions unrelated to those of RscS. For example, FlrC regulates flagellar synthesis (10,12), whereas RscS does not appear to control motility (37). Furthermore, 9 of the 12 RRs are linked to a putative SK in the genome.…”

“…Recently, we identified and disrupted 35 of 40 putative RRs in the V. fischeri genome (10). Twelve of these genes were required for competitive colonization, a phenotype expected for the RR that functions downstream of RscS.…”

“…One candidate partner already known to affect syp transcription is SypG, a syp-encoded regulator that is also required for colonization (10,43). SypG is predicted to be an RR in the NtrC-like family of 54 -dependent transcriptional activators.…”

“…At least 14 of the 40 putative RRs within the V. fischeri genome are required for efficient colonization. These include the luminescence regulator LuxO (15)(16)(17), the metabolic regulator ArcA (3), the global regulator GacA (39), the extracellular polysaccharide regulator SypG (10), and several lesscharacterized RRs (10).…”