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Two-component signal transduction systems, composed of sensor kinase (SK) and response regulator (RR) proteins, allow bacterial cells to adapt to changes such as environmental flux or the presence of a host. RscS is an SK required for Vibrio fischeri to initiate a symbiotic partnership with the Hawaiian squid Euprymna scolopes, likely due to its role in controlling the symbiosis polysaccharide (syp) genes and thus biofilm formation. To determine which RR(s) functions downstream of RscS, we performed epistasis experiments with a library of 35 RR mutants. We found that several RRs contributed to RscS-mediated biofilm formation in V. fischeri. However, only the syp-encoded symbiosis regulator SypG was required for both biofilm phenotypes and syp transcription induced by RscS. These data support the hypothesis that RscS functions upstream of SypG to induce biofilm formation. In addition, this work also revealed a role for the syp-encoded RR SypE in biofilm formation. To our knowledge, no other study has used a large-scale epistasis approach to elucidate two-component signaling pathways. Therefore, this work both contributes to our understanding of regulatory pathways important for symbiotic colonization by V. fischeri and establishes a paradigm for evaluating two-component pathways in the genomics era.

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.