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

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.