Two-component response regulators of Vibrio fischeri: their identification, mutagenesis and characterization

Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153; Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706

^* To whom correspondence should be addressed. Email: kvisick{at}lumc.edu.

	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 in either 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 6 as NarL-type, 13 as OmpR-type, and 6 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 3 novel regulators of V. fischeri luminescence and 7 regulators that altered motility. Furthermore, we found 11 regulators with a previously undescribed effect on competitive colonization of the host squid. Interestingly, 5 of the newly characterized regulators each affected two or more of the phenotypes examined, strongly suggesting inter-connectivity 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 bacterial-host interactions.