| The bioluminescent marine bacterium Vibrio fischeri exists as both a free-living microorganism, and as a mutualistic symbiont of the squid Euprymna scolopes. This symbiotic association is monospecific, providing for a good model system to study host-microbe interactions. Because juvenile squid must acquire symbionts upon hatching, V. fischeri cells must be able to adapt quickly from the free-living to the symbiotic state. Bacteria typically utilize two-component signal transduction systems to sense and response such environmental stimuli. Therefore, I chose to investigate the role of V. fischeri two-component signaling (TCS) systems in squid colonization.;TCS systems are comprised of two proteins: a sensor kinase (SK) and a response regulator (RR). SKs become phosphorylated upon receipt of a signal, at which point the RR partner acquires the phosphate and becomes activated to elicit a cellular response. I chose to identify all of the putative RRs in the V. fischeri genome, and target them for mutagenesis. Based upon the presence of a number of amino acid residues conserved amongst all RRs, I elucidated 40 putative V. fischeri RRs. We mutated 35 of these 40 by a vector-integration approach. I next chose to characterize the 35 RR mutants with respect their ability to compete with wild-type V. fischeri for colonization of the host squid. By this approach, I determined that 12 of the 35 RRs examined were required for competitive colonization of squid; 11 of these had not been previously identified as symbiotic determinants.;One RR mutant, lacking sypG, exhibited a severe squid colonization defect, indicating its importance in communication with the host. My subsequent analyses established that SypG serves as a sigma54 -dependent transcriptional activator of the syp cluster of genes, involved in polysaccharide synthesis and transport. Epistasis experiments determined that SypG functions downstream of at least two SK proteins, RscS and SypF, to promote V. fischeri biofilm formation. I also determined that an additional RR, SypE, functions to inhibit SypG-mediated phenotypes, possibly as a fine-tuning mechanism of syp cluster regulation. Thus, this work provides greater insight into the roles of various TCS systems in the symbiotic association V. fischeri and E. scolopes. |
