| Animal epithelial tissue becomes reproducibly colonized by specific environmental bacteria. The bacteria (microbiota) perform critical functions for the host's tissue development, immune system development, and nutrition; yet the processes by which bacterial diversity in the environment is selected to assemble the correct communities in the host are unclear. To understand the molecular determinants of microbiota selection, I examined colonization of a simplified model in which the light organ of Euprymna scolopes squid forms an exclusive association with Vibrio fischeri bacteria. I applied high-throughput insertion sequencing (INSeq) to identify which bacterial genes are required during host colonization. A library of over 41,000 unique transposon insertions was analyzed before and after colonization of 1,500 squid hatchlings. Mutants that were reproducibly depleted following squid colonization represented 380 genes, including 37 that encode for known colonization determinants.;Validation of mutants in defined competitions against the wild-type strain revealed nine mutants that exhibited reproducible colonization defects. Some of the colonization factors identified included genes predicted to influence copper regulation and export. Other mutants exhibited defects in biofilm development, which is required for aggregation in the host mucus and initiation of colonization. Biofilm formation in culture and in vivo was abolished in a strain lacking DnaJ, suggesting an important role for protein quality control during elaboration of bacterial biofilm in the context of the intact host immune system.;The gene, binK (first described from my work, encoding biofilm inhibitor kinase BinK [VF_A0360]) exhibited a substantial colonization advantage in the global genetic screen, a phenotype that was confirmed in defined competitions. Biofilm aggregates in the host were substantially larger in strains lacking BinK, whereas overexpression of BinK suppressed biofilm formation and squid colonization. Furthermore, I present evidence that BinK functions upstream of SypG, the 54-dependent transcriptional regulator of the syp locus, required for biofilm development in the host mucus. Additional genetic studies describe novel determinants of biofilm factors, including a role for cysteine biosynthesis. Overall, these data suggest that cellular stress responses and biofilm formation are critical processes underlying the reproducible colonization of animal hosts by specific microbial symbionts. |
