Planktonic environmental microorganisms can switch to an aggregated, biofilm-associated state upon animal colonization. To understand the processes by which host-microbe interaction directs bacteria to adopt a sessile lifestyle, it is useful to study a model system in which individual stages can be dissected through genetics and imaging approaches. The light organ of the bobtail squid, Euprymna scolopes, is colonized exclusively by one bacterial species, Vibrio fischeri. Colonization is robust: it occurs within four hours via the native route and in the presence of the host immune system. Biofilm formation is required for initiating a successful colonization, and bacteria do not progress past this initial phase if they fail to aggregate in host mucus. We conducted a genome-wide screen for colonization factors in V. fischeri during squid colonization and biofilm factors were enriched among the colonization-defective mutants. Furthermore we identified a hybrid histidine kinase, BinK, that has dramatic effects on the ability of V. fischeri to aggregate and colonize squid. Here we present five projects that represent active efforts in the laboratory to pursue these results to identify principles underlying reproducible colonization: (Project 1) BinK is a strong negative regulator of biofilm aggregation in vivo. We will identify the genes regulated by the BinK histidine kinase and examine their dynamics during entry to and exit from squid colonization. (2) We have shown that BinK regulates c-di-GMP accumulation. We will dissect the role played by c-di-GMP in regulating aggregation downstream of the BinK histidine kinase. (3) BinK is a predicted to act in a phosphorelay and requires its conserved site of histidine phosphorylation to function. We will characterize the BinK phosphorelay in vitro and in vivo. (4) Through in vivo and in vitro analyses we have identified hundreds of new colonization factors and dozens of new biofilm regulators genes. We will define the genes that play the most important role in biofilm formation and in novel behaviors in the mucus, and we will characterize the signal transduction pathways that act at the squid interface. (5) We will expand from our analyses in V. fischeri to examine genes that are broadly essential for bacterial colonization. Using a combination of ortholgous gene analysis and modeling of signaling and metabolic pathways, we will use the V. fischeri model to examine genes that act broadly in bacterial colonization with a focus on hypothetical proteins.
|Effective start/end date||9/1/16 → 8/31/17|
- National Institute of General Medical Sciences (5R35GM119627-02 REVISED)