Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen of wounds, cornea, and the Cystic Fibrosis lung. P. aeruginosa is also a model organism for the study of bacterial biofilm formation. Biofilms are multicellular communities that form from bacterial growth concomitant with the production of extracellular polymeric substances (EPS). EPS includes polymers such as polysaccharides, DNA, and proteins; these polymers provide structure and protection to the biofilm cells. Proteomics experiments by the Parsek Lab and others have demonstrated that a notable component of the biofilm matrix are the secreted proteases. Secreted proteases have defined roles in virulence and nutrient acquisition, but their role in the biofilm matrix of P. aeruginosa has not been explored. I hypothesize that these secreted proteases recycle nutrients, remove cell waste, and protect cells from host immunity. To test my hypothesis, I generated a mutant strain of P. aeruginosa that lacks the six major secreted proteases. While we see that loss of the proteases does not impact planktonic growth, preliminary data suggests that loss of proteolytic activity results in moderately increased biofilm formation. Using a general proteolysis assay relying on casein hydrolysis, I have determined the relative contribution of each of the six proteases to the total proteolytic capacity of P. aeruginosa in planktonic growth. I will further test the impact of the proteases on biofilm growth in different growth environments, including under flow conditions and in artificial sputum medium. I will also assess which proteases contribute the most to proteolysis during biofilm growth. My work fits into a growing body of literature that suggests that the biofilm matrix is not an inert scaffold, but is instead a dynamic and active network.

Plainfin midshipman (Porichthys notatus) are seasonally reproducing teleost fish found along the Pacific Coast of North America. During the non-reproductive months (December-February), midshipman live offshore in deep waters. Between March and July, they migrate to the intertidal zone to reproduce. During the reproductive period, dominant (type I) males build and defend nests and hum to attract female mates. Unlike most vertebrates, courtship (humming to attract mates) and parental care (egg cleaning, nest defense) are both conducted only by type I males. These males therefore face energetic and temporal trade-offs between courtship to parental care within a single reproductive season. Our research investigates the neural mechanisms underlying the transition from courtship to paternal care in type I males during the reproductive period. We propose that this behavioral shift is regulated by steroid hormones (i.e. testosterone and estrogen) and neuropeptides (i.e. galanin). We are measuring hormone levels in blood and brain of midshipman fish across various conditions of courtship and parental care. We show the relationship between changes in steroid and neuropeptide levels in both blood and brain and changes in courtship and parental care behavior in type I males. Understanding these mechanisms in midshipman fish is crucial, as the steroid and neuropeptide pathways that regulate social behaviors share similar pathways across vertebrates. Therefore, studying this in teleost fish can provide valuable insights into the broader regulatory mechanisms of reproductive life-stage transitions in vertebrates.

Bacteria face a variety of threats, including antagonistic killing by other bacteria in competition for space and resources. In response to this antagonism, many bacteria have evolved specific defense systems. One pertinent example is the Pseudomonas aeruginosa Response to Antagonism (PARA) in P. aeruginosa, which provides defense against various antagonists by activating a suite of genes, mediated by two-component pathway Gac/Rsm, in response to kin cell lysis. The Gac/Rsm machinery is conserved across the Pseudomonas genus, but its function in defense has not been studied outside of P. aeruginosa. Here, we investigate whether two divergent Pseudomonas species, P. putida (KT2440) and P. protegens (Pf-5), similarly use Gac/Rsm in defense. To do this, we performed competitive growth assays against an antagonistic competitor, Enterobacter cloacae, comparing Gac/Rsm deletion mutants against wild-type, and quantified relative survival as an indicator of competitive fitness. Preliminary data indicate that the deletion of the core Gac/Rsm gene gacS results in dramatically decreased competitive fitness for Pf-5, but not for KT2440. This indicates that Pf-5 uses the Gac/Rsm system in a similar manner to P. aeruginosa and that, while Gac/Rsm is conserved, it may differ in function between species. To identify additional specific genes involved in defense systems, we set up a genome-wide screen. The screen indicated that genes related to the flagellum and lipopolysaccharide biosynthesis may be involved in defense against antagonism, which was surprising because these well-characterized structures have never before been implicated in defense. Work is currently underway to validate these genes as true defense factors and determine the mechanism by which they confer survival. Our findings advance the understanding of defense systems among Pseudomonas species by shedding light on their conservation and complexity, thus providing a foundation for future work on defense systems across bacterial phyla.