| dc.description.abstract | Flagellinolysins are a family of novel enzymes that act both as structural flagellins and proteases. Although previously identified and shown to be both structurally integrated into filaments and proteolytically active, flagellinolysin’s biological function has remained unknown. The size of the bacterial flagellum and its prevalence in the natural world mean an enzymatic flagellin could have a significant effect in shaping the microscopic and macroscopic environments in ways that have as of yet gone undiscovered. This thesis represents a multipronged approach to characterize flagellinolysin in the marine bacterium Pseudoalteromonas tunicata, elaborate upon its context, and attempt to uncover both its role and possible substrates.
The first avenue of exploration relies upon building an understanding of flagellinolysin’s context in the natural world. This includes a phylogenetic survey of flagellinolysin’s presence across the bacterial tree of life, the identification of a likely regulatory method, and its presence within a flagellar gene cluster. Flagellinolysin is also identified as being expressed through both qrtPCR and proteomics, and shown to be integrated into mature filaments by antibody-based techniques. Flagellinolysin’s nature in P. tunicata as a metalloprotease is also affirmed here by demonstrating abolition of activity through mutation.
With this information in hand, two mutant strains were generated: a catalytically inert point mutant (E238A) and a whole gene knockout mutant (ΔFlaMP). These mutants were shown to be capable of motility and to produce full-length flagella capable of generating movement, but were found to also produce defective biofilms that produce hyperaggregated architecture, indicative of difficulties in spreading across surfaces.
Defective biofilms led to an investigation to try and identify a potential substrate and determine a mechanism for the biofilm defect phenotype. Multiple proteomics-based methods were deployed, eventually pointing to a putative surface adhesin (VCBS) as a promising candidate. This has culminated in the development of a possible model of flagellinolysin function similar to the LapG/A system and opened the possibility of an entirely new chapter in flagellar adhesion, flagellar sheaths, and biofilm regulation. | en |
