Discovery and characterization of novel biofilm-associated proteins in Pseudoalteromonas tunicata

Abstract

Pseudoalteromonas tunicata is a marine bacterium that is a useful model for studying mechanisms of biofilm development due to its ability to form, colonize, and inhibit growth of other microorganisms in marine and eukaryotic host-associated biofilms. However, the pathways responsible for P. tunicata biofilm formation are still incompletely understood, in part due to a lack of functional information for a large proportion of its proteome. In this thesis, I use comparative shotgun proteomics to explore P. tunicata biofilm development from the planktonic phase to three stages (early, middle, late) of biofilm development. Proteomic analysis identified 232 proteins that were up regulated during different stages of biofilm development, including proteins known to be important for P. tunicata biofilm development (e.g., autocidal enzyme AlpP, violacein proteins, and various pili proteins) as well as many hypothetical proteins of unknown function. I then characterized two novel, biofilm-associated hypothetical proteins, labeled EAR28894 and EAR30327. Functional characterization of EAR28894 revealed that it is the major S-layer protein of P. tunicata. Bioinformatic methods predicted a beta-helical structure for EAR28894 similar to the Caulobacter S-layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S-layer was found surrounding the outer membrane in wild-type cells and completely removed from cells in an EAR28894 deletion mutant. S-layer material also appeared to be “shed” from wild-type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S-layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio and found exclusively in marine metagenomes. This novel protein family was given the name Slr4. Functional investigation of the uncharacterized protein, EAR30327, revealed its function as a novel biofilm adhesin. This protein, which I designated as BapP, was the top identified biofilm-associated protein by proteomic analysis. BapP showed partial homology to outer membrane adhesins containing repeats of bacterial cadherin-like and immunoglobulin (Ig) domains. A ΔbapP mutant strain was unable to form proper pellicle biofilms in liquid media. The Δ bapP mutant also had a significantly reduced ability to form biofilms in crystal violet assays, which was rescued by re-insertion of the bapP gene into the genome. As predicted by the identification of putative Ca2+-binding motifs in BapP, biofilm formation in the wild-type strain was demonstrated to be Ca2+-dependent, which was significantly reduced in the ΔbapP mutant. This study provides a unique proteomic dataset of biofilm development and identifies BapP as a Ca2+-dependent adhesin responsible for biofilm formation in P. tunicata. The occurrence of BapP-related homologs in other species suggests that this protein family represents a broadly conserved mechanism for biofilm adhesion in marine Gammaproteobacteria species. This thesis research establishes a proteomics-based pipeline for biofilm protein discovery and new directions for biofilm research in P. tunicata and related bacteria, and offers insights into potential targets for biofilm management and control.