Comparative proteomics of biofilm development in Pseudoalteromonas tunicata discovers a distinct family of Ca ²⁺ -dependent adhesins

The marine bacterium, Pseudoalteromonas tunicata , is a useful model for studying mechanisms of biofilm development due to its ability to colonize and form biofilms on a variety of marine and eukaryotic host-associated surfaces. 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. Here, we used comparative shotgun proteomics to examine P. tunicata biofilm development throughout the planktonic phase to three stages of biofilm development at 24, 48, and 72 h. Proteomic analysis identified 232 proteins that were up-regulated during different stages of biofilm development, including many hypothetical proteins as well as proteins known to be important for P. tunicata biofilm development such as the autocidal enzyme AlpP, violacein proteins, S-layer protein SLR4, and various pili proteins. We further investigated the top identified biofilm-associated protein (Bap), a previously uncharacterized 1600 amino acid protein (EAR30327), which we designated as “BapP”. Based on AlphaFold modeling and genomic context analysis, we predicted BapP as a distinct Ca ²⁺ -dependent biofilm adhesin. Consistent with this prediction, a Δ bapP knockout mutant was defective in forming both pellicle and surface-associated biofilms, which was rescued by re-insertion of bapP into the genome. Similar to mechanisms of RTX adhesins, BapP-mediated biofilm formation was influenced by Ca ²⁺ levels, and BapP is likely exported by a type 1 secretion system. Ultimately, our work not only provides a useful proteomic dataset for studying biofilm development in an ecologically relevant organism, but it also adds to our knowledge of bacterial adhesin diversity, emphasizing Bap-like proteins as widespread determinants of biofilm formation in bacteria.