Biofilm formation in Flavobacterium johnsoniae
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Abstract
Cells of Flavobacterium johnsoniae belong to the phylum Bacteroidetes, and many of the bacteria in this group crawl over a variety of surfaces in a process known as gliding motility. The exact mechanism underlying gliding motility remains unknown; however, recent studies suggest that the large protein adhesin, SprB, is propelled around the cell surface and is required for gliding on agar and glass surfaces (1). Inspection of the genome revealed the presence of eight other large proteins with conserved domains predicted to be involved in cell-to-cell adhesion (e.g. hyaline repeat domain, fibronectin-binding domain, and polycystic kidney disease domain), and we hypothesize that these proteins strengthen the contact between cells and/or mediate motility on substrates other than agar and glass. Deletion of several of these genes has not resulted in an observable motility phenotype; however, expression of these genes under the current growth conditions is unknown. Therefore, we plan to investigate the expression of these genes under various growth conditions using RT-PCR, and evaluate the effect on biofilm formation using a previously described microplate assay (2). Additionally, we plan to coat the microplates with substrates predicted to interact with the conserved domains or with substrates demonstrated to mediate adhesion in other microorganisms (e.g. fibronectin, vitronectin and laminin) (3). For gene expression studies, RNA will be extracted from cells cultured overnight and gene specific primers will be used to generate cDNA to be used as a template in PCR reactions. For biofilm assays, cells cultured overnight will be diluted 1:100 in growth medium and seeded in an uncoated microplate or a microplate coated with the appropriate substrate. Microplates will be incubated for 24 hours, washed to remove unbound cells, and stained with crystal violet. This work will be conducted during the 2015-2016 academic year by Luke Shrum and Mariah Tugel in partial fulfillment of their requirements (BIO499) to graduate with University Honors. The work is important for understanding the mechanisms underlying attachment and motility in F. johnsoniae, and the findings can be extended to these processes in other important organisms in the phylum, including the fish pathogens F. columnare and F. psychrophilum as well as the human periodontal pathogen Porphyromonas gingivalis. Additionally, this work has the potential to open new avenues of study for future undergraduates in the lab interested in characterizing the role of adhesins in the bacterial outer membrane.
