The focus of this work is to unravel the regulatory networks that participate in the surface-adapted lifestyle in order to gain insight into how bacteria form and maintain communities on surfaces. The hypothesis is that different gene sets are turned on and off during the development of these communities.
V. parahaemolyticus is adapted to communal life on surfaces. When grown on a surface or in a viscous layer, the organism induces a large gene system and differentiates into a swarmer cell. Differentiation to the swarmer cell allows movement over surfaces and through viscous environments. Cells recognize each other, and movement is coordinated or social in nature, resulting in complex multicellular behavior and growth in organized communities. The swarmer cell is well-equipped to move over and colonize surfaces: it is elongated and hyperflagellated. </p>
One large set, encoding the lateral flagellar motility system, is known. </p>
The first aim will be to identify genes induced by growth on surfaces; a novel transposon and delivery system has been designed to implement search for surface-induced genes. Completion of this aim will result in the identification of genes involved in the lateral flagellar hierarchy, which has not been systematically dissected, and new genes not required for motility but pertinent to the surface-adapted lifestyle. A comprehensive picture of gene activity (gene identity and timing of expression) of surface-attached bacteria will be developed; this will likely reveal the existence of interconnecting regulatory circuits to coordinate gene expression. </p>
The second aim focuses on one potential circuit that has recently been discovered. Mutations in two linked operons cause uninducible expression of swarmer cell genes. One operon (fim) encodes a fimbrial-like chaperone-usher pair and proteins that may be outer-membrane associated. The second operon (fli) encodes an ABC periplasmic-type binding protein (similar to Escherichia coli FliY) and a product that resembles a number of proteins in the GGDEF-type sensor family. The interactions between these gene sets and swarmer cell differentiation will be dissected by using a combination of mutagenesis, overexpression, and protein localization experiments. </p>
These studies should define gene activity pertinent to growth on surfaces and trace the network of regulatory circuitry that enables bacteria to establish growth and develop communities on surfaces. Understanding the pattern of surface-induced gene expression should reveal important principles about how bacteria adapt to their environment, coordinate behavior, and develop structured communities or biofilms. Moreover, this project will have significant educational impact for genetic analysis in Vibrio, specifically transposon mutagenesis and the genome-wide search for surface-controlled genes, will be used as an undergraduate and graduate level teaching tool.</p>