Abstract 2198 Biofilm Regulation in Vibrio fischeri: Uncovering the Structural and Functional Mechanism of SypA and SypE

Approximately 80% of all bacterial populations reside in biofilmstightly regulated communities of matrix-associated bacteria.It is estimated that over 70% of all human bacterial infections are associated with biofilms.We are studying the protein regulatory network controlling biofilm formation.To do this, we focus on two Vibrio fischeri regulatory proteins, SypA and SypE.These proteins play an essential role in the symbiotic relationship between V. fischeri and its host, the Hawaiian bobtail squid.Based on sequence analysis, SypA is a single domain anti-sigma factor antagonist (STAS) protein.Single domain STAS proteins typically bind and are phosphorylated by their cognate anti-sigma factor to regulate various cellular functions.SypE contains an anti-sigma factorlike kinase domain, but also uniquely has a phosphatase and a receiver (REC) domain, suggesting a more complex regulatory function.Here, we use a structure-function based approach to understand the molecular mechanisms of SypA and SypE.Based on the presence of a REC domain, we suspected SypE oligomerizes.Through analytical ultracentrifugation, we confirmed SypE dimerization.Computational modeling suggests SypE dimerizes through its REC domain.We speculate that dimerization may affect the regulation of SypE's kinase vs. phosphatase activity.Small angle x-ray scattering coupled with size exclusion chromatography and light scattering shows a large amount of flexibility between SypE's functional domains.This flexibility and dimerization points towards SypE having a complex regulatory mechanism.To better determine how SypA and SypE interact to regulate biofilm formation, we employed molecular dynamic simulations and affinity purification mass spectrometry.Our simulations suggest SypA has uncharacteristic flexible regions compared to homologs.This may be imperative to protein-protein interactions beyond SypE.Therefore, we are working toward identifying additional binding partners using affinity purification and mass spectrometry.Together, these findings will allow us to reconstruct the regulatory pathway controlling V. fischeri's biofilm formation.