Glucose-specific enzyme IIA has unique binding partners in the vibrio cholerae biofilm

Glucose-specific enzyme IIA (EIIA(Glc)) is a central regulator of bacterial metabolism and an intermediate in the phosphoenolpyruvate phosphotransferase system (PTS), a conserved phosphotransfer cascade that controls carbohydrate transport. We previously reported that EIIA(Glc) activates transcription of the genes required for Vibrio cholerae biofilm formation. While EIIA(Glc) modulates the function of many proteins through a direct interaction, none of the known regulatory binding partners of EIIA(Glc) activates biofilm formation. Therefore, we used tandem affinity purification (TAP) to compare binding partners of EIIA(Glc) in both planktonic and biofilm cells. A surprising number of novel EIIA(Glc) binding partners were identified predominantly under one condition or the other. Studies of planktonic cells revealed established partners of EIIA(Glc), such as adenylate cyclase and glycerol kinase. In biofilms, MshH, a homolog of Escherichia coli CsrD, was found to be a dominant binding partner of EIIA(Glc). Further studies revealed that MshH inhibits biofilm formation. This function was independent of the Carbon storage regulator (Csr) pathway and dependent on EIIA(Glc). To explore the existence of multiprotein complexes centered on EIIA(Glc), we also affinity purified the binding partners of adenylate cyclase from biofilm cells. In addition to EIIA(Glc), this analysis yielded many of the same proteins that copurified with EIIA(Glc). We hypothesize that EIIA(Glc) serves as a hub for multiprotein complexes and furthermore that these complexes may provide a mechanism for competitive and cooperative interactions between binding partners.

EIIA(Glc) is a global regulator of microbial physiology that acts through direct interactions with other proteins. This work represents the first demonstration that the protein partners of EIIA(Glc) are distinct in the microbial biofilm. Furthermore, it provides the first evidence that EIIA(Glc) may exist in multiprotein complexes with its partners, setting the stage for an investigation of how the multiple partners of EIIA(Glc) influence one another. Last, it provides a connection between the phosphoenolpyruvate phosphotransferase (PTS) and Csr (Carbon storage regulator) regulatory systems. This work increases our understanding of the complexity of regulation by EIIA(Glc) and provides a link between the PTS and Csr networks, two global regulatory cascades that influence microbial physiology.