서강대학교

초록

Bacterial biofilms are bacteria communities attached to living or non-living surfaces that play a crucial role in bacterial survival from various environments. Biofilms include self-produced polymers mainly composed of polysaccharides, proteins, extracellular DNAs, and lipids. Biofilm formation is divided into three major steps and finely regulated at transcriptional, translational, and post-translational levels. These steps include an initial attachment to surfaces, maturation of the extra polymeric matrix, and dispersal/detachment. Three distinct polysaccharides are involved in each step. Among them, exopolysaccharide (EPS) plays a key role in the maturation step and has been well-studied in gram-negative bacteria. EPS expressions are usually regulated at the transcriptional level in response to environmental parameters. In Vibrio vulnificus, previous studies have demonstrated that intracellular levels of c-di-GMP, the well-known bacterial signal molecule, induced the transcription of EPS-gene cluster II via a transcription factor cascade composed of BrpR and BrpT. In the presence of dicarboxylic acids, the two-component system (TCS) NtrB/C activated the transcription of another TCS DctB/D. DctD, a member of the bacterial enhancer binding protein (bEBP) family, was phosphorylated in response to directly sensing dicarboxylic acids via its cognate sensor kinase DctB, resulting in a gain of transcription activity, thus activating the transcription of EPS-gene clusters II and III through direct binding. Unlike other bEBPs that bind to only one upstream activator sequence, phosphorylated form of DctD (p-DctD) bound to two distinct upstream activator sequences designated as binding site 1 for a remote site from promoter region and binding site 2 for site close to the promoter by forming a hexamer. After binding, the two hexameric form of DctD directly interacted while simultaneously interacting with sigma factor N. Moreover, two DctD-binding sites possessed different conformation of hexameric DctD. DctD was also able to activate the transcription of its regulons in the dephosphorylated form (d-DctD) by binding with the dephosphorylated form of the glucose-specific enzyme IIA (d-IIAGlc), a member of the phosphoenolpyruvate transferase system (PTS). The binding of d-IIAGlc to d-DctD provided DNA-binding affinity and, thus, transcription activity. However, the DNA-binding characteristic of the d-IIAGlc/d-DctD complex was distinctively different from that of p-DctD. This complex only bound to the binding site 1 of p-DctD. In addition, binding of this complex required not only the binding site 1, but also the ‘downstreamly’ extended regions of binding site 1. This binding characteristic was different from that of typical bEBP. Finally, the intracellular protein stabilities of p-DctD and d-DctD seemed to be regulated by heat-shock protease HslUV. This protease appeared to degrade only d-DctD. However, in the presence of glucose, the binding of d-IIAGlc to DctD protected DctD from proteolysis by HslUV. Taken together, the bacterial enhancer binding protein DctD of Vibrio vulnificus shows distinct characteristics in regulating its regulon in either phosphorylation through TCS or binding with the PTS component. Moreover, this binding imparts not only the transcriptional activity, but also the protein stability.