H-057. PGA Accumulation and Biofilm Formation in Escherichia coli: Modulation by c-di-GMP

C. C. Goller1, A. Pannuri1, T. Romeo1, Y. Itoh2, K. Suzuki3;
1Emory Univ., Atlanta, GA, 2Hokkaido Univ., Sapporo, JAPAN, 3Niigata Univ., Niigata, JAPAN.

The pgaABCD operon is necessary for biosynthesis of β-1,6-N-acetyl-D-glucosamine (PGA), a polymer that promotes biofilm formation by Escherichia coli K-12 and various other eubacteria. Cyclic diguanylate (c-di-GMP) is a widespread bacterial second messenger that regulates a host of functions, including biofilm formation. We have observed that genes for certain proteins involved in synthesis (containing the GGDEF domain) or degradation (EAL domain) of c-di-GMP affect biofilm formation and PGA levels without altering expression of a pgaA’-‘lacZ fusion. We have tested a panel of single gene deletions of all GGDEF and EAL domain proteins found in E. coli to determine which proteins are most important for biofilm formation. Our studies indicate that only a few of these genes significantly affected biofilm formation in our strain. Immunoblot assays with anti-PGA were used to determine the effects of two of the mutant genes on accumulation of PGA and we plan to test additional mutations. Plasmidic expression of the gene for the GGDEF protein YdeH was found to increase both biofilm formation and PGA accumulation, while the gene for the EAL protein YhjH decreased biofilm and PGA levels. We aim to determine how c-di-GMP affects PGA synthesis. Our working model is one in which c-di-GMP acts allosterically to enhance activity of the enzyme responsible for synthesis of PGA, PgaC. To pursue this hypothesis, we have adapted an in vitro assay to measure incorporation of [14C] UDP-GlcNAc into PGA using crude membrane extracts. The goal is to reconstitute PGA synthesis and c-di-GMP stimulation using this approach and to identify target molecules for c-di-GMP-mediated stimulation of PGA synthesis. Because PgaC and the other proteins of the PGA system do not appear to contain a classical c-di-GMP binding domain (e.g. PilZ domain), and the known PilZ domain proteins of E. coli do not appear to account for effects of c-di-GMP on PGA, these studies may identify a novel response system for c-di-GMP.