H-051. Serratia marcescens Biofilm Formation and Fimbriae Production Are Regulated by Intracellular Levels of cAMP

E. J. Kalivoda1, N. A. Stella1, D. M. O'Dee2, G. J. Nau2, R. M. Q. Shanks1,2;
1UPMC Eye Center, Pittsburgh, PA, 2Univ. of Pittsburgh Sch. of Med., Pittsburgh, PA.

Biofilm formation has been associated with Serratia marcescens pathogenesis. An adenylate cyclase (cyaA) homolog was identified in a transposon mutagenesis screen for genes that modulate biofilm formation. This cyaA mutant demonstrated drastic biofilm increases (>10-fold) that could be complemented by cyaA on a plasmid in trans. The catabolite repression system is known to function in the utilization of environmental carbon sources, and is mediated through intracellular levels of the cyclic nucleotide cAMP. Classically, glucose uptake inversely mediates intracellular synthesis of cAMP by negative regulation of CyaA activity. Growth of wild-type S. marcescens in glucose-rich medium or site-directed mutagenesis of other catabolite repression machinery components, EIIA-Glc (crr) and cAMP-receptor protein (crp), strongly stimulated biofilm formation, further suggesting that decreased intracellular cAMP levels positively regulate biofilm formation. An ELISA immunoassay confirmed that cyaA and crr mutants exhibited ~5-fold decreases in cAMP levels. Suppressor mutation analysis of crp revealed that hyper-biofilm phenotypes require the fimABCD operon, which is responsible for type 1 fimbriae production. Quantitative real-time PCR demonstrated that fimA transcript levels were upregulated (~4-fold) in a cyaA mutant background. TEM analysis revealed hyper-fimbriae phenotypes in the cyaA and crp mutants, which was absent in double mutants with fimC. The regulatory pathway by which the catabolite repression system influences cAMP concentrations to alter production of type 1 fimbrial adhesins establishes a novel mechanism by which bacteria control biofilm development.