| Bacteria cause persistent infections and serious damage to industrial systems. Understanding the structural and regulatory determinants of biofilm development is important for designing strategies to combat detrimental biofilms. The Csr system (c&barbelow;arbon s&barbelow;torage r&barbelow;egulatory system) exhibits profound effect on biofilm formation by Escherichia coli and its pathogenic relatives. The RNA binding protein CsrA represses biofilm formation, while the noncoding RNAs CsrB and CsrC activate this process by sequestering CsrA. Previous studies suggest that biofilm repression by CsrA involves an unidentified adhesin. To test this idea, a library of transposon insertion biofilm mutants was created using a simple screening assay. Analysis of the flanking regions of transposon insertions revealed 101 genes with diverse functions, many of which were not previously known to affect E. coli biofilm formation.; Mutations in the ycdSRQP locus severely impaired cell adherence to abiotic surfaces and biofilm formation under a variety of growth conditions. This locus, renamed as pgaABCD, is responsible for the synthesis of cell-bound unbranched poly-beta-1,6-N-acetyl-D-glucosamine (PGA), which is previously unknown in the gram-negative bacteria. Evidence establishes a role of PGA in intercellular adhesion and stabilization of E. coli biofilm structure. Wide distribution of the pgaABCD operon in plant and mammalian pathogens implies a potential role of PGA-like polysaccharide in biofilm formation by these organisms.; The Csr system affects E. coli biofilm formation though its influence on PGA biosynthesis. Disruption of csrA resulted in ∼3-fold increase in PGA production and ∼7-fold increase in expression of a pgaA'-'lacZ translational fusion. A csrB csrC double mutant exhibited a modest but significant decrease in pgaA'-'lacZ expression, while overexpression of CsrB or the response regulator UvrY, a transcription activator of csrB and csrC, activates pgaA'-' lacZ expression. CsrA represses pga gene expression by specifically binding to pgaA mRNA, interfering with ribosome binding and promoting the turnover of pga transcript. CsrA binds to six sites in the leader sequence of pgaA transcript. Substitution mutations in CsrA binding sites overlapping the Shine-Dalgarno sequence and pgaA initiation colon partially relieve CsrA repression, establishing a role of these sites in vivo. |
