The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the ompR gene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgD promoter region and stimulating its transcription. The csgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBA operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234 mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via rpoS -dependent transcription of the transcription regulator cpxR ; cpxR -mediated repression of csgB and csgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.

The σ ^S subunit of RNA polymerase, the product of the rpoS gene, controls the expression of genes responding to starvation and cellular stresses. Using gene array technology, we investigated rpoS -dependent expression at the onset of stationary phase in Escherichia coli grown in rich medium. Forty-one genes were expressed at significantly lower levels in an rpoS mutant derived from the MG1655 strain; for 10 of these, we also confirmed rpoS and stationary-phase dependence by reverse transcription-PCR. Only seven genes ( dps , osmE , osmY , sodC , rpsV , wrbA , and yahO ) had previously been recognized as rpoS dependent. Several newly identified rpoS -dependent genes are involved in the uptake and metabolism of amino acids, sugars, and iron. Indeed, the rpoS mutant strain shows severely impaired growth on some sugars such as fructose and N -acetylglucosamine. The rpoS gene controls the production of indole, which acts as a signal molecule in stationary-phase cells, via regulation of the tnaA -encoded tryptophanase enzyme. Genes involved in protein biosynthesis, encoding the ribosome-associated protein RpsV ( sra ) and the initiation factor IF-1 ( infA ), were also induced in an rpoS -dependent fashion. Using primer extension, we determined the promoter sequences of a selection of rpoS -regulated genes representative of different functional classes. Significant fractions of these promoters carry sequence features specific for Eσ ^S recognition of the −10 region, such as cytosines at positions −13 (70%) and −12 (30%) as well as a TG motif located upstream of the −10 region (50%), thus supporting the TGN _0-2 C(C/T)ATA(C/A)T consensus sequence recently proposed for σ ^S .

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Curli fibers, encoded by thecsgBACgenes, promote biofilm formation inEscherichia coliand other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain ofE. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from thecsgBACpromoter; however,csgBACexpression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. ThecspAandfecRgenes, encoding regulatory proteins responding to cold shock and to iron, respectively, andyoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of theyoaDgene negatively affects cell aggregation, whileyoaDinactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation offecRresults in significant increases in both cell aggregation and biofilm formation, while the effects ofcspAare not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of theyoaDgene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of thefecRgene.