The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA-lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.

Biofilm formation was repressed by glucose in several species of Enterobacteriaceae. In Escherichia coli, this effect was mediated at least in part by cyclic AMP (cAMP)-cAMP receptor protein. A temporal role for cAMP in biofilm development was indicated by the finding that glucose addition after ϳ24 h failed to repress and generally activated biofilm formation.In the natural environment, bacteria predominantly exist in matrix-enclosed, sessile communities referred to as biofilms (4). Biofilms protect cells from deleterious conditions, such as attack by the mammalian immune system (5). Biofilms are complex assemblages of cells which exhibit channels and pillars that are thought to permit the exchange of nutrients and wastes. A recent model for biofilm development proposes that it is initiated by the attachment of individual cells to a surface, followed by their migration and replication to form microcolonies that eventually produce the mature biofilm (20,22). A variety of extracellular molecules and surface organelles participate in E. coli biofilm formation (6,7,23,33).Central carbon flux and its regulation may represent key features of bacterial biofilm development. We recently reported that the RNA binding protein CsrA of Escherichia coli represses biofilm formation and activates biofilm dispersal (13). The effect of CsrA on biofilm formation is mediated largely through its regulatory role in central carbon flux and intracellular glycogen synthesis and catabolism (17,18,24,25,28,34). The influence of CsrA is substantially greater than that of other regulators of E. coli biofilm formation, OmpR, RpoS, or the Cpx two-component system (1,8,33). Studies with other species have revealed that the global regulator Crc (catabolite repression control) of Pseudomonas aeruginosa activates biofilm formation (21), and the expression of the staphylococcal biofilm polysaccharide PIA (polysaccharide intracellular adhesin) requires a functional glucose phosphoenolpyruvate:sugar phosphotransferase system (15).During studies of biofilm formation, we noted that the addition of glucose to media was inhibitory. To substantiate this observation, E. coli K-12 parental strains MG1655, MC4100, and W3110 and their isogenic csrA mutants (Table 1) were grown in microtiter wells in colony-forming antigen (CFA) medium (9) with or without glucose (0.2% wt/vol), and biofilm was quantitated after 24 h of growth using crystal violet staining (A 630 ), as described previously (13) (Fig. 1A). Essentially identical results were observed in Luria-Bertani (LB) medium (19) (data not shown). These and other biofilm experiments described in this article were performed at least in triplicate with three samples per experiment, and data were analyzed by Tukey multigroup analysis (Stat View; SAS Institute Inc., Cary, N.C.). Glucose caused a statistically significant decrease in biofilm formation in every case, which varied from ϳ30 to 95% reduction, depending primarily on the strain background but also on the medium. Biofilm formation by related clinical i...

The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression. In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P. aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P. aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid. Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes. Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2). An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1. Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P. aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain. AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557. Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant. This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb 3 -type cytochrome PA1557.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was ϳ3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 ؎ 0.85 mol of HCN/mg of protein versus 2.4 ؎ 0.40 mol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in individuals suffering from the genetic disorder cystic fibrosis. In P. aeruginosa, the transcriptional regulator AlgR controls a variety of virulence factors, including alginate production, twitching motility, biofilm formation, quorum sensing, and hydrogen cyanide (HCN) production. In this study, the regulation of HCN production was examined. Strains lacking AlgR or the putative AlgR sensor AlgZ produced significantly less HCN than did a nonmucoid isogenic parent. In contrast, algR and algZ mutants showed increased HCN production in an alginate-producing (mucoid) background. HCN production was optimal in a 5% O 2 environment. In addition, cyanide production was elevated in bacteria grown on an agar surface compared to bacteria grown in planktonic culture. A conserved AlgR phosphorylation site (aspartate at amino acid position 54), which is required for surfacedependent twitching motility but not alginate production, was found to be critical for cyanide production. Nuclease protection mapping of the hcnA promoter identified a new transcriptional start site required for HCN production. A subset of clinical isolates that lack this start site produced small amounts of cyanide. Taken together, these data show that the P. aeruginosa hcnA promoter contains three transcriptional start sites and that HCN production is regulated by AlgZ and AlgR and is maximal under microaerobic conditions when the organism is surface attached.