Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.Pseudomonas aeruginosa ͉ GGDEF domain ͉ exopolysaccharide ͉ EAL domain B iofilms are surface-associated multicellular communities encased in a self-produced extracellular matrix. Existence in a biofilm provides many advantages over a planktonic (single-cell) existence, including increased resistance to predation and antimicrobial agents (1-4). Biofilms of P. aeruginosa cause chronic infections of humans with underlying predispositions (5).A number of genes involved in adherence and aggregation of P. aeruginosa, properties that are thought to be important during biofilm formation, have been identified (6-10). A mutation in one such gene, wspF, has been shown to result in increased cell aggregation and a wrinkled colony morphology (7). wspF is part of a gene cluster predicted to encode a signal transduction system similar to that which regulates swimming-mediated chemotaxis in bacteria (7). The WspF protein is homologous to CheB, a methylesterase involved in adaptation to chemotactic stimuli. Chemotaxis signal transduction systems also include response regulators called CheY. The response regulator of the Wsp chemosensory system, WspR, is a CheY homolog that contains the conserved GGDEF domain (7). These domains have been implicated in the formation of the intracellular signaling molecule cyclic diguanylate (c-diGMP) (11-15).c-diGMP was first identified in Gluconacetobacter xylinus, where it regulates production of cellulose through modulation of cellulose synthase a...

The beta-ketoadipate pathway is a chromosomally encoded convergent pathway for aromatic compound degradation that is widely distributed in soil bacteria and fungi. One branch converts protocatechuate, derived from phenolic compounds including p-cresol, 4-hydroxybenzoate and numerous lignin monomers, to beta-ketoadipate. The other branch converts catechol, generated from various aromatic hydrocarbons, amino aromatics, and lignin monomers, also to beta-ketoadipate. Two additional steps accomplish the conversion of beta-ketoadipate to tricarboxylic acid cycle intermediates. Enzyme studies and amino acid sequence data indicate that the pathway is highly conserved in diverse bacteria, including Pseudomonas putida, Acinetobacter calcoaceticus, Agrobacterium tumefaciens, Rhodococcus erythropolis, and many others. The catechol branch of the beta-ketoadipate pathway appears to be the evolutionary precursor for portions of the plasmid-borne ortho-pathways for chlorocatechol degradation. However, accumulating evidence points to an independent and convergent evolutionary origin for the eukaryotic beta-ketoadipate pathway. In the face of enzyme conservation, the beta-ketoadipate pathway exhibits many permutations in different bacterial groups with respect to enzyme distribution (isozymes, points of branch convergence), regulation (inducing metabolites, regulatory proteins), and gene organization. Diversity is also evident in the behavioral responses of different bacteria to beta-ketoadipate pathway-associated aromatic compounds. The presence and versatility of transport systems encoded by beta-ketoadipate pathway regulons is just beginning to be explored in various microbial groups. It appears that in the course of evolution, natural selection has caused the beta-ketoadipate pathway to assume a characteristic set of features or identity in different bacteria. Presumably such identities have been shaped to optimally serve the diverse lifestyles of bacteria.

SummaryHigh levels of the intracellular signalling molecule cyclic diguanylate (c-di-GMP) supress motility and activate exopolysaccharide (EPS) production in a variety of bacterial species. In many bacteria part of the effect of c-di-GMP is on gene expression, but the mechanism involved is not known for any species. We have identified the protein FleQ as a c-di-GMPresponsive transcriptional regulator in Pseudomonas aeruginosa. FleQ is known to activate expression of flagella biosynthesis genes. Here we show that it also represses transcription of genes including the pel operon involved in EPS biosynthesis, and that this repression is relieved by c-di-GMP. Our in vivo data indicate that FleQ represses pel transcription and that pel transcription is not repressed when intracellular c-di-GMP levels are high. FleN, a known antiactivator of FleQ also participates in control of pel expression. In in vitro experiments we found that FleQ binds to pel promoter DNA and that this binding is inhibited by c-di-GMP. FleQ binds radiolabelled c-di-GMP in vitro. FleQ does not have amino acid motifs that resemble previously defined c-di-GMP binding domains. Our results show that FleQ is a new type of c-di-GMP binding protein that controls the transcriptional regulation of EPS biosynthesis genes in P. aeruginosa.

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Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.Pseudomonas aeruginosa is responsible for chronic infections in the airways of cystic fibrosis (CF) patients (13). During the course of chronic infection, P. aeruginosa forms biofilms, which are thought to promote persistence by protecting the bacterium from antibiotics and host clearance. P. aeruginosa also undergoes phenotypic and genotypic diversification. A manifestation of this diversification is the appearance of colony morphology variants among CF sputum sample isolates. One clear example of this phenomenon, which has been termed "dissociative" behavior (42), is the appearance of mucoid colonies. Mucoidy is characterized by overproduction of the exopolysaccharide (EPS) alginate, a polymer of 1,4-␤-linked mannuronic acid and its epimer, guluronic acid (13). The appearance of mucoid colonies is thought to correlate with a downturn in the p...

The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO 2 . A less appreciated function of CO 2 fixation is as an electronaccepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO 2 fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with 13 C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO 2 and then fixed 68% of this CO 2 into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO 2 fixation plays a major role in cofactor recycling. When H 2 production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO 2 was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N 2 fixation and CO 2 fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO 2 fixation to recycle reduced cofactors deserves closer scrutiny.Calvin cycle | hydrogen gas | metabolic flux analysis | nitrogenase | RhodopseudomonasA t least 170 y ago, it became widely recognized that CO 2 could serve as the sole carbon source for plants and certain bacteria (autotrophic growth). Using experimental results collected by a number of scientists in the late 1700s and early 1800s, the German chemist von Liebig successfully argued that plants obtain carbon from CO 2 gas rather than from carbon in the soil (the prevailing theory at the time) (1). At this time, photosynthetic bacteria were still considered to be plants and thus were also recognized to fix CO 2 for growth (2).Nearly a century later, it was proposed that CO 2 fixation also plays a role in maintaining redox balance in the group of anoxygenic phototrophs known as purple nonsulfur bacteria (PNSB). PNSB obtain energy from light and carbon from organic substrates to support growth under anaerobic conditions (photoheterotrophic growth). In 1933, Muller reported that CO 2 had to be supplied for PNSB bacteria to grow photoheterotrophically on butyrate, a substrate more reduced than biomass (Table 1) (3). Muller proposed a hypothesis that CO 2 is needed to accept excess electrons and allow butyrate to be oxidized to the redox state of biomass ( Fig. 1) (3). This hypothesis went untested for nearly 50 y. In 1977 Hillmer and Gest demonstrated that Rhodobacter capsulatus would grow on butyrate without CO 2 if growth conditions permitted N 2 ...

SummaryIn Escherichia coli and some other g g g g -Proteobacteria, the alternative s s s s factor RpoS functions as a regulator of the general stress response. The role of RpoS in Pseudomonas aeruginosa is not clear. Although P. aeruginosa RpoS contributes to the resistance to several environmental stresses, its role appears to be less pivotal than in E. coli . In P. aeruginosa , RpoS also regulates the production of several virulence factors and influences the expression of individual genes that are controlled by quorum sensing. Some quorum-controlled genes are induced by RpoS, whereas others are repressed. To gain insights about RpoS function in P. aeruginosa and to understand better the regulation of quorum-controlled genes, we used transcript profiling to define an RpoS regulon. We identified 772 genes regulated by RpoS in stationary but not in logarithmic growth phase (504 were induced and 268 were repressed), and we identified putative RpoS promoter sequence elements with similarity to the E. coli RpoS consensus in several of these genes. Many genes in the regulon, for example a set of chemotaxis genes, have assigned functions that are distinct from those in E. coli and are not obviously related to a stress response. Furthermore, RpoS affects the expression of more than 40% of all quorum-controlled genes identified in our previous transcriptome analysis. This highlights the significance of RpoS as a global factor that controls quorum-sensing gene expression at the onset of stationary phase. The transcription profiling results have allowed us to build a model that accommodates previous seemingly conflicting reports.

Quorum sensing is a term used to describe cell-to-cell communication that allows cell-density-dependent gene expression. Many bacteria use acyl-homoserine lactone (acyl-HSL) synthases to generate fatty acyl-HSL quorum-sensing signals, which function with signal receptors to control expression of specific genes. The fatty acyl group is derived from fatty acid biosynthesis and provides signal specificity, but the variety of signals is limited. Here we show that the photosynthetic bacterium Rhodopseudomonas palustris uses an acyl-HSL synthase to produce p-coumaroyl-HSL by using environmental p-coumaric acid rather than fatty acids from cellular pools. The bacterium has a signal receptor with homology to fatty acyl-HSL receptors that responds to p-coumaroyl-HSL to regulate global gene expression. We also found that p-coumaroyl-HSL is made by other bacteria including Bradyrhizobium sp. and Silicibacter pomeroyi. This discovery extends the range of possibilities for acyl-HSL quorum sensing and raises fundamental questions about quorum sensing within the context of environmental signalling.