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 studies reported here identify propionyl coenzyme A (propionyl-CoA) as the common intermediate in the 1,2-propanediol and propionate catabolic pathways of Salmonella enterica serovar Typhimurium LT2. Growth on 1,2-propanediol as a carbon and energy source led to the formation and excretion of propionate, whose activation to propionyl-CoA relied on the activities of the propionate kinase (PduW)/phosphotransacetylase (Pta) enzyme system and the CobB sirtuin-controlled acetyl-CoA and propionyl-CoA (Acs, PrpE) synthetases. The different affinities of these systems for propionate ensure sufficient synthesis of propionyl-CoA to support wildtype growth of S. enterica under low or high concentrations of propionate in the environment. These redundant systems of propionyl-CoA synthesis are needed because the prpE gene encoding the propionyl-CoA synthetase enzyme is part of the prpBCDE operon under the control of the PrpR regulatory protein, which needs 2-methylcitrate as a coactivator. Because the synthesis of 2-methylcitrate by PrpC (i.e., the 2-methylcitrate synthase enzyme) requires propionyl-CoA as a substrate, the level of propionyl-CoA needs to be raised by the Acs or PduW-Pta system before 2-methylcitrate can be synthesized and prpBCDE transcription can be activated.A question of great interest to cell physiologists in the postgenomic era is how the cell integrates the large number of metabolic pathways that make up its intermediary metabolism. The wealth of genomic information and new technologies developed in recent years has attracted attention to this area of metabolism research.In the recent past, work in several laboratories has shown that the 2-methylcitric acid pathway for propionate catabolism is widely distributed among prokaryotes (5,6,11,15,(22)(23)(24)(25)39). Studies of the role of sirtuin function (encoded by the cobB gene) in propionate catabolism in Salmonella enterica serovar Typhimurium uncovered an interaction between the pathways required for the degradation of propionate and 1,2-propanediol (1,2-PDL) (41). Briefly, growth conditions were reported that restored the growth of a cobB mutant strain of S. enterica serovar Typhimurium on propionate. Key to this phenotypic correction was the induction of the genes encoding functions required for the catabolism of 1,2-PDL, i.e., the 1,2-PDL utilization (pdu) operon, which contains 21 genes (pduABCDEGHJKLMNOPQSTUVWX) (3); catabolism of 1,2-PDL was not required to correct the inability of the cobB mutant strain to grow on propionate.Studies reported here identify pduW as the gene encoding a propionate kinase enzyme that can compensate for the lack of CobB during the growth of a cobB pduW mutant strain on propionate. PduW activity is important for the synthesis of propionyl coenzyme A (Pro-CoA) during growth on 1,2-PDL. Pro-CoA is the precursor of 2-methylcitrate, the coactivator needed for transcriptional activation of the prpBCDE operon by the PrpR activator protein (23, 31, 42). The PrpB, -C, -D, and -E and aconitase enzymes (AcnA, AcnB) convert P...

The genes required for the catabolism of propionate in Salmonella enterica serovar Typhimurium are organized as two transcriptional units (prpR and prpBCDE) that are divergently transcribed from one another. Sequence homology to genes encoding members of the sigma-54 family of transcriptional activators and the identification of a consensus sigma-54 promoter 5 to the prpBCDE operon suggested that PrpR was required to activate expression of this operon. We isolated insertions in prpR and showed that prpR function was needed for growth on propionate as a carbon and energy source. A medium-copy-number plasmid carrying the lacZ gene under the control of the native sigma-54 promoter of prpBCDE was used to study prpBCDE operon expression. Transcription of the lacZ reporter in prpR, ntrA, and ihfB mutants was 85-, 83-, and 15-fold lower than the level of transcription measured in strains carrying the wild-type allele of the gene tested. These data indicated that PrpR, IHF, and transcription sigma factor RpoN were required for the expression of the prpBCDE operon. Further analysis of the involvement of the integration host factor (IHF) protein in the expression of this operon is required due to the well-documented pleiotropic effect the lack of this global regulator has on gene expression. Deletion of the 5 615-bp portion of the prpR gene resulted in a PrpR c mutant protein that activated prpBCDE transcription regardless of the ability of the strain to synthesize 2-methylcitrate, the putative coactivator of PrpR. These results indicate that the N terminus of PrpR is the coactivatorsensing domain of the protein. When placed under the control of the arabinose-inducible promoter P araBAD , expression of prpR c allele by arabinose had a strong negative effect on growth of the cell. It is proposed that this deleterious effect of PrpR c may be due to an uncontrolled ATPase activity of PrpR or to cross-activation of genes whose functions negatively affect cell growth under the conditions tested.

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The function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied in vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica. prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpRc) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpRA162T was as active as the PrpRc protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the PprpBCDE promoter.