The sigma factor RpoS (ςS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS inPseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50°C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. TherpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoSmutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.

Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.Bacterial biofilms are communities of microorganisms residing within a polysaccharide matrix that have been imaged in dental plaques, medical prostheses, and contact lenses (7,31,33). It is well accepted that biofilms play important roles in bacterial persistence and antibiotic resistance in chronic infections, such as cystic fibrosis and otitis media (3,8,9,30). However, the existence and/or roles of biofilms in acute infections, which are defined by short time courses and high severity, have not been examined. The opportunistic gram-negative pathogen Pseudomonas aeruginosa causes both chronic and acute infections and is one of the leading causes of morbidity and mortality in thermally injured patients (27,37). In this study we examined the production of P. aeruginosa biofilms in the thermally injured mouse model of acute infections.The differentiation or maturation of P. aeruginosa biofilms in vitro depends on intercellular signaling systems or quorum sensing (QS) (5,22). QS systems in many gram-negative bacteria rely on acylated homoserine lactones (AHLs), which are produced at high levels when cell density is high and act as ligands for transcriptional regulators. The P. aeruginosa synthases LasI and RhlI synthesize two AHLs, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL) and N-butyryl-homoserine lactone (C 4 -HSL), which bind and modulate the activity of the transcriptional regulators LasR and RhlR, respectively (28). These transcriptional regulators then regulate the transcription of many genes whose products, including proteases, elastases, toxins, and hemolysins, are thought to be crucial for virulence (28). P. aeruginosa strains lacking functional QS systems are less virulent than wild-type strains (29) and form flat, undifferentiated biofilms on glass surfaces (5). These undifferentiated biofilms are less stable than the differentiated biofilms formed by wild-type P. aeruginosa as they can be easily disrupted by the detergent sodium dodecyl sulfate (5). However, the role of QS in biofilm formation has not previously been examined in vivo. Therefore, in this study we have also examined the role of QS in P. aeruginosa biofilm formation in the acute infection model. ...

Vfr of Pseudomonas aeruginosa is 91 % similar to the cAMP receptor protein (CRP) of Escherichia coli. Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P. aeruginosa as CRP did for E. coli. This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P. aeruginosa. In a vfr101 ::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain. In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101 ::aacC1 mutant. Thus, a mutation in vfr affected production of at least 60 proteins in P. aeruginosa. In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed. To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (K d ) for binding to cAMP were determined. The K d values were 16 µM for Vfr and 04 µM for CRP, suggesting that these proteins have a similar affinity for cAMP. Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E. coli. This study presents evidence that Vfr binds to the E. coli lac promoter and that this binding requires the presence of cAMP. Finally, the possible involvement of Vfr in catabolite repression control in P. aeruginosa was investigated. It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants. This implied that catabolite repression control was not affected by the vfr null mutation. In support of this, the cloned vfr gene failed to complement a mutation in the P. aeruginosa crc gene. Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P. aeruginosa, Vfr is not required for catabolite repression control in this bacterium.

The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP: co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A eutT ؉ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe 2؉ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe 2؉ . We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.The biosynthesis of AdoCbl (coenzyme B 12 ) is unique to some prokaryotes. Multiple functions (encoded by Ͼ25 genes) are required for the assembly of this complex molecule (36). The chief structural feature of AdoCbl is the presence of an adenosyl moiety liganded to the Co ion of cobalamin via a covalent Co-C bond. In the enterobacterium Salmonella enterica, corrinoid adenosylation is required for de novo synthesis and for the assimilation of incomplete precursors such as cobinamide (12). Insights into the corrinoid adenosylation pathway in S. enterica ( Fig. 1) were recently reported, including the three-dimensional crystal structure of the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme responsible for the formation of the Co-C bond (2,(13)(14)(15)35). The cobA gene in S. enterica is not part of the 17-gene cob operon (27, 33) and appears to be constitutively expressed (34). In this bacterium, CobA is the housekeeping adenosyltransferase needed for de novo biosynthesis and for the assimilation of exogenous corrinoids. However, CobA is not the only corrinoid adenosyltransferase present in S. enterica. Other large operons like the 1,2-propanediol utilization (pdu) and ethanolamine utilization (eut) operons appear to encode their own corrinoid adenosyltransferases. Johnson et al. recently reported evidence that the pduO gene of S. enterica encodes an ATP:cobalamin adenosyltransferase (19). The eutT gene of the eut operon was suggested to encode the corrinoid adenosyltransferase enzyme for this pathway, but support for this assignment was inconclusive (20). Interestingly, CobA, PduO, and EutT do not have an ancestor in common, suggesting ...