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Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

ABSTRACT: The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemical basis of conversion to mucoidy. MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy. These data support a model in which a multicomponent system, encompassing an anti-delta factor and elements in the periplasmic compartment, modulates activity of AlgU. Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P. aeruginosa may be applicable to the regulation of AlgU (sigma E) equivalents in other organisms.

SUBMITTER: Schurr MJ

PROVIDER: S-EPMC178285 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

Schurr M J MJ Yu H H Martinez-Salazar J M JM Boucher J C JC Deretic V V

Journal of bacteriology 19960801 16

The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemic ...[more]

PMID: 8759866

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Project description:Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with sigma E from Escherichia coli and Salmonella typhimurium. sigma E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P1 through P5) that fall into three categories: (i) the P1 and P3 promoters, which display strong similarity to the -35 and -10 canonical sequences of sigma E promoters and were found to be absolutely dependent on AlgU; (ii) the P2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tcr cells; and (iii) the transcripts corresponding to P4 and P5, which were not affected by inactivation of algU. Introduction of E. coli rpoE (encoding sigma E) or algU into P. aeruginosa algU::Tcr strains restored P1 and P3 transcription and brought the P2 signal back to the wild-type level. The AlgU-dependent promoters P1 and P3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two-dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock. Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., sigma E in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions.

Project description:Alginate overproducition by mucoid Pseudomonas aeruginosa is a critical pathogenic determinant expressed by this organism during chronic infections in cystic fibrosis. Conversion to mucoidy and a subsequent loss of mucoid character can occur via different mutations in the algU mucA mucB gene cluster. The algU gene encodes a 22.2-kDa putative alternative sigma factor required for expression of the critical alginate biosynthetic gene algD. In this work, algU transcription was studied by S1 nuclease protection analysis. Transcription from the promoter proximal to the algU coding region was found to be dependent on AlgU. The -35 and -10 sequences of this newly mapped promoter showed strong similarity ot the promoters of two other critical alg genes: algD and algR. The proximal promoter of algR was also shown to depend on algU. Interestingly, the putative -35 and -10 regions of all three promoters displayed striking similarity to the consensus sequence of the sigma E-dependent promoters in Escherichia coli and Salmonella typhimurium. This 24-kDa sigma factor, controlling genes participating in resistance to high temperatures and oxidative stress, has been previously biochemically characterized, but the gene for sigma E remained unidentified. To examine whether AlgU is related to sigma E, the effect of algU inactivation on the sensitivity of P. aeruginosa to killing by heat and reactive oxygen intermediates was tested. Two isogenic pairs of algU+ and algU mutant strains were compared. The algU mutants, irrespective of the mucoid status of the parental strains, displayed increased sensitivity to killing by paraquat, known to generate intracellular superoxide radicals, and heat. Further lgobal homology searches revealed the presence of a previously unrecognized E. coli gene with the predicted gene product showing a striking 66% identity to AlgU. The corresponding gene from S. typhimurium was cloned and sequenced, and it is displayed one amino acid substitution relative to its E. coli equivalent. AlgU and its close homologs in E. coli and S. typhimurium may be functionally related.

Project description:Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.

Project description:Conversion to a mucoid, exopolysaccharide alginate-overproducing phenotype in Pseudomonas aeruginosa is associated with chronic respiratory infections in cystic fibrosis. Mucoidy is caused by muc mutations that derepress the alternative sigma factor AlgU, which in turn activates alginate biosynthetic and ancillary regulatory genes. Here we report the molecular characterization of two newly identified genes, algW and mucD, that affect expression of mucoidy. The algW gene, mapping at 69 min, was isolated on the basis of its ability to suppress mucoidy and reduce transcription of the alginate biosynthetic gene algD. The predicted primary structure of AlgW displayed similarity to HtrA (DegP), a serine protease involved in proteolysis of abnormal proteins and required for resistance to oxidative and heat stress in enteric bacteria. Inactivation of algW on the chromosome of the wild-type nonmucoid strain PAO1 caused increased sensitivity to heat, H2O2, and paraquat, a redox cycling compound inducing intracellular levels of superoxide. This mutation also permitted significant induction of alginate production in the presence of subinhibitory concentrations of paraquat. Two new genes, mucC and mucD, were identified immediately downstream of the previously characterized portion (algU mucA mucB) of the gene cluster at 67.5 min encoding the alternative sigma factor AlgU and its regulators. Interestingly, the predicted gene product of mucD also showed similarities to HtrA. Inactivation of mucD on the PAO1 chromosome resulted in conversion to the mucoid phenotype. The mutation in mucD also caused increased sensitivity to H2O2 and heat killing. However, in contrast to algW mutants, no increase in susceptibility to paraquat was observed in mucD mutants. These findings indicate that algW and mucD play partially overlapping but distinct roles in P. aeruginosa resistance to reactive oxygen intermediates and heat. In addition, since mutations in mucD and algW cause conversion to mucoidy or lower the threshold for its induction by reactive oxygen intermediates, these factors may repress alginate synthesis either directly by acting on AlgU or its regulators or indirectly by removing physiological signals that may activate this stress response system.

Project description:Mucoidy due to alginate overproduction by the Gram-negative bacterium Pseudomonas aeruginosa facilitates chronic lung infections in patients with cystic fibrosis (CF). We previously reported that disruption in de novo synthesis of pyrimidines resulted in conversion to a nonmucoid small-colony variant (SCV) in the mucoid P. aeruginosa strain (PAO581), which has a truncated anti-sigma factor, MucA25, that cannot sequester sigma factor AlgU (AlgT). Here, we showed that supplementation with the nitrogenous bases uracil or cytosine in growth medium complemented the SCV to normal growth, and nonmucoidy to mucoidy, in these mucA25 mutants. This conversion was associated with an increase in intracellular levels of UMP and UTP suggesting that nucleotide restoration occurred via a salvage pathway. In addition, supplemented pyrimidines caused an increase in activity of the alginate biosynthesis promoter (P algD ), but had no effect on P algU , which controls transcription of algU Cytosolic levels of AlgU were not influenced by uracil supplementation, yet levels of RpoN, a sigma factor that regulates nitrogen metabolism, increased with disruption of pyrimidine synthesis and decreased after supplementation of uracil. This suggested that an elevated level of RpoN in SCV may block alginate biosynthesis. To support this, we observed that overexpressing rpoN resulted in a phenotypic switch to nonmucoidy in PAO581 and in mucoid clinical isolates. Furthermore, transcription of an RpoN-regulated promoter increased in the mutants and decreased after uracil supplementation. These results suggest that the balance of RpoN and AlgU levels may regulate growth from SCV to mucoidy through sigma factor competition for P algD IMPORTANCE Chronic lung infections with P. aeruginosa are the main cause of morbidity and mortality in patients with cystic fibrosis. This bacterium overproduces a capsular polysaccharide called alginate (also known as mucoidy), which aids in bacterial persistence in the lungs and in resistance to therapeutic regimens and host immune responses. The current study explores a previously unknown link between pyrimidine biosynthesis and mucoidy at the level of transcriptional regulation. Identifying/characterizing this link could provide novel targets for the control of bacterial growth and mucoidy. Inhibiting mucoidy may improve antimicrobial efficacy and facilitate host defenses to clear the noncapsulated P. aeruginosa bacteria, leading to improved prognosis for patients with cystic fibrosis.

Dataset Information

Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

Publications

Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

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