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In silico analysis and molecular modeling of RNA polymerase, sigma S (RpoS) protein in Pseudomonas aeruginosa PAO1.

ABSTRACT: BACKGROUND:Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (?38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection). METHODS:The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. RESULTS:The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace. CONCLUSION:Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions.

SUBMITTER: Sedighi M

PROVIDER: S-EPMC4757095 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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In silico analysis and molecular modeling of RNA polymerase, sigma S (RpoS) protein in Pseudomonas aeruginosa PAO1.

Sedighi Mansour M Moghoofei Mohsen M Kouhsari Ebrahim E Pournajaf Abazar A Emadi Behzad B Tohidfar Masoud M Gholami Mehrdad M

Reports of biochemistry & molecular biology 20151001 1

<h4>Background</h4>Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it a ...[more]

PMID: 26989748

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Project description:The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT-minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS-minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS-minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS-minus phenotypes and help explain the selection of mexT-minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.

Project description:UnlabelledAlthough genes encoding enzymes and proteins related to ethanolamine catabolism are widely distributed in the genomes of Pseudomonas spp., ethanolamine catabolism has received little attention among this metabolically versatile group of bacteria. In an attempt to shed light on this subject, this study focused on defining the key regulatory factors that govern the expression of the central ethanolamine catabolic pathway in Pseudomonas aeruginosa PAO1. This pathway is encoded by the PA4022-eat-eutBC operon and consists of a transport protein (Eat), an ethanolamine-ammonia lyase (EutBC), and an acetaldehyde dehydrogenase (PA4022). EutBC is an essential enzyme in ethanolamine catabolism because it hydrolyzes this amino alcohol into ammonia and acetaldehyde. The acetaldehyde intermediate is then converted into acetate in a reaction catalyzed by acetaldehyde dehydrogenase. Using a combination of growth analyses and β-galactosidase fusions, the enhancer-binding protein PA4021 and the sigma factor RpoN were shown to be positive regulators of the PA4022-eat-eutBC operon in P. aeruginosa PAO1. PA4021 and RpoN were required for growth on ethanolamine, and both of these regulatory proteins were essential for induction of the PA4022-eat-eutBC operon. Unexpectedly, the results indicate that acetaldehyde (and not ethanolamine) serves as the inducer molecule that is sensed by PA4021 and leads to the transcriptional activation of the PA4022-eat-eutBC operon. Due to its regulatory role in ethanolamine catabolism, PA4021 was given the name EatR. Both EatR and its target genes are conserved in several other Pseudomonas spp., suggesting that these bacteria share a mechanism for regulating ethanolamine catabolism.ImportanceThe results of this study provide a basis for understanding ethanolamine catabolism and its regulation in Pseudomonas aeruginosa PAO1. Interestingly, expression of the ethanolamine-catabolic genes in this bacterium was found to be under the control of a positive-feedback regulatory loop in a manner dependent on the transcriptional regulator PA4021, the sigma factor RpoN, and the metabolite acetaldehyde. Previously characterized regulators of ethanolamine catabolism are known to sense and respond directly to ethanolamine. In contrast, PA4021 (EatR) appears to monitor the intracellular levels of free acetaldehyde and responds through transcriptional activation of the ethanolamine-catabolic genes. This regulatory mechanism is unique and represents an alternative strategy used by bacteria to govern the acquisition of ethanolamine from their surroundings.

Dataset Information

In silico analysis and molecular modeling of RNA polymerase, sigma S (RpoS) protein in Pseudomonas aeruginosa PAO1.

Publications

In silico analysis and molecular modeling of RNA polymerase, sigma S (RpoS) protein in Pseudomonas aeruginosa PAO1.

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