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Overproduction and purification of highly active recombinant Pseudomonas aeruginosa str. PAO1 RNA polymerase holoenzyme complex.

ABSTRACT: The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is the engine of gene expression. Despite global conservation in their structures and function, RNAPs from different bacteria can have unique features in promoter and transcription factor recognition. Therefore, availability of purified RNAP from different bacteria is key to understanding these species-specific aspects and will be valuable for antibiotic drug discovery. Pseudomonas aeruginosa is one of the leading causes of hospital and community acquired infections worldwide - making the organism an important public health pathogen. We developed a method for producing high quantities of highly pure and active recombinant P. aeruginosa str. PAO1 RNAP core and holoenzyme complexes that employed two-vector systems for expressing the core enzyme (?, ?, ?', and ? subunits) and for expressing the holoenzyme complex (core + ?70). Unlike other RNAP expression approaches, we used a low temperature autoinduction system in E. coli with T7 promoters that produced high cell yields and stable protein expression. The purification strategy comprised of four chromatographic separation steps (metal chelate, heparin, and ion-exchange) with yields of up to 11?mg per 500?mL culture. Purified holoenzyme and reconstituted holoenzyme from core and ?70 were highly active at transcribing both small and large-sized DNA templates, with a determined elongation rate of ~18?nt/s for the holoenzyme. The successful purification of the P. aeruginosa RNAP provides a gateway for studies focusing on in vitro transcriptional regulation in this pathogen.

SUBMITTER: Afful D

PROVIDER: S-EPMC6688924 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Overproduction and purification of highly active recombinant Pseudomonas aeruginosa str. PAO1 RNA polymerase holoenzyme complex.

Afful Derrick D Cai Liming L Momany Cory C

Protein expression and purification 20190704

The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is the engine of gene expression. Despite global conservation in their structures and function, RNAPs from different bacteria can have unique features in promoter and transcription factor recognition. Therefore, availability of purified RNAP from different bacteria is key to understanding these species-specific aspects and will be valuable for antibiotic drug discovery. Pseudomonas aeruginosa is one of the leading cau ...[more]

PMID: 31279833

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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.

Dataset Information

Overproduction and purification of highly active recombinant Pseudomonas aeruginosa str. PAO1 RNA polymerase holoenzyme complex.

Publications

Overproduction and purification of highly active recombinant Pseudomonas aeruginosa str. PAO1 RNA polymerase holoenzyme complex.

Similar Datasets

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