Biochemical Characterization and Posttranslational Modification of Algu, a Regulator of Stress Response in Pseudomonas aeruginosa

Schurr, Michael J.; Martínez-Salazar, Jaime M.; Yu, Hongwei D.; Hibler, N S; Deretic, Vojo

doi:10.1006/bbrc.1995.2703

Cited by 35 publications

(42 citation statements)

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“…pRK2013 is the plasmid that contains the tra functions used in triparental conjugations (Figurski and Helinski, 1979). pETU-1610 is the AlgU expression clone that produced the purified AlgU as previously described (Schurr et al, 1995b). P. aeruginosa was grown in Pseudomonas isolation agar (PIA) supplemented with 300 g of carbenicillin or 300 g of tetracycline when required.…”

Section: Methodsmentioning

confidence: 99%

“…In order to examine whether AlgU directs transcription of the rpoH P 3 promoter, purified AlgU was preincubated with core RNA polymerase and added to the standard in vitro transcriptional run-off assay (Schurr et al, 1995b) with rpoH as a template. The predicted distance from P 3 initiation to the end of the truncated rpoH is 77 nucleotides and a transcript of this size was observed upon addition of AlgU to the mixture (Fig.…”

Section: S1mentioning

confidence: 99%

“…AlgU was purified from E. coli BL21(DE3) pLysS (Studier and Moffat, 1986) using the expression vector pETU-1610 as previously described (Schurr et al, 1995b). E. coli BL21(DE3) pLysS cells containing pETU-1610 were grown in LB supplemented with 30 g of chloramphenicol and 40 g of ampicillin to an OD 590 of 0.4.…”

Section: Algu Purificationmentioning

confidence: 99%

“…Biochemical evidence that algU encodes a sigma factor has been reported recently (Hershberger et al, 1995;Schurr et al, 1995b). AlgU activity is controlled at a post-translational level by the products of the downstream genes (mucABCD ) that encode its negative regulators.…”

Section: Introductionmentioning

confidence: 99%

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Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa

Schurr

Deretić

1997

Molecular Microbiology

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SummaryConversion of Pseudomonas aeruginosa to the mucoid phenotype plays a major role in the pathogenesis of respiratory infections in cystic fibrosis (CF). One mechanism responsible for mucoidy is based on mutations that inactivate the anti-factor, MucA, which normally inhibits the alternative sigma factor, AlgU. The loss of MucA allows AlgU to freely direct transcription of the genes responsible for the production of the exopolysaccharide alginate resulting in mucoid colony morphology. In Escherichia coli, a close homologue of AlgU, E , directs transcription of several genes under conditions of extreme heat shock. Here we examined whether AlgU, besides its role in controlling alginate production, affects the heat-shock response in P. aeruginosa. The P. aeruginosa rpoH gene encoding a homologue of the major heat-shock sigma factor, 32 , was found to be transcribed by AlgU containing RNA polymerase from one of its promoters (P 3 ) identified in this study. Transcription of rpoH from P 3 was elevated upon exposure to extreme heat shock in an algU-dependent manner. Importantly, the AlgU-dependent promoter of rpoH was found to be activated in mucoid mucA mutants. In keeping with this observation, introduction of a wild-type mucA gene abrogated AlgU-dependent rpoH transcription in mucoid P. aeruginosa laboratory isolates and CF isolates. These results suggest that conversion to mucoidy and the heat-shock response are co-ordinately regulated in P. aeruginosa. The simultaneous activation of both systems in mucA mutants, selected in the lungs of CF patients, may have significance for the inflammatory processes characteristic of the establishment of chronic infection and ensuing clinical deterioration in CF.

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Section: Methodsmentioning

confidence: 99%

Section: S1mentioning

confidence: 99%

Section: Algu Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa

Schurr

Deretić

1997

Molecular Microbiology

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“…Operons encoding other ECF sigmas have previously been shown to also encode regulators of the sigma-factor activity. For example, the operon encoding AlgU/T, an alternative sigma factor required for alginate biosynthesis in Pseudomonas aeruginosa (Hershberger et al, 1995;Schurr et al, 1995), includes four downstream genes, mucA-mucB/ algN-mucC-mucD, involved in regulating AlgU/T activity (Flynn and Ohman, 1988;Goldberg et al, 1993;Martin et al, 1993a,b,c). MucA inhibits AlgU/T activity in vitro Xie et al, 1996) and previous work in E. coli had identified a partial ORF immediately downstream of rpoE, termed mclA (mucA-like), that showed significant homology to mucA .…”

Section: Identification Of Three Open Reading Frames (Orfs) Downstreamentioning

confidence: 99%

The σ^E‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σ^E

Peñas

Connolly

Gross

1997

Molecular Microbiology

300

313

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SummaryThe extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, E . E activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that E activity is under the control of three genes, rseABC (

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Complementing genomics with proteomics: The membrane subproteome ofPseudomonas aeruginosa PAO1

Nouwens

Cordwell

Larsen³

et al. 2000

Electrophoresis

178

128

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With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.

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Biochemical Characterization and Posttranslational Modification of Algu, a Regulator of Stress Response in Pseudomonas aeruginosa

Cited by 35 publications

References 0 publications

Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa

Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa

The σ^E‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σ^E

Complementing genomics with proteomics: The membrane subproteome ofPseudomonas aeruginosa PAO1

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Biochemical Characterization and Posttranslational Modification of Algu, a Regulator of Stress Response in Pseudomonas aeruginosa

Cited by 35 publications

References 0 publications

Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa

Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa

The σE‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σE

Complementing genomics with proteomics: The membrane subproteome ofPseudomonas aeruginosa PAO1

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The σ^E‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σ^E