The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product

MacGregor, C H; Arora, Shiwani K.; Hager, Paul W.; Dail, Mary Beth; Phibbs, Paul V.

doi:10.1128/jb.178.19.5627-5635.1996

Cited by 75 publications

(84 citation statements)

References 41 publications

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“…2), which was in agreement with previous studies on catabolite repression in this strain (19). Mucoid PDO300 (a mucA22 derivative of PAO1) contained approximately 1.5-fold more zwf-lacZ activity than the parent when grown on glycerol but still showed strong catabolite repression of zwf-lacZ with succinate (Fig.…”

Section: Resultssupporting

confidence: 91%

Adaptations of Pseudomonas aeruginosa to the Cystic Fibrosis Lung Environment Can Include Deregulation of zwf , Encoding Glucose-6-Phosphate Dehydrogenase

et al. 2005

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Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid Pseudomonas aeruginosa strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in zwf, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a ϳ90% reduction in alginate production in the mucoid, CF isolate, P. aeruginosa FRD1. The main regulator of alginate, sigma-22 encoded by algT (algU), plays a small but demonstrable role in the induction of zwf expression in P. aeruginosa. However, G6PDH activity and zwf expression were higher in FRD1 strains than in PAO1 strains. In PAO1, zwf expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1 zwf expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of zwf was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident P. aeruginosa strain underwent an adaptive change that deregulated zwf expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to P. aeruginosa within the lung environment. Interestingly, zwf expression in P. aeruginosa was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by P. aeruginosa can include altered regulation of basic metabolic activities, including carbon catabolism.Pseudomonas aeruginosa is an important opportunistic and nosocomial bacterial pathogen that contributes to a high rate of fatality among cystic fibrosis (CF) patients. During pathogenesis, the CF lung environment promotes and selects for multiple phenotypic and genotypic alterations in P. aeruginosa (29). The most obvious and predominant alteration selected within the CF lung is overproduction of the exopolysaccharide alginate (7). The appearance of these alginate-overproducing strains, also known as mucoid variants, correlates with the establishment of a chronic lung infection and a poor prognosis for the CF patient (8). The roles for alginate in virulence are varied and include neutralization of oxygen radicals (38), inhibition of phagocytosis (30), inhibition of antibiotic penetration (10), and inhibition of complement activation (32). Although a significant number of P. aeruginosa isolates from adult CF patients are mucoid (7), presentations of the mucoid phenotype in each isolate differ with respect to amount of alginate produced, stability of the phenotype, and growth conditions that promote alginate production. These variations imply that there are multiple factors that affect alginate production, which may be influenced by the CF lung environment. Given the correlation of alginate overproduction with pulmonary deterioration in CF patients, a better understanding of alg...

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Section: Resultssupporting

confidence: 91%

Adaptations of Pseudomonas aeruginosa to the Cystic Fibrosis Lung Environment Can Include Deregulation of zwf , Encoding Glucose-6-Phosphate Dehydrogenase

et al. 2005

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“…When Crc − mutants were grown in the presence of succinate, production of glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase, aldolase, amidase, and the enzymes involved in glucose and mannitol transport were not repressed . The deduced amino acid sequence of the Crc protein showed that it was similar to a family of DNA repair enzymes composed of apurinic\apyrimidinic endonucleases (MacGregor et al, 1996). However, MacGregor et al (1996) were not able to demonstrate in vitro nuclease or DNA-binding activity with purified Crc protein.…”

Section: Introductionmentioning

confidence: 98%

“…The deduced amino acid sequence of the Crc protein showed that it was similar to a family of DNA repair enzymes composed of apurinic\apyrimidinic endonucleases (MacGregor et al, 1996). However, MacGregor et al (1996) were not able to demonstrate in vitro nuclease or DNA-binding activity with purified Crc protein. Thus, the mechanism by which Crc controls catabolite repression in P. aeruginosa is still unclear.…”

Section: Introductionmentioning

confidence: 98%

Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa

et al. 2002

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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.

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“…In addition to the above-mentioned pathways for aromatic compounds, it regulates the expression of genes involved in the assimilation of sugars, nitrogenated compounds, and several hydrocarbons (2,8,14,15,22,45,47). Crc represses gene expression posttranscriptionally (15,27,32,47).…”

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confidence: 99%

The Target for the Pseudomonas putida Crc Global Regulator in the Benzoate Degradation Pathway Is the BenR Transcriptional Regulator

2008

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Crc protein is a global regulator involved in catabolite repression control of several pathways for the assimilation of carbon sources in pseudomonads when other preferred substrates are present. In Pseudomonas putida cells growing exponentially in a complete medium containing benzoate, Crc strongly inhibits the expression of the benzoate degradation genes. These genes are organized into several transcriptional units. We show that Crc directly inhibits the expression of the peripheral genes that transform benzoate into catechol (the ben genes) but that its effect on genes corresponding to further steps of the pathway (the cat and pca genes of the central catechol and ␤-ketoadipate pathways) is indirect, since these genes are not induced because the degradation intermediates, which act as inducers, are not produced. Crc inhibits the translation of target genes by binding to mRNA. The expression of the ben, cat, and pca genes requires the BenR, CatR, and PcaR transcriptional activators, respectively. Crc significantly reduced benABCD mRNA levels but did not affect those of benR. Crc bound to the 5 end of benR mRNA but not to equivalent regions of catR and pcaR mRNAs. A translational fusion of the benR and lacZ genes was sensitive to Crc, but a transcriptional fusion was not. We propose that Crc acts by reducing the translation of benR mRNA, decreasing BenR levels below those required for the full expression of the benABCD genes. This strategy provides great metabolic flexibility, allowing the hierarchical assimilation of different structurally related compounds that share a common central pathway by selectively regulating the entry of each substrate into the central pathway.

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The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product

Cited by 75 publications

References 41 publications

Adaptations of Pseudomonas aeruginosa to the Cystic Fibrosis Lung Environment Can Include Deregulation of zwf , Encoding Glucose-6-Phosphate Dehydrogenase

Adaptations of Pseudomonas aeruginosa to the Cystic Fibrosis Lung Environment Can Include Deregulation of zwf , Encoding Glucose-6-Phosphate Dehydrogenase

Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa

The Target for the Pseudomonas putida Crc Global Regulator in the Benzoate Degradation Pathway Is the BenR Transcriptional Regulator

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