Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO

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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“…However, the catabolite control of zwf was relaxed in the CF strain, FRD1, and its derivatives. This was a phenotype much like a crc mutant (46).…”

Section: Resultsmentioning

confidence: 99%

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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“…rather than sugars. This phenomenon is known as 'reverse carbon catabolite repression' [3]. One of the major reasons for the inefficiency of pseudomonads in the process of bioremediation, despite their metabolic versatility, may be due to carbon catabolite repression, which is poorly understood [4].…”

Section: Introductionmentioning

confidence: 99%

Periplasmic glucose‐binding protein from Pseudomonas putida CSV86 – identification of the glucose‐binding pocket by homology‐model‐guided site‐specific mutagenesis

2013

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Glucose transport in Pseudomonas putida CSV86 is mediated via a periplasmic glucose-binding protein (GBP)-dependent putative glucose ABC transporter. Here we describe a homology model and functional characterization of GBP from CSV86 (ppGBP). A whole-cell [ 14 C]-glucose uptake study revealed that glucose is transported by the high-affinity intracellular phosphorylative pathway. ppGBP was cloned, over-expressed in Escherichia coli and purified to apparent homogeneity. The purified ppGBPs from both E. coli and CSV86 were found to be specific for glucose. A homology model of ppGBP was constructed that resembles the class II family of periplasmic binding proteins. The model showed highest structural similarity to GBP of Thermus thermophilus (ttGBP, rmsd 0.64 A). Structural analysis and molecular docking studies predicted W35, W36, E41, K92, K339 and H379 of ppGBP as putative glucose-binding residues. Alanine substitution of these residues resulted in significantly reduced [ 14 C]-glucose binding activity. Analysis of the operonic arrangement and structural comparative studies suggested that ppGBP and ttGBP probably originated from a common ancestor. Structural adaptations that inhibit binding of di-or trisaccharides at the glucose-binding pocket of ppGBP were also identified.

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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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Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO

Cited by 118 publications

References 27 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

Periplasmic glucose‐binding protein from Pseudomonas putida CSV86 – identification of the glucose‐binding pocket by homology‐model‐guided site‐specific mutagenesis

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

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