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P V Phibbs

Publications and source records attributed to P V Phibbs.

At least 19 recordsLinked to original sources
PubMed2001

Isolation and phenotypic characterization of Pseudomonas aeruginosa pseudorevertants containing suppressors of the catabolite repression control-defective crc-10 allele.

The amiE gene encodes an aliphatic amidase capable of converting fluoroacetamide to the toxic compound fluoroacetate and is one of many genes whose expression is subject to catabolite repression control in Pseudomonas aeruginosa. The protein product of the crc gene, Crc, is required for repression of amiE and most other genes subject to catabolite repression control in this bacterium. When grown in a carbon source such as succinate, wild-type P. aeruginosa is insensitive to fluoroacetamide (due to repression of amiE expression). In contrast, mutants harboring the crc-10 null allele cannot grow in the presence of fluoroacetamide (due to lack of repression of amiE). Selection for succinate-dependent, fluoroacetamide-resistant derivatives of the crc-10 mutant yielded three independent pseudorevertants containing suppressors that restored a degree of catabolite repression control. Synthesis of Crc protein was not reestablished in these pseudorevertants. All three suppressors of crc-10 were extragenic, and all three also suppressed a Delta crc::tetA allele. In each of the three pseudorevertants, catabolite repression control of amidase expression was restored. Catabolite repression control of mannitol dehydrogenase production was also restored in two of the three isolates. None of the suppressors restored repression of glucose-6-phosphate dehydrogenase or pyocyanin production.

Amidohydrolases↗
PubMed2000

The Pseudomonas aeruginosa devB/SOL homolog, pgl, is a member of the hex regulon and encodes 6-phosphogluconolactonase.

A cyclic version of the Entner-Doudoroff pathway is used by Pseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form the hex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOL family of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5' end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgl and propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 (hexR). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl, a structural gene of the hex regulon.

Aldehyde-Lyases↗
PubMed2000

The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa.

The transition from a planktonic (free-swimming) existence to growth attached to a surface in a biofilm occurs in response to environmental factors, including the availability of nutrients. We show that the catabolite repression control (Crc) protein, which plays a role in the regulation of carbon metabolism, is necessary for biofilm formation in Pseudomonas aeruginosa. Using phase-contrast microscopy, we found that a crc mutant only makes a dispersed monolayer of cells on a plastic surface but does not develop the dense monolayer punctuated by microcolonies typical of the wild-type strain. This is a phenotype identical to that observed in mutants defective in type IV pilus biogenesis. Consistent with this observation, crc mutants are defective in type IV pilus-mediated twitching motility. We show that this defect in type IV pilus function is due (at least in part) to a decrease in pilA (pilin) transcription. We propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.

Bacterial Proteins↗
PubMed2000

Crc is involved in catabolite repression control of the bkd operons of Pseudomonas putida and Pseudomonas aeruginosa.

Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

Cloning and characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat).

In this study, we cloned the Pseudomonas aeruginosa zwf gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD+- or NADP+-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted zwf gene product is 490 residues, which could form a tetramer with a molecular mass of approximately 220 kDa. G6PDH activity and zwf transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and zwf transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be approximately 220 kDa by size exclusion chromatography. Estimated Km values of purified G6PDH acting on glucose-6-phosphate, NADP+, and NAD+ were 530, 57, and 333 microM, respectively. The specific activities with NAD+ and NADP+ were calculated to be 176 and 69 micromol/min/mg. An isogenic zwf mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.

Amino Acid Sequence↗
PubMed1997

Glucose stimulates alginate production and algD transcription in Pseudomonas aeruginosa.

A previous study [DeVault et al. (1991) Mol. Microbiol. 5, 2503-2509] suggested that growth of Pseudomonas aeruginosa in glucose-containing medium represses algD gene transcription. In this study, growth of P. aeruginosa in rich medium containing glucose or gluconate increased alginate production and algD transcription at concentrations ranging from 1 to 5%.

Alginates↗
PubMed1996

Characterization and genetic mapping of phosphoglucoisomerase mutations in mannitol-negative mutants of Pseudomonas aeruginosa.

Phosphoglucoisomerase (pgi) mutations in a number of independently isolated mannitol-negative mutants of Pseudomonas aeruginosa PAO1 were mapped on the chromosome by plasmid FP5-mediated conjugation and by cotransduction with the generalized transducing phages G101 and F116L. Mutant allele pgi-9001 exhibited linkage to ilvB, C-9059 (46-85%), car-9003 (93-100%), and pur-9047 (70%), but not with met-9011, in FP5-mediated conjugational crosses. All known pgi mutations and several previously uncharacterized mannitol-negative mutations exhibited transductional linkage to two independent car mutations at frequencies ranging from 13% to 42% and 53% to 99%, in transductional crosses mediated by phages G101 and F116L respectively. These pgi and mannitol-negative mutations also were cotransducible at very low frequencies (<1%) with two independent ilv mutations. Cotransduction of the car and ilv loci could not be detected. These data suggest the location of pgi within the first minute of the P. aeruginosa chromosome closely linked to the car marker and probably between the ilv and car loci. All of the mannitol-negative mutations that exhibited linkage to the car and ilv loci were characterized as pgi mutations by enzyme assays. A phenotypically similar, mannitol-negative mutant was shown to contain a mutation in glucose-6-phosphate dehydrogenase (zwf-9012) that maps to a different region on the chromosome.

Chromosome Mapping↗
PubMed1996

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

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.

Amino Acid Sequence↗
PubMed1996

A two-component response regulator, gltR, is required for glucose transport activity in Pseudomonas aeruginosa PAO1.

A 729-bp open reading frame (gltR) was identified in Pseudomonas aeruginosa PAO1 that encodes a product homologous to the two-component response regulator family of proteins. Disruption of gltR caused loss of glucose transport activity. Restoration of gltR resulted in wild-type levels of glucose transport. These findings indicate that gltR is required for expression of the glucose transport system in P. aeruginosa.

Amino Acid Sequence↗
PubMed1994

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1.

The hexC locus of Pseudomonas aeruginosa PAO1 was localized to a 247-bp segment of chromosomal DNA on the multicopy broad-host-range vector pRO1614. The presence of this plasmid (pPZ196) in strain PAO1 produced the so-called "hexC effect," a two- to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. The extent of the hexC effect was restricted, since three independently regulated metabolic enzymes were not affected by the presence of the hexC plasmid. Furthermore, the hexC-containing plasmid did not suppress catabolite repression control. Nucleotide sequence analysis of the segment of DNA encompassing hexC revealed a 128-bp region rich in adenosine-plus-thymine (AT) content separating two divergent open reading frames (ORFs). Transcriptional start sites for these two genes were mapped to the intergenic region, demonstrating that this sequence contained overlapping divergent promoters. The intergenic region contained potential regulatory sequences such as dyad symmetry motifs, polydeoxyadenosine tracts, and a sequence matching the integration host factor recognition site in Escherichia coli. One of the ORFs encoded a 610-amino-acid protein with 55 to 60% identity to 6-phosphogluconate dehydratase from E. coli and Zymomonas mobilis. The second ORF coded for a protein of 335 amino acids that displayed 45 to 60% identity to the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) family of enzymes. The NAD-dependent GAP gene on the P. aeruginosa chromosome was previously unmapped. GAP was found to exhibit the hexC-dependent increase in its basal activity, establishing it as a fifth catabolic enzyme in the multioperonic hex regulon.

Amino Acid Sequence↗
PubMed1993

Reconstitution of glucose uptake and chemotaxis in Pseudomonas aeruginosa glucose transport defective mutants.

Wild-type glucose uptake and glucose chemotaxis activities were restored in glucose transport defective Pseudomonas aeruginosa strains PFB360 and PFB362 after introduction of plasmid pPZ129, containing a 1.1-kilobase DNA fragment that is essential for the expression of the P. aeruginosa periplasmic glucose binding protein. The restoration of glucose uptake and chemotaxis to wild-type levels in these strains was also achieved by reconstitution with cold-shock fluid and purified glucose binding protein isolated from P. aeruginosa PA01 wild-type strain H103 grown in conditions resulting in the induction of the high-affinity glucose transport system. Glucose uptake was determined by whole cell uptake and shock fluid binding of D-[U-14C]glucose, using standard filter binding assays. Positive chemotaxis towards glucose was assessed by capillary assays using 10 mM glucose, the amount required for optimal chemotaxis, and judged by plating capillary contents accumulated after 30 min.

Biological Transport↗
PubMed1991

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

Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the 11-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc- mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc- mutants but exhibited wild-type inducible expression.

Amides↗
PubMed1991

Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa.

Mutants which are defective in catabolite repression control (CRC) of multiple independently regulated catabolic pathways have been previously described. The mutations were mapped at 11 min on the Pseudomonas aeruginosa chromosome and designated crc. This report describes the cloning of a gene which restores normal CRC to these Crc- mutants in trans. The gene expressing this CRC activity was subcloned on a 2-kb piece of DNA. When this 2-kb fragment was placed in a plasmid behind a phage T7 promoter and transcribed by T7 RNA polymerase, a soluble protein with a molecular weight (MW) of about 30,000 was produced in Escherichia coli. A soluble protein of identical size was overproduced in a Crc- mutant when it contained the 2-kb fragment on a multicopy plasmid. This protein could not be detected in the mutant containing the vector without the 2-kb insert or with no plasmid. When a 0.3-kb AccI fragment was removed from the crc gene and replaced with a kanamycin resistance cassette, the interrupted crc gene no longer restored CRC to the mutant, and the mutant containing the interrupted gene no longer overproduced the 30,000-MW protein. Pools of intracellular cyclic AMP and the activities of adenylate cyclase and phosphodiesterase were measured in mutant and wild-type strains with and without a plasmid containing the crc gene. No consistent differences between any strains were found in any case. These results provide original evidence for a 30,000-MW protein encoded by crc+ that is required for wild-type CRC in P. aeruginosa and confirms earlier reports that the mode of CRC is cyclic AMP independent in this bacterium.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO.

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.

Carbohydrate Metabolism↗
PubMed1985

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.

Aldehyde-Lyases↗
PubMed1985

Chromosomal mapping of mutations affecting glycerol and glucose catabolism in Pseudomonas aeruginosa PAO.

Mutations causing deficiencies in the inducible, membrane-associated sn-glycerol-3-phosphate dehydrogenase (glpD) and in inducible glucose transport (glcT) were mapped on the Pseudomonas aeruginosa PAO1 chromosome by using the generalized transducing phages F116L and G101. These mutations, in separate catabolic regulatory units, were cotransducible with a previously described cluster of carbohydrate catabolic gene loci (zwf-1 eda-9001 edd-1) that maps at ca. 50 to 53 min on the chromosome. Mutant strain PFB362 (glcT1) did not transport glucose and did not produce a functional, periplasmic, glucose-binding protein that is required for glucose transport. This mutation was cotransducible with zwf-1 (70%), nalA (29%), and phe-2 (19%) but not with glpD1 or leu-10. The glpD1 mutation in strain PRP408 was cotransducible with zwf-1 (5%), eda-9001 (4%), and edd-1 (1%) and also with ami-151 (17%) and phe-2 (33%). These results expand the number of known carbohydrate catabolism genes that are clustered in the 50- to 55-min region of the PAO1 chromosome and allow us to propose the following relative gene order: ami-151 glpD1 phe-2 nalA zwf-1 eda-9001 edd-1 glcT1 leu-10. Three independently obtained nal determinants for high-level resistance to nalidixic acid, which were employed in these studies, exhibited similar cotransduction frequencies with several flanking marker mutations.

Biological Transport↗