Dataset Information

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

ABSTRACT: Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.

SUBMITTER: Schweizer HP

PROVIDER: S-EPMC205338 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Schweizer H P HP Po C C

Journal of bacteriology 19940401 8

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. ...[more]

PMID: 8157588

Similar Datasets

Project description:Pseudomonas aeruginosa is an important opportunistic pathogen that is lethal to cystic fibrosis (CF) patients. Glycerol generated during the degradation of phosphatidylcholine, the major lung surfactant in CF patients, could be utilized by P. aeruginosa. Previous studies have indicated that metabolism of glycerol by this bacterium contributes to its adaptation to and persistence in the CF lung environment. Here, we investigated the metabolic mechanisms of glycerol and its important metabolic intermediate glycerol 3-phosphate (G3P) in P. aeruginosa PAO1. We found that G3P homeostasis plays an important role in the growth and virulence factor production of P. aeruginosa PAO1. The G3P accumulation caused by the mutation of G3P dehydrogenase (GlpD) and exogenous glycerol led to impaired growth and reductions in pyocyanin synthesis, motilities, tolerance to oxidative stress, and resistance to kanamycin. Transcriptomic analysis indicates that the growth retardation caused by G3P stress is associated with reduced glycolysis and adenosine triphosphate (ATP) generation. Furthermore, two haloacid dehalogenase-like phosphatases (PA0562 and PA3172) that play roles in the dephosphorylation of G3P in strain PAO1 were identified, and their enzymatic properties were characterized. Our findings reveal the importance of G3P homeostasis and indicate that GlpD, the key enzyme for G3P catabolism, is a potential therapeutic target for the prevention and treatment of infections by this pathogen. IMPORTANCE In view of the intrinsic resistance of Pseudomonas aeruginosa to antibiotics and its potential to acquire resistance to current antibiotics, there is an urgent need to develop novel therapeutic options for the treatment of infections caused by this bacterium. Bacterial metabolic pathways have recently become a focus of interest as potential targets for the development of new antibiotics. In this study, we describe the mechanism of glycerol utilization in P. aeruginosa PAO1, which is an available carbon source in the lung environment. Our results reveal that the homeostasis of glycerol 3-phosphate (G3P), a pivotal intermediate in glycerol catabolism, is important for the growth and virulence factor production of P. aeruginosa PAO1. The mutation of G3P dehydrogenase (GlpD) and the addition of glycerol were found to reduce the tolerance of P. aeruginosa PAO1 to oxidative stress and to kanamycin. The findings highlight the importance of G3P homeostasis and suggest that GlpD is a potential drug target for the treatment of P. aeruginosa infections.

Project description:We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5alphaF'(pOD22) and DH5alphaF'(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (beta-ISP) and 48,243 Da (alpha-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.

Project description:The enzyme L-ornithine N5-oxygenase catalyzes the hydroxylation of L-ornithine (L-Orn), which represents an early step in the biosynthesis of the peptidic moiety of the fluorescent siderophore pyoverdin in Pseudomonas aeruginosa. A gene bank of DNA from P. aeruginosa PAO1 (ATCC 15692) was constructed in the broad-host-range cosmid pLAFR3 and mobilized into the L-Orn N5-oxygenase-defective (pvdA) P. aeruginosa mutant PALS124. Screening for fluorescent transconjugants made it possible to identify the trans-complementing cosmid pPV4, which was able to restore pyoverdin synthesis and L-Orn N5-oxygenase activity in the pvdA mutant PALS124. The 17-kb PAO1 DNA insert of pPV4 contained at least two genetic determinants involved in pyoverdin synthesis, i.e., pvdA and pvdC4, as shown by complementation analysis of a set of mutants blocked in different steps of the pyoverdin biosynthetic pathway. Deletion analysis, subcloning, and transposon mutagenesis enabled us to locate the pvdA gene in a minimum DNA fragment of 1.7 kb flanked by two SphI restriction sites. Complementation of the pvdA mutation was under stringent iron control; both pyoverdin synthesis and L-Orn N5-oxygenase activity were undetectable in cells of the trans-complemented mutant which had been grown in the presence of 100 microM FeCl3. The entire nucleotide sequence of the pvdA gene, from which the primary structure of the encoded polypeptide was deduced, was determined. The pvdA structural gene is 1,278 bp; the cloned DNA fragment contains at the 5' end of the gene a putative ribosome-binding site but apparently lacks known promoterlike sequences. The P. aeruginosa L-Orn N5-oxygenase gene codes for a 426-amino-acid peptide with a predicted molecular mass of 47.7 kDa and an isoelectric point of 8.1. The enzyme shows approximately 50% homology with functional analogs, i.e., L-lysine N6-hydroxylase of aerobactin-producing Escherichia coli and L-Orn N5-oxygenase of ferrichrome-producing Ustilago maydis. The pvdA gene was expressed in P. aeruginosa under the control of the T7 promoter. Induction of the T7 RNA polymerase system resulted in parallel increases of the L-Orn N5-oxygenase activity and of the amount of a 47.7-kDa polypeptide. We also constructed a site-specific pvdA mutant by insertion of a tetracycline-resistance cassette in the chromosomal pvdA gene of P. aeruginosa PAO1. Similarly to strain PALS124, the pvdA mutant obtained by gene disruption also disclosed no pyoverdin synthesis, lacked L-Orn N5-oxygenase activity, was complemented by the cloned pvdA gene, and produced pyoverdin at wild-type levels when fed with the biosynthetic precursor L-N5-OH-Orn. Southern blot analysis indicated that genes homologous to pvdA could be located within a 1.7-kb DNA fragment from SphI-digested genomic DNA of different hydroxamate-producing Pseudomonas spp. Our results suggest that omega-amino acid oxygenases have been conserved over a wide evolutionary range and probably evolved from a common ancestor.

Dataset Information

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Publications

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets