Gene_locus Report for: psepf-PHAZ

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.

Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To clone the PHA synthase gene(s) (phaC), the genomic library of P. aureofaciens was constructed using a cosmid vector. The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P. putida GPp104. The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert. Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD). The heterologous expression of pha genes from P. aureofaciens was confirmed. P. putida GPp104 regained the ability to accumulate PHA on introduction of pVK6. Wild-type strains P. oleovorans and P. fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6.

Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens.

To shed light on the genetic equipment of the beneficial plant-associated bacterium Pseudomonas brassicacearum, we sequenced the whole genome of the strain NFM421. Its genome consists of one chromosome equipped with a repertoire of factors beneficial for plant growth. In addition, a complete type III secretion system and two complete type VI secretion systems were identified. We report here the first genome sequence of this species.

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.

Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To clone the PHA synthase gene(s) (phaC), the genomic library of P. aureofaciens was constructed using a cosmid vector. The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P. putida GPp104. The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert. Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD). The heterologous expression of pha genes from P. aureofaciens was confirmed. P. putida GPp104 regained the ability to accumulate PHA on introduction of pVK6. Wild-type strains P. oleovorans and P. fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6.

Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens.

Two polyhydroxyalkanoate synthase genes, phaC1 from Pseudomonas pseudoalcaligenes HBQ06 and phaC2 from Pseudomonas nitroreducens 0802, were cloned using a PCR cloning strategy based on the type II pha loci property of Pseudomonas strains. The complete open reading frames (ORFs) of phaC1 (P. nitroreducens HBQ06) and phaC2 (P. nitroreducens 0802) were identified from the PCR products. Using the sequence information, the complete PHA synthase genes were PCR cloned directly from the genomic DNA and expressed in Escherichia coli as confirmed by Fourier transform-infrared spectroscopy and gas chromatography. The differences between PhaC1 and PhaC2 were analyzed and the two proteins were suggested to contain different functions and evolution history.

Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]) consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), beta-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs. Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of beta-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.