Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans.
The phaZ gene of Pseudomonas resinovorans codes for a poly(hydroxyalkanoates) (PHA) depolymerase. Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZ(FOAC001) (239 amino-acid residues) and PhaZ(FOAC002) (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZ(FOAC001) might still retain a partial PHA depolymerization activity, while PhaZ(FOAC002) is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source. The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of beta-hydroxyoctanoate and higher beta-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up. These phaZ mutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.
Solaiman
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Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA. dsolaiman@arserrc.gov
Ashby
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Foglia
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D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D016254:Mutagenesis, Insertional; D009584:Nitrogen / Q000378:metabolism; D011091:Polyesters / Q000032:analysis* / Q000378:metabolism*; D011549:Pseudomonas / Q000166:cytology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
D011091:Polyesters; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase; D009584:Nitrogen