Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans
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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 PhaZFOAC001 (239 amino-acid residues) and PhaZFOAC002 (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZFOAC001 might still retain a partial PHA depolymerization activity, while PhaZFOAC002 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 β-hydroxyoctanoate and higher β-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.
KeywordsPHAs Pseudomonas Oleovorans Energy Reserve Material
The authors thank Dr. Peter Cooke of the Microscopic Imaging Group of Eastern Regional Research Center for acquiring the transmission electron micrographs, and Nicole Cross and Marshall Reed for technical assistance. Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.
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