Applied Microbiology and Biotechnology

, Volume 62, Issue 5–6, pp 536–543 | Cite as

Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans

  • D. K. Y. SolaimanEmail author
  • R. D. Ashby
  • T. A. Foglia
Original Paper


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.


PHAs Pseudomonas Oleovorans Energy Reserve Material 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



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.


  1. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472PubMedGoogle Scholar
  2. Ashby RD, Foglia TA, Solaiman DKY, Liu C-K, Nunez A, Eggink G (2000) Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing. Int J Biol Macromol 27:355–361PubMedGoogle Scholar
  3. Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly(β-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982Google Scholar
  4. Cromwick A-M, Foglia T, Lenz RW (1996) The microbial production of poly(hydroxyalkanoates) from tallow. Appl Microbiol Biotechnol 46:464–469CrossRefGoogle Scholar
  5. De Smet MJ, Eggink G, Witholt B, Kingma J, Wynberg H (1983) Characterization of intracellular inclusions formed by Pseudomonas oleovovans during growth on octane. J Bacteriol 154:870-878PubMedGoogle Scholar
  6. Doi Y, Segawa A, Kawaguchi Y, Kunioka M (1990) Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol Lett 67:165–170CrossRefGoogle Scholar
  7. Foster LJR, Lenz RW, Fuller RC (1994) Quantitative determination of intracellular depolymerase activity in Pseudomonas oleovorans inclusions containing poly-3-hydroxyalkanoates with long alkyl substitutents. FEMS Microbiol Lett 118:279–282CrossRefPubMedGoogle Scholar
  8. Goryshin IY, Reznikoff WS (1998) Tn5 in vitro transposition. J Biol Chem 273:7367–7374PubMedGoogle Scholar
  9. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580Google Scholar
  10. Huisman GW, Wonink E, Meima R, Kazemier B, Terpstra P, Witholt B (1991) Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. J Biol Chem 266:2191–2198PubMedGoogle Scholar
  11. Huisman GW, Wonink E, de Koning G, Preusting H, Witholt B (1992) Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains. Appl Microbiol Biotechnol 38:1–5Google Scholar
  12. Jendrossek D, Handrick R (2002) Microbial degradation of polyhydroxyalkanoates. Annu. Rev. Microbiol. 56:403–432Google Scholar
  13. Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B (1988) Formation of polyesters by Pseudomonas oleovorans: Effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54:2924–2932Google Scholar
  14. Poirier Y, Nawrath C, Somerville C (1995) Production of polyhydroxyalkanoate, a family of biodegradable plastics and elastomers, in bacteria and plants. Bio/Technol 13:142–150Google Scholar
  15. Rehm BHA, Steinbüchel A (1999) Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 25:3–19PubMedGoogle Scholar
  16. Ruiz JA, Lopez NI, Fernandez RO, Mendez BS (2001) Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms. Appl Environ Microbiol 67:225–230CrossRefPubMedGoogle Scholar
  17. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  18. Solaiman DKY (1998) Genetic transformation of Pseudomonas oleovorans by electroporation. Biotechnol Techniques 12:829–832CrossRefGoogle Scholar
  19. Solaiman DKY (2002) Polymerase-chain-reaction-based detection of individual polyhydroxyalkanoate synthase phaC1 and phaC2 genes. Biotechnol Lett 24:245–250CrossRefGoogle Scholar
  20. Solaiman DKY, Ashby RD, Foglia TA (2000) Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction. Appl Microbiol Biotechnol 53:690–694CrossRefPubMedGoogle Scholar
  21. Steinbüchel A (1991) Polyhydroxyalkanoic acids. In: Byrom D (ed) Biomaterials. Macmillan, New York, pp 123–213Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • D. K. Y. Solaiman
    • 1
    Email author
  • R. D. Ashby
    • 1
  • T. A. Foglia
    • 1
  1. 1. Eastern Regional Research Center, Agricultural Research ServiceU.S. Department of AgricultureWyndmoorUSA

Personalised recommendations