Skip to main content
Log in

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

  • Original Paper
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1A, B.
Fig. 2.

Similar content being viewed by others

References

  • Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472

    CAS  PubMed  Google Scholar 

  • 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 27:355–361

    CAS  PubMed  Google Scholar 

  • 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 54:1977–1982

    CAS  Google Scholar 

  • Cromwick A-M, Foglia T, Lenz RW (1996) The microbial production of poly(hydroxyalkanoates) from tallow. Appl Microbiol Biotechnol 46:464–469

    Article  CAS  Google Scholar 

  • 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 154:870-878

    PubMed  Google Scholar 

  • Doi Y, Segawa A, Kawaguchi Y, Kunioka M (1990) Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol Lett 67:165–170

    Article  CAS  Google Scholar 

  • 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 118:279–282

    Article  CAS  PubMed  Google Scholar 

  • Goryshin IY, Reznikoff WS (1998) Tn5 in vitro transposition. J Biol Chem 273:7367–7374

    CAS  PubMed  Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Google Scholar 

  • 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 266:2191–2198

    CAS  PubMed  Google Scholar 

  • 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 38:1–5

    CAS  Google Scholar 

  • Jendrossek D, Handrick R (2002) Microbial degradation of polyhydroxyalkanoates. Annu. Rev. Microbiol. 56:403–432

    Google Scholar 

  • 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 54:2924–2932

    CAS  Google Scholar 

  • Poirier Y, Nawrath C, Somerville C (1995) Production of polyhydroxyalkanoate, a family of biodegradable plastics and elastomers, in bacteria and plants. Bio/Technol 13:142–150

    Google Scholar 

  • Rehm BHA, Steinbüchel A (1999) Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 25:3–19

    CAS  PubMed  Google Scholar 

  • 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 67:225–230

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Solaiman DKY (1998) Genetic transformation of Pseudomonas oleovorans by electroporation. Biotechnol Techniques 12:829–832

    Article  CAS  Google Scholar 

  • Solaiman DKY (2002) Polymerase-chain-reaction-based detection of individual polyhydroxyalkanoate synthase phaC1 and phaC2 genes. Biotechnol Lett 24:245–250

    Article  CAS  Google Scholar 

  • 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 53:690–694

    Article  CAS  PubMed  Google Scholar 

  • Steinbüchel A (1991) Polyhydroxyalkanoic acids. In: Byrom D (ed) Biomaterials. Macmillan, New York, pp 123–213

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to D. K. Y. Solaiman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Solaiman, D.K.Y., Ashby, R.D. & Foglia, T.A. Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans . Appl Microbiol Biotechnol 62, 536–543 (2003). https://doi.org/10.1007/s00253-003-1317-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-003-1317-4

Keywords

Navigation