1887

Abstract

Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20·0, 20·2, 19·6 and 20·2 kDa, respectively. RT-PCR analysis showed that , and were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of had previously been shown by others. Although PhaP2 could not be localized on poly(3HB) granules, experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in , and . These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

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2004-07-01
2024-03-29
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References

  1. Asrar J., Gruys K. J. 2002; Biodegradable polymer (Biopol®. In Biopolymers, Volume 4: Polyesters III - Applications and Commercial Products pp. 53–90 Edited by Doi Y., Steinbüchel A. Weinheim: Wiley-VCH;
    [Google Scholar]
  2. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 277:1513–1523
    [Google Scholar]
  3. Dennis D., Liebig C., Holley T., Thomas K. S., Khosla A., Wilson D., Augustine B. 2003; Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy. FEMS Microbiol Lett 226:113–119 [CrossRef]
    [Google Scholar]
  4. Fukui T., Kichise T., Iwata T., Doi Y. 2001; Characterization of 13 kDa granule-associated protein in Aeromonas caviae and biosynthesis of polyhydroxyalkanoates with altered molar composition by recombinant bacteria. Biomacromolecules 2:148–153 [CrossRef]
    [Google Scholar]
  5. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
    [Google Scholar]
  6. Handrick R., Reinhardt S., Jendrossek D. 2000; Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha. J Bacteriol 182:5916–5918 [CrossRef]
    [Google Scholar]
  7. Hanley S. Z., Pappin D. J. C., Rahmann D., White A. J., Elborough K. M., Slabas A. R. 1999; Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding. FEBS Lett 447:99–105 [CrossRef]
    [Google Scholar]
  8. Heukeshofen J., Dernick R. 1985; A simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6:103–112 [CrossRef]
    [Google Scholar]
  9. Jendrossek D. 2002; Extracellular polyhydroxyalkanoate depolymerases: the key enzymes of PHA degradation. In Biopolymers, Volume 3b: Polyesters II - Properties and Chemical Synthesis pp. 41–84 Edited by Doi Y., Steinbüchel A. Weinheim: Wiley-VCH;
    [Google Scholar]
  10. Jendrossek D., Handrick R. 2002; Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 56:403–432 [CrossRef]
    [Google Scholar]
  11. Jendrossek D., Knoke I., Habibian R. D., Steinbüchel A., Schlegel H. G. 1993; Degradation of poly(3-hydroxybutyrate), PHB, by bacteria and purification of a novel PHB depolymerase from Comamonas sp. J Environ Polym Degrad 1:53–63 [CrossRef]
    [Google Scholar]
  12. Jendrossek D., Schirmer A., Schlegel H. G. 1996; Biodegradation of polyhydroxyalkanoic acids. Appl Microbiol Biotechnol 46:451–463 [CrossRef]
    [Google Scholar]
  13. Jurasek L., Marchessault R. H. 2002; The role of phasins in the morphogenesis of poly(3-hydroxybutyrate) granules. Biomacromolecules 3:256–261 [CrossRef]
    [Google Scholar]
  14. Jurasek L., Nobes G. A. R., Marchessault R. H. 2001; Computer simulation of in vitro formation of PHB granules: particulate polymerization. Macromol Biosci 1:258–265 [CrossRef]
    [Google Scholar]
  15. Kobayashi T., Shiraki M., Abe T., Sugiyama A., Saito T. 2003; Purification and properties of an intracellular 3-hydroxybutyrate oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase. J Bacteriol 185:3485–3490 [CrossRef]
    [Google Scholar]
  16. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  17. Lee S. Y., Park S. Y. 2002; Fermentative production of SCL-PHAs. In Biopolymers, Volume 3a: Polyesters I - Biological Systems and Biotechnological Production pp. 263–290Edited by Doi Y., Steinbüchel A. Weinheim: Wiley-VCH;
    [Google Scholar]
  18. Lütke-Eversloh T., Steinbüchel A. 2003; Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha. FEMS Microbiol Lett 221:191–196 [CrossRef]
    [Google Scholar]
  19. Lütke-Eversloh T., Bergander K., Luftmann H., Steinbüchel A. 2001a; Biosynthesis of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages. Microbiology 147:11–19
    [Google Scholar]
  20. Lütke-Eversloh T., Bergander K., Luftmann H., Steinbüchel A. 2001b; Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate) (PHB. Biomacromolecules 2:1061–1065 [CrossRef]
    [Google Scholar]
  21. Lütke-Eversloh T., Fischer A., Remminghorst U. & 8 other authors; 2002; Biosynthesis of polythioesters by engineered Escherichia coli as novel thermoplastic materials. Nature Materials 1:236–240 [CrossRef]
    [Google Scholar]
  22. Lutter P., Meyer H. E., Langer M., Witthohn K., Dormeyer W., Sickmann A., Bluggel M. 2001; Investigation of charge variants of rViscumin by two-dimensional gel electrophoresis and mass spectrometry. Electrophoresis 22:2888–2897 [CrossRef]
    [Google Scholar]
  23. Marmur J. 1961; A procedure for the isolation of desoxyribonucleic acids from microorganisms. J Mol Biol 3:208–218 [CrossRef]
    [Google Scholar]
  24. Mayer F., Hoppert M. 1997; Determination of the thickness of the boundary layer surrounding bacterial PHA inclusion bodies, and implications for models describing the molecular architecture of this layer. J Basic Microb 37:45–52 [CrossRef]
    [Google Scholar]
  25. Poirier Y., Gruys K. J. 2002; Production of polyhydroxyalkanoates in transgenic plants. In Biopolymers, Volume 3a: Polyesters I - Biological Systems and Biotechnological Production pp. 401–436Edited by Doi Y., Steinbüchel A. Weinheim: Wiley-VCH;
    [Google Scholar]
  26. Pötter M., Madkour M. H., Mayer F., Steinbüchel A. 2002; Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology 148:2413–2426
    [Google Scholar]
  27. Preusting H., Kingma J., Huisman G., Steinbüchel A., Witholt B. 1993; Formation of polyester blends by a recombinant strain of Pseudomonas oleovorans: different poly(3-hydroxyalkanoates) are stored in separate granules. J Environ Polym Degrad 1:11–21 [CrossRef]
    [Google Scholar]
  28. Prieto M. A., Jung K., Witholt B., Kessler B., Bühler B. 1999; PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes. J Bacteriol 181:858–868
    [Google Scholar]
  29. Saegusa H., Shiraki M., Kanai C., Saito T. 2001; Cloning of an intracellular poly[d(−)-3-hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product. J Bacteriol 183:94–100 [CrossRef]
    [Google Scholar]
  30. Saegusa H., Shiraki M., Saito T. 2002; Cloning of an intracellular d(−)-3-hydroxybutyrate-oligomer hydrolase gene from Ralstonia eutropha H16 and identification of the active site serine residue by site-directed mutagenesis. J Biosci Bioeng 94:106–112 [CrossRef]
    [Google Scholar]
  31. Saito T., Kobayashi T. 2002; Intracellular degradation of PHA. In Biopolymers, Volume 3b: Polyesters II - Properties and Chemical Synthesis pp. 23–40 Edited by Doi Y., Steinbüchel A. Weinheim: Wiley-VCH;
    [Google Scholar]
  32. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  33. Schlegel H. G., Gottschalk G., Bartha V. 1961a; Formation of and utilization of poly-β-hydroxybutyric acid by knallgas bacteria (Hydrogenomonas. Nature 29:463–465
    [Google Scholar]
  34. Schlegel H. G., Kaltwasser H., Gottschalk G. 1961b; Ein submersverfahren zur kultur wasserstoffoxidierender bakterien: wachstumsphysiologische untersuchungen. Arch Mikrobiol 38:209–222 [CrossRef]
    [Google Scholar]
  35. Schubert P., Schlegel H. G., Steinbüchel A. 1988; Cloning of the Alcaligenes eutrophus genes for synthesis of poly-b-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol 170:5837–5847
    [Google Scholar]
  36. Schwartz E., Friedrich B. 2001; A physical map of the megaplasmid pHG1, one of three genomic replicons in Ralstonia eutropha H16. FEMS Microbiol Lett 201:213–219 [CrossRef]
    [Google Scholar]
  37. Schwartz E., Henne A., Cramm R., Eitinger T., Friedrich B., Gottschalk G. 2003; Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H2-based lithoautotrophy and anaerobiosis. J Mol Biol 332:369–383 [CrossRef]
    [Google Scholar]
  38. Shevchenko A., Wilm O., Vorm O., Mann M. 1996; Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858 [CrossRef]
    [Google Scholar]
  39. Slater T., Houmiel K. L., Tran M., Mitsky T. A., Taylor N. B., Padgette S. R., Gruys K. J. 1998; Multiple β-ketothiolases mediate poly(β-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 180:1979–1987
    [Google Scholar]
  40. Srinivasan S., Barnard G. C., Gerngross T. U. 2002; A novel high-cell-density protein expression system based on Ralstonia eutropha. Appl Environ Microbiol 68:5925–5932 [CrossRef]
    [Google Scholar]
  41. Steinbüchel A. 2001; Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. Macromol Bioscience 1:1–24 [CrossRef]
    [Google Scholar]
  42. Steinbüchel A., Valentin H. E. 1995; Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett 128:219–228 [CrossRef]
    [Google Scholar]
  43. Steinbüchel A., Aerts K., Babel W. & 8 other authors; 1995; Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Can J Microbiol 41:94–105 [CrossRef]
    [Google Scholar]
  44. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. 1997; The clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 [CrossRef]
    [Google Scholar]
  45. Towbin H., Straehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354 [CrossRef]
    [Google Scholar]
  46. Vazquez G. J., Wieczoreck R., Mendez B. S., Steinbüchel A. 1996; Poly-(3-hydroxybutyrate) granules in Bacillus megaterium. Isolation and analysis of associated proteins. Rev Argent Microbiol 28:118–122
    [Google Scholar]
  47. Veith P. D., Talbo G. H., Slakeski N., Reynolds E. C. 2001; Identification of a novel heterodimeric outer membrane protein of Porphyromonas gingivalis by two-dimensional gel electrophoresis and peptide mass fingerprinting. Eur J Biochem 268:4748–4757 [CrossRef]
    [Google Scholar]
  48. Weber K., Osborn M. 1969; The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
    [Google Scholar]
  49. Wieczorek R., Steinbüchel A. 1996; Occurrence of PHA granule-associated proteins related to the Alcaligenes eutrophus H16 GA24-protein in other bacteria. FEMS Microbiol Lett 135:23–30 [CrossRef]
    [Google Scholar]
  50. Wieczorek R., Pries A., Mayer F., Steinbüchel A. 1995; Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus. J Bacteriol 177:2425–2435
    [Google Scholar]
  51. Williams S. F., Martin D. P. 2002; Applications of PHAs in medicine and pharmacy. In Biopolymers, Volume 4: Polyesters III - Applications and Commercial Products pp. 91–128Edited by Doi Y., Steinbüchel A. Weinheim: Wiley-VCH;
    [Google Scholar]
  52. York G. M., Junker B. H. H., Stubbe J. A., Sinskey A. J. 2001; Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells. J Bacteriol 183:4217–4226 [CrossRef]
    [Google Scholar]
  53. York G. M., Lupberger J., Tian J., Lawrence A. G., Stubbe J., Sinskey A. J. 2003; Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[d-(−)-3-hydroxybutyrate] depolymerase genes. J Bacteriol 185:3788–3794 [CrossRef]
    [Google Scholar]
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