1887

Abstract

Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by in The occurrence of PhaR homologues and the organization of genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of , as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and binding studies employing artificial PHA granules. PhaR binds upstream of at two sites comprising the transcriptional start site plus the −10 region and a region immediately upstream of the −35 region of the σ promoter of , where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the translational start codon, where the σ-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a ΩKm mutant of H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules and the transcription of is again repressed. In addition to this, expression is subject to autoregulation. Excess PhaR that has not bound to the upstream region or to PHA granules binds to the upstream region, thereby repressing its own transcription.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-8-2413
2002-08-01
2022-01-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/8/1482413a.html?itemId=/content/journal/micro/10.1099/00221287-148-8-2413&mimeType=html&fmt=ahah

References

  1. Anderson A. J., Dawes E. A. 1990; Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1987 Current Protocols in Molecular Biology New York: Wiley;
    [Google Scholar]
  3. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 277:1513–1523
    [Google Scholar]
  4. Brandl H., Gross R. A., Lenz R. W., Fuller R. C. 1988; Pseudomonas oleovorans as a source of poly(β-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 66:2117–2124
    [Google Scholar]
  5. Cabrera J. E., Panzetta-Dutari G., Pruneda J. L., Genti-Raimondi S. 1997; A new Comamonas testosteroni steroid-inducible gene: cloning and sequence analysis. J Steroid Biochem Mol Biol 63:91–98 [CrossRef]
    [Google Scholar]
  6. Doi Y., Segawa A., Nakamura S., Kunioka M. T. 1990; Production of biodegradable copolyesters by Alcaligenes eutrophus . In New Biosynthetic Biodegradable Polymers of Industrial Interest from Microorganisms pp 37–48 Edited by Dawes E. A. Dordrecht: Kluwer;
    [Google Scholar]
  7. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
    [Google Scholar]
  8. Hawley D. K., McClure W. R. 1983; Compilation and analysis of Escherichia coli promoter sequences. Nucleic Acids Res 11:2234–2255
    [Google Scholar]
  9. 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]
  10. Hjelm H., Hjelm K., Sjöquist J. 1972; Protein A from Staphylococcus aureus . Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Lett 28:73–76 [CrossRef]
    [Google Scholar]
  11. Hocking P. J., Marchessault R. H. 1994; Biopolyesters. In Chemistry and Technology of Biodegradable Polymers pp 48–96 Edited by Griffin G. J. L. London: Chapman & Hall;
    [Google Scholar]
  12. Holmes P. A., Wright L. F., Collins S. H. 1981; Betahydroxybutyrate polymers. European Patent Application 0052459:
    [Google Scholar]
  13. Jendrossek D., Knoke I., Habidian 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]
  14. Kunioka M., Nakamura Y., Doi Y. 1988; New bacterial copolyesters produced in Alcaligenes eutrophus from organic acids. Polym Commun 29:174–176
    [Google Scholar]
  15. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  16. Lemoigne M. 1926; Produits de deshydration et de polymerisation de lácide β-oxybutyrique. Bull Soc Chim Biol 8:770–782
    [Google Scholar]
  17. Lütke-Eversloh T., Bergander K., Luftmann H., Steinbüchel A. 2001a; Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages. Microbiology 147:11–19
    [Google Scholar]
  18. 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]
  19. Maehara A., Ueda S., Nakano H., Yamane T. 1999; Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans . J Bacteriol 181:2914–2921
    [Google Scholar]
  20. Maehara A., Doi Y., Nishiyama T., Takagi Y., Ueda S., Nakano H., Yamane T. 2001; PhaR, a protein of unknown function conserved among short-chain-length polyhydroxyalkanoic acids producing bacteria, is a DNA-binding protein and represses Paracoccus denitrificans phaP expression in vitro . FEMS Microbiol Lett 200:9–15 [CrossRef]
    [Google Scholar]
  21. Marmur J. 1961; A procedure for the isolation of deoxyribonucleic acids from microorganisms. J Mol Biol 3:208–218 [CrossRef]
    [Google Scholar]
  22. Müller H. M., Seebach D. 1993; Poly(hydroxyfettsäureester), eine fünfte Klasse von physiologisch bedeutsamen organischen Biopolymeren?. Angew Chem 105:483–509 [CrossRef]
    [Google Scholar]
  23. Olmsted J. B. 1981; Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem 256:11955–11957
    [Google Scholar]
  24. Overhage J., Priefert H., Rabenhorst J., Steinbüchel A. 1999; Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase ( vdh ) gene. Appl Microbiol Biotechnol 52:820–828 [CrossRef]
    [Google Scholar]
  25. Peoples O. P., Sinskey A. J. 1989a; Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding β-ketothiolase and acetoacetyl-CoA reductase. J Biol Chem 264:15293–15297
    [Google Scholar]
  26. Peoples O. P., Sinskey A. J. 1989b; Poly-β-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene ( phbC . J Biol Chem 264:15298–15303
    [Google Scholar]
  27. Pieper-Fürst U., Madkour M. H., Mayer F., Steinbüchel A. 1994; Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber . J Bacteriol 176:4328–4337
    [Google Scholar]
  28. Pieper-Fürst U., Madkour M. H., Mayer F., Steinbüchel A. 1995; Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules. J Bacteriol 177:2513–2523
    [Google Scholar]
  29. Povolo S., Casella S. 2000; A critical role for aniA in energy–carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti . Arch Microbiol 174:42–49 [CrossRef]
    [Google Scholar]
  30. 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]
  31. Rehm B. H. A., Steinbüchel A. 1999; Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 25:3–19 [CrossRef]
    [Google Scholar]
  32. Reitzer L. J., Magasanik B. 1985; Expression of glnA in Escherichia coli is regulated at tandem promotors. Proc Natl Acad Sci USA 82:1979–1983 [CrossRef]
    [Google Scholar]
  33. Rosenberg M., Court D. 1979; Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353 [CrossRef]
    [Google Scholar]
  34. Roth J., Bendayan M., Carlemalm E., Villiger W., Garavito M. 1981; Enhancement of structural preservation and immunocytochemical straining in low temperature embedded pancreatic tissue. J Histochem Cytochem 29:663–671 [CrossRef]
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Schlegel H. G., Kaltwasser H., Gottschalk G. 1961; Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38:209–222 [CrossRef]
    [Google Scholar]
  37. Schubert P., Steinbüchel A., Schlegel H. G. 1988; Cloning of the Alcaligenes eutrophus genes for synthesis of poly-β-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli . J Bacteriol 170:5837–5847
    [Google Scholar]
  38. Simon R., Priefer U., Pühler A. 1983a; Vector plasmids for in vivo and in vitro manipulations of Gram negative bacteria. In Molecular Genetics of the Bacteria–Plant Interaction pp 98–106 Edited by Pühler A. Berlin: Springer;
    [Google Scholar]
  39. Simon R., Priefer U., Pühler A. 1983b; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784–791 [CrossRef]
    [Google Scholar]
  40. Slater S. C., Voige W. H., Dennis D. E. 1988; Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-β-hydroxybutyrate biosynthetic pathway. J Bacteriol 170:4431–4436
    [Google Scholar]
  41. Slater S., 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]
  42. Spurr A. R. 1969; A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43 [CrossRef]
    [Google Scholar]
  43. Steinbüchel A. 1991; Polyhydroxyalkanoic acids. In Biomaterials pp 123–213 Edited by Byrom D. Basingstoke, UK: Macmillan;
    [Google Scholar]
  44. Steinbüchel A., Schlegel H. G. 1991; Physiology and molecular genetics of poly(β-hydroxy-alkanoic acid) synthesis in Alcaligenes eutrophus . Mol Microbiol 5:535–542 [CrossRef]
    [Google Scholar]
  45. Steinbüchel A., Valentin H. E. 1995; Diversity of microbial polyhydroxyalkanoic acids. FEMS Microbiol Lett 128:219–228 [CrossRef]
    [Google Scholar]
  46. Steinbüchel A., Aerts K., Babel W. 8 other authors 1995; Consideration on the structural and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Can J Microbiol 41:94–105 [CrossRef]
    [Google Scholar]
  47. Steinbüchel A., Wieczorek R., Krüger N. 1996; PHA biosynthesis, its regulation and application of C1-utilizing microorganisms for polyester production. In Microbial Growth on Compounds pp 237–244 Edited by Lidstrom M. E., Tabita F. R. Dordrecht: Kluwer;
    [Google Scholar]
  48. Timm A., Steinbüchel A. 1990; Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol 56:3360–3367
    [Google Scholar]
  49. Tombolini R., Povolo S., Buson A., Squartini A., Nuti M. P. 1995; Poly-β-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41. Microbiology 141:2553–2559 [CrossRef]
    [Google Scholar]
  50. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354 [CrossRef]
    [Google Scholar]
  51. Walther-Mauruschat A., Aragno M., Mayer F., Schlegel H. G. 1977; Micromorphology of Gram-negative hydrogen bacteria. II. Cell envelope, membranes, and cytoplasmic inclusions. Arch Microbiol 114:101–110 [CrossRef]
    [Google Scholar]
  52. 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]
  53. Wieczorek R., Pries A., Steinbüchel A., Mayer F. 1995; Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus . J Bacteriol 177:2425–2435
    [Google Scholar]
  54. York G. M., Junker B. H., Stubbe J. A., Sinskey A. J. 2001a; Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells. J Bacteriol 183:4217–4226 [CrossRef]
    [Google Scholar]
  55. York G. M., Stubbe J., Sinskey A. J. 2001b; New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate. J Bacteriol 183:2394–2397 [CrossRef]
    [Google Scholar]
  56. York G. M., Stubbe J., Sinskey A. J. 2002; The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J Bacteriol 184:59–66 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-8-2413
Loading
/content/journal/micro/10.1099/00221287-148-8-2413
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error