Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16.

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (DeltaphaP1, DeltaphaP2, DeltaphaP3, DeltaphaP4, DeltaphaP12, DeltaphaP123 and DeltaphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (DeltaphaP2, DeltaphaP3 and DeltaphaP4), with the exception of DeltaphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single DeltaphaP1 mutant and the DeltaphaP12, DeltaphaP123 and DeltaphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single DeltaphaP2, DeltaphaP3 or DeltaphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

[1]  K. Gruys,et al.  Biodegradable Polymer (Biopol , 2005 .

[2]  Y. Poirier,et al.  Production of polyhydroxyalkanoates in transgenic plants. , 2005 .

[3]  A. Steinbüchel,et al.  The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. , 2004, Microbiology.

[4]  F. Mayer,et al.  Micromorphology of Gram-negative hydrogen bacteria , 1977, Archives of Microbiology.

[5]  F. Mayer,et al.  Micromorphology of Gram-negative hydrogen bacteria , 1977, Archives of Microbiology.

[6]  H. Schlegel,et al.  Ein Submersverfahren zur Kultur wasserstoffoxydierender Bakterien: Wachstumsphysiologische Untersuchungen , 2004, Archiv für Mikrobiologie.

[7]  G. Gottschalk,et al.  Complete Nucleotide Sequence of pHG1: A Ralstonia eutropha H16 Megaplasmid Encoding Key Enzymes of H2-based Lithoautotrophy and Anaerobiosis , 2003 .

[8]  T. Gerngross,et al.  A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha , 2002, Applied and Environmental Microbiology.

[9]  A. Steinbüchel,et al.  Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. , 2002, Microbiology.

[10]  A. Sinskey,et al.  The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production , 2002, Journal of bacteriology.

[11]  Björn H. Junker,et al.  Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells , 2001, Journal of bacteriology.

[12]  A. Sinskey,et al.  New Insight into the Role of the PhaP Phasin of Ralstonia eutropha in Promoting Synthesis of Polyhydroxybutyrate , 2001, Journal of bacteriology.

[13]  A. Steinbüchel,et al.  PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi , 2000, Applied and Environmental Microbiology.

[14]  A. Steinbüchel,et al.  Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene , 1999, Applied Microbiology and Biotechnology.

[15]  A. Steinbüchel,et al.  A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds , 1999, Archives of Microbiology.

[16]  K. Houmiel,et al.  Multiple beta-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. , 1998, Journal of bacteriology.

[17]  C. Föllner,et al.  Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. , 1995, Canadian journal of microbiology.

[18]  A. Steinbüchel,et al.  Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus , 1995, Journal of bacteriology.

[19]  A. Steinbüchel,et al.  Diversity of bacterial polyhydroxyalkanoic acids , 1995 .

[20]  A. Steinbüchel,et al.  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 , 1995, Journal of bacteriology.

[21]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[22]  M. Hynes,et al.  Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. , 1993, Gene.

[23]  A. Huang,et al.  Oil bodies and oleosins in seeds , 1992 .

[24]  A. Anderson,et al.  Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. , 1990, Microbiological reviews.

[25]  A. Steinbüchel,et al.  Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads , 1990, Applied and environmental microbiology.

[26]  A. Sinskey,et al.  Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). , 1989, The Journal of biological chemistry.

[27]  A. Steinbüchel,et al.  Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli , 1988, Journal of bacteriology.

[28]  S. Slater,et al.  Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway , 1988, Journal of bacteriology.

[29]  G. W. Haywood,et al.  The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus , 1988 .

[30]  R. Gross,et al.  Pseudomonas oleovorans as a Source of Poly(β-Hydroxyalkanoates) for Potential Applications as Biodegradable Polyesters , 1988, Applied and environmental microbiology.

[31]  G. W. Haywood,et al.  Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus , 1988 .

[32]  D. Römermann,et al.  Alcaligenes eutrophus hydrogenase genes (Hox) , 1984, Journal of bacteriology.

[33]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[34]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[35]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[36]  A. Pühler,et al.  Vector Plasmids for in-Vivo and in-Vitro Manipulations of Gram-Negative Bacteria , 1983 .

[37]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[38]  S. Falkow,et al.  Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. , 1977, Gene.

[39]  Hans G. Schlegel,et al.  β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism , 1973 .

[40]  A. Spurr.  A low-viscosity epoxy resin embedding medium for electron microscopy. , 1969, Journal of ultrastructure research.

[41]  J. Marmur.  A procedure for the isolation of deoxyribonucleic acid from micro-organisms , 1961 .