Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber

The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a protein with a calculated M(r) of 14,175 which was recently localized downstream of the PHA synthase gene (U. Pieper and A. Steinbüchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies indicates that a tetrameric structure of the recombinant, native GA14 protein is most likely. Immunoelectron microscopy demonstrated a localization of the GA14 protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that expression of the GA14 protein depended strongly on PHA synthesis.

[1]  A. Sinskey,et al.  Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules , 1993, Journal of bacteriology.

[2]  I. Maxwell,et al.  Biosynthesis of poly-(R)-3-hydroxyalkanoate: an emulsion polymerization , 1993 .

[3]  R. Lenz,et al.  The supramolecular architecture of the polyhydroxyalkanoate inclusions in Pseudomonas oleovorans , 1992 .

[4]  F. Mayer.  Structural aspects of poly‐β‐hydroxybutyrate granules , 1992 .

[5]  A. Steinbüchel,et al.  Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D. , 1992, FEMS microbiology letters.

[6]  U. Pieper,et al.  Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria. , 1992, FEMS microbiology reviews.

[7]  U. Pieper,et al.  Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the gram-positive bacterium Rhodococcus ruber. , 1992, FEMS microbiology letters.

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

[9]  G. W. Haywood,et al.  Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. , 1991, International journal of biological macromolecules.

[10]  A. Steinbüchel,et al.  Physiology and molecular genetics of poly(β‐hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus , 1991, Molecular microbiology.

[11]  David Byrom.  Biomaterials: Novel Materials from Biological Sources , 1991 .

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

[13]  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.

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

[15]  W. Bullock.  XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. , 1987 .

[16]  D. Johnson,et al.  Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates. , 1985, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[17]  P A Holmes,et al.  Applications of PHB - a microbially produced biodegradable thermoplastic , 1985 .

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

[19]  W. Konigsberg,et al.  Removal of sodium dodecyl sulfate from proteins by ion-pair extraction. , 1983, Methods in enzymology.

[20]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[21]  J. Olmsted,et al.  Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. , 1981, The Journal of biological chemistry.

[22]  J. Roth,et al.  Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. , 1981, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[23]  J. Wallace,et al.  Further electron microscope studies on pyruvate carboxylase. , 1980, European journal of biochemistry.

[24]  B. Vogelstein,et al.  Preparative and analytical purification of DNA from agarose. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[25]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[26]  K. Weber,et al.  The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. , 1969, The Journal of biological chemistry.

[27]  J. Merrick,et al.  Metabolism of poly-beta-hydroxybutyrate. I. Purification, composition, and properties of native poly-beta-hydroxybutyrate granules from Bacillus megaterium. , 1968, Biochemistry.

[28]  E. Stadtman,et al.  Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. , 1968, Biochemistry.

[29]  F. Kaudewitz.  Inaktivierende und mutagene Wirkung salpetriger Säure auf Zellen von Escherichia coli , 1959 .