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.
Institut für Mikrobiologie, Georg-August-Universität zu Göttingen, Germany.
U
U
Pieper-Fürst
M H
MH
Madkour
F
F
Mayer
A
A
Steinbüchel
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D001483:Base Sequence; D003594:Cytoplasmic Granules / Q000737:chemistry*; D006880:Hydroxy Acids / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D012240:Rhodococcus / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
D001426:Bacterial Proteins; C088136:GA14 protein, Rhodococcus ruber; D006880:Hydroxy Acids; D008565:Membrane Proteins