RESUMO
Electrophoretic analysis of the proteins bound to poly(3-hydroxybutyric acid), PHB-, granules in Methylobacterium extorquens, M. rhodesianum as well as the PHB-leaky mutants Mu 1 and Mu 11, which were isolated from the latter, resulted in two dominant low-molecular weight proteins, which were referred to as GA11 and GA20. After purification of these proteins antibodies against the GA11 and GA20 protein of M. extorquens were obtained. Both proteins bound to the surface of PHB granules as revealed by immunoelectron microscopy of whole cells of M. extorquens and M. rhodesianum. With cells of the PHB-leaky mutants Mu 1 and Mu 11 no specific labeling was observed. The N-terminal amino acid sequences of the GA11 and the GA20 protein were determined. We found significant homologies between the sequences of the investigated strains. The use of oligonucleotide probes based on the N-terminal sequences of the GA20 protein from M. rhodesianum to identify the corresponding structural genes in various genomic libraries failed.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Bactérias Aeróbias Gram-Negativas/química , Acetil-CoA C-Aciltransferase/metabolismo , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Anticorpos Antibacterianos/isolamento & purificação , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Western Blotting , Clonagem Molecular , Sondas de DNA/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Biblioteca Gênica , Bactérias Aeróbias Gram-Negativas/genética , Bactérias Aeróbias Gram-Negativas/ultraestrutura , Hidroxibutiratos/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Mutagênese , Hibridização de Ácido Nucleico , Plasmídeos , Homologia de Sequência de AminoácidosRESUMO
Some mathematical calculations were done that provided information about the structure and biochemistry of polyhydroxyalkanoic acid (PHA) granules and about the amounts of the different constituents that contribute to the PHA granules. The data obtained from these calculations are compared with data from the literature, which show that PHA granules consist not only of the polyester but also of phospholipids and proteins. The latter are referred to as granule-associated proteins, and they are always located at the surface of the PHA granules. A concept is proposed that distinguishes four classes of structurally and functionally different granule-associated proteins: (i) class I comprises the PHA synthases, which catalyze the formation of ester linkages between the constituents; (ii) class II comprises the PHA depolymerases, which are responsible for the intracellular degradation of PHA, (iii) class III comprises a new type of protein, which is referred to as phasins and which has most probably a function analogous to that of oleosins in oilseed plants, and (iv) class IV comprises all other proteins, which have been found to be associated with the granules but do not belong to classes I-III. Particular emphasis is placed on the phasins, which constitute a significant fraction of the total cellular protein. Phasins are assumed to form a close protein layer at the surface of the granules, providing the interface between the hydrophilic cytoplasm and the much more hydrophobic core of the PHA inclusion.