The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of was identified, and its own matching gene was analyzed and cloned on the molecular level. for -ketothiolase, acetoacetyl-CoA reductase, and PHA synthase genes, respectively. Lately, we’ve examined and cloned these three PHA synthesis genes within a facultative methylotrophic bacterium, (31, 32), that was in a position to synthesize the P(3HB), poly(3-hydroxyvaleric acidity), and poly(3-hydroxybutyric acid-and produced PF-4136309 enzyme inhibitor an operon, while was located at a remote control locus over the chromosomal DNA. Gene medication dosage results for PHA synthesis had been looked into in recombinants of whose level of expression of each PHA-synthetic enzyme was improved. Among these recombinants, a self-cloning recombinant of showed the highest PHA content material and the highest rate of PHA build PF-4136309 enzyme inhibitor up (16). In the recombinant-enhancing (36), the GA14 protein of (17, 18), the GA13 protein of spp. (21), and the GA14 protein of (15). These proteins are called phasins, a new class of protein that forms a coating at the surface of a PHA granule, and they have been shown to influence the size and quantity of PHA granules (9, 18, 36). In addition, production of phasins is definitely suggested to depend on the presence of an intact PHA biosynthesis apparatus and to become regulated by unfamiliar mechanisms (36). In this study, we statement the recognition of a new granule-associated 16-kDa protein from PHA granules of and the cloning, sequencing, and characterization of its gene (named (ATCC 17741) and XL1-Blue (3). The plasmids used in this study are outlined in Table ?Table1.1. Inorganic salt medium comprising 1% (vol/vol) methanol or 0.1% (vol/vol) was grown at 37C in Luria-Bertani (LB) medium (19). When needed, kanamycin (50 g/ml) and/or ampicillin (100 g/ml) was added to FLJ13165 the medium. TABLE 1 Plasmids used in this?study were from cells which were cultivated inside a 5-liter fermentor in inorganic salt medium maintained at 0.02% were from cells which were cultivated in LB medium containing 2% sodium lactate as an excess carbon resource. After 30 h of cultivation, the cells were harvested by centrifugation (8,000 was isolated according to the process of Wilson (38). Plasmid DNA isolation, agarose gel electrophoresis, and transformation of were carried out as explained by Sambrook et al. (19). All DNA-manipulating enzymes were used as recommended from the manufacturers. Southern or dot blot hybridization analysis. Hybridization was carried out as explained by Southern (23). DNA fragments were transferred from agarose gels or from bacterial colonies to nylon membranes after alkali denaturation. Preparation of a digoxigenin-labeled probe and its visualization within the membrane were carried out having a digoxigenin nucleic acid labeling and detection kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Cloning of and offered two positive signals at sizes of 0.7 and 7.2 kb for the transformants with the probe revealed the presence of one positive clone, which contained a 7.2-kb fragment of DNA, referred to as PP72. In order to clone an upstream region of DNA, referred to as PP26. Nucleotide PF-4136309 enzyme inhibitor sequence analysis. The DNA fragments to be sequenced were subcloned into pBluescript II SK(+), which was used for making serial deletions. DNA sequencing was carried out having a model PF-4136309 enzyme inhibitor 373A automatic DNA sequencer and a dye terminator cycle-sequencing FS ready-reaction kit (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.). The nucleotide sequence was analyzed with genetic information-processing software (SDC-GENETYX; Software Development Co., Tokyo, Japan). Electrophoresis of proteins. Proteins were separated by sodium dodecyl sulfate (SDS)C12.5% polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie brilliant blue (CBB) R-250 as explained by Laemmli (11). N-terminal amino acid sequence analysis. PHA granule-associated proteins were PF-4136309 enzyme inhibitor separated on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After proteins within the membrane were stained with CBB R-250, those protein.