Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans

The polyhydroxyalkanoic acid (PRA) granule-associated 16-kDa protein (GA16 protein) of Paracoccus denitrificans was identified, and its corresponding gene was cloned and analyzed at the molecular level. The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (phaC(Pd)) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774-779, 1996). Gene walking around phaC(Pd) revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the phaP(Pd) gene, encoding GA16 protein, and the other was the phaR(Pd) gene, encoding a protein that is putatively involved in the regulation of the expression of phaP(Pd) Overproduction of Pha(Pd) was observed in Escherichia coli carrying phaP(Pd), but the overproduction was not observed in the presence of phaR(Pd). Coexpression of phaP(Pd) and PHA biosynthesis genes in E. coli caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB. GA16 protein was considered a phasin protein. The phaR(Pd) gene had significant similarities to stdC, a possible transcriptional factor of Comamonas testosteroni,as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria.