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Journal of Bacteriology, May 1999, p. 2914-2921, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Analyses of a Polyhydroxyalkanoic Acid Granule-Associated 16-Kilodalton Protein and Its Putative Regulator in the pha Locus of Paracoccus denitrificans

Akira Maehara,1 Shunsaku Ueda,2 Hideo Nakano,1 and Tsuneo Yamane1,*

Laboratory of Molecular Biotechnology, Division of Molecular Cell Mechanisms, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601,1 and Laboratory of Applied Microbiology, Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505,2 Japan

Received 3 September 1998/Accepted 10 February 1999

The polyhydroxyalkanoic acid (PHA) 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 (phaCPd) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774-779, 1996). Gene walking around phaCPd revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the phaPPd gene, encoding GA16 protein, and the other was the phaRPd gene, encoding a protein that is putatively involved in the regulation of the expression of phaPPd. Overproduction of PhaPPd was observed in Escherichia coli carrying phaPPd, but the overproduction was not observed in the presence of phaRPd. Coexpression of phaPPd 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 phaRPd 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.


* Corresponding author. Mailing address: Laboratory of Molecular Biotechnology, Division of Molecular Cell Mechanisms, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Phone: 81-52-789-4142. Fax: 81-52-789-4145. E-mail: yamanetu{at}agr.nagoya-u.ac.jp.


Journal of Bacteriology, May 1999, p. 2914-2921, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.



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