Open AccessPhaR, a protein of unknown function conserved among short‐chain‐length polyhydroxyalkanoic acids producing bacteria, is a DNA‐binding protein and represses Paracoccus denitrificans phaP expression in vitro
Author(s) -
Maehara Akira,
Doi Yoshiharu,
Nishiyama Tatsuaki,
Takagi Yasuo,
Ueda Shunsaku,
Nakano Hideo,
Yamane Tsuneo
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb10685.x
Subject(s) - biology , electrophoretic mobility shift assay , escherichia coli , microbiology and biotechnology , biochemistry , binding protein , recombinant dna , gel electrophoresis , flag tag , molecular mass , gene , affinity chromatography , gene expression , fusion protein , enzyme
A putative regulatory protein, PhaR, which was identified in the polyhydroxyalkanoic acid synthetic locus ( phaZCPR ) in Paracoccus denitrificans , was investigated. The PhaR protein purified from a recombinant Escherichia coli was estimated to be 22 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, being consistent with the mass calculated from the nucleotide sequence. The molecular mass was determined to be 93 kDa by size‐exclusion chromatography, suggesting that the protein formed a tetramer. A gel mobility shift assay showed that PhaR specifically bound to the intergenic region of phaC – phaP . In a cell‐free protein synthesis system using E. coli S30 extract, the expression of the phaP gene was repressed by the addition of purified PhaR. These results suggest that PhaR is a DNA‐binding protein and may play a role in the regulation of phaP gene expression.