Versatile transcription of biphenyl catabolic bph operon in Pseudomonas pseudoalcaligenes KF707.
Pseudomonas pseudoalcaligenes KF707 possesses a chromosomally encoded bph gene cluster responsible for the catabolism of biphenyl/polychlorinated biphenyls. The gene cluster consists of (orf0)bphA1A2(orf3)bphA3A4BCX0X1X2X3D. We studied the role of orf0 and transcription in the KF707 bph operon. Primer extension analyses revealed that at least as many as six transcriptional initiation sites exist upstream of orf0, bphA1, bphX0, bphX1, and bphD, including two upstream of bphD. The orf0-disruptant failed to grow on biphenyl but accumulated large amounts of the biphenyl ring meta-cleavage yellow compound (2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate). Western blot analysis revealed that ORF0 protein is inducibly expressed in KF707 in the presence of biphenyl. Gel shift assay revealed that ORF0 directly binds to the orf0 operator region. This binding was greatly enhanced by addition of the biphenyl ring meta-cleavage yellow compound. These results indicated that orf0, bphA1A2(orf3)bphA3A4BC and bphX0X1X2X3D are independently transcribed, and that ORF0 protein belonging to the GntR family is involved in the regulation of the bph operon in KF707 and is absolutely required for the expression of orf0 and bphX0X1X2X3D.
Watanabe
T
T
Laboratory of Applied Microbiology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1 Fukuoka 812-8581, Japan.
Inoue
R
R
Kimura
N
N
Furukawa
K
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001713:Biphenyl Compounds / Q000378:metabolism; D015139:Blotting, Southern; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D049308:Dioxygenases; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006867:Hydrolases / Q000235:genetics*; D007506:Iron-Sulfur Proteins; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D010105:Oxygenases / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001713:Biphenyl Compounds; D004247:DNA; D017931:DNA Primers; D004268:DNA-Binding Proteins; D049308:Dioxygenases; D029968:Escherichia coli Proteins; D006867:Hydrolases; D007506:Iron-Sulfur Proteins; D010088:Oxidoreductases; D010105:Oxygenases; D012313:RNA; D012097:Repressor Proteins; D012260:Ribonucleases; D014157:Transcription Factors; C081598:GntR protein, E coli; C415364:orf0 protein, Pseudomonas pseudoalcaligenes; C010574:biphenyl; C074272:2,3-dihydroxybiphenyl oxygenase; C074161:biphenyl-2,3-dioxygenase; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; C062907:2,6-dioxo-6-phenylhexa-3-enoate hydrolase