Versatile Transcription of Biphenyl Catabolic bphOperon in Pseudomonas pseudoalcaligenes KF707

Takahito Watanabe,Ryuichi Inoue,Nobutada Kimura,Kensuke Furukawa

doi:10.1074/jbc.m003023200

Abstract

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.

Highlights

The biphenyl-utilizing bacteria have been widely isolated from various environmental samples, which include both Gram-negative and Gram-positive bacteria
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
E. coli S17–1 cells carrying pSUPB101 were filter-mated with KF707 overnight. pSUPB101 cannot replicate in Pseudomonas strains; single cross-over recombinants were first screened on basal salt medium (BSM) agar plates supplemented with 0.1% (w/v) succinate, Tc (30 ␮g/ml), and gentamicin (20 ␮g/ml)

Summary

EXPERIMENTAL PROCEDURES

The biphenyl-utilizing strain P. pseudoalcaligenes KF707 was grown in basal salt medium (BSM) supplemented with 0.2% (w/v) biphenyl as a sole source of carbon as described previously [1]. Biphenyl-utilizing defective derivatives of KF707 such as KF730 (bphA1::Tn5-B21), KF748 (bphB::Tn5-B21), and KF744 (bphC::Tn5-B21) were previously constructed [20], and KF7095 (orf0::TcR) was constructed in this study. They were grown in BSM supplemented with 0.1% (w/v) sodium succinate and tetracycline (Tc)

The abbreviations used are

RESULTS

DISCUSSION