Analysis of transcription of the bph locus of Burkholderia sp. strain LB400 and evidence that the ORF0 gene product acts as a regulator of the bphA1 promoter

Abstract

Although gene clusters for the degradation of biphenyls and polychlorobiphenyls have been extensively characterized, comparatively little is known about the regulation of their expression. In the present work, different aspects of transcription of the bph locus of the potent polychlorobiphenyl degrader Burkholderia sp. strain LB400 were investigated. An RNA blot analysis of the entire gene cluster revealed that the transcription of all genes encoding biphenyl catabolic enzymes responded similarly to the presence of biphenyl, succinate or a mixture of the two. One region of the locus, encompassing ORF0, was separately transcribed and differently regulated. A single start position was mapped for this monocistronic transcript. Synthesis of the adjacent RNA, encoding subunits of biphenyl dioxygenase, was strongly biphenyl-inducible. In this case, four major 5'-ends were mapped between 25 and 70 bp upstream of the start codon of gene bphA1. Sequence elements between approximately positions 710 and 1080 upstream were required in cis for full functioning of the respective promoter(s) (P bphA1 ). ORF0 N mutants of strain LB400 retained the ability to grow on biphenyl, but showed decreased concentrations of bphA1A2 RNA and decreased lacZ expression in strains harbouring a reporter system with a bphA1-lacZ transcriptional fusion. This effect was compensated by the introduction of an intact ORF0 in trans, indicating that the ORF0 gene product mediates activation of P bphA1 .

Growth Substrate- and Phase-Specific Expression of Biphenyl, Benzoate, and C 1 Metabolic Pathways in Burkholderia xenovorans LB400

“…Moreover, the studies confirmed the bph pathway (Fig. 1B) was induced during growth on biphenyl, consistent with one published report, but not another (2,24). Finally, it was revealed that C 1 metabolic pathways were induced upon transition towards stationary phase.…”

Biphenyl and Benzoate Metabolism in a Genomic Context: Outlining Genome-Wide Metabolic Networks in Burkholderia xenovorans LB400

“…These authors also suggested that there are two constitutive 70 -dependent promoters upstream of bphA1. However, mutational studies and Northern blot analyses performed by Beltrametti et al (2), who proposed that ORF0 is a positive regulator of bphA1A2, and our observation of increased expression ratios of the bph genes with increased ORF0 expression indicate that there is a positive regulatory function of ORF0 induced by biphenyl. The similarity in expression patterns among ORF0, bphJ, and bphI and possibly bphD suggests that ORF0 is an inducer of bphJ, bphI, and bphD.…”

“…Burkholderia xenovorans strain LB400 (17) is one of the most-studied and effective aerobic PCB degraders known and is able to cometabolically degrade up to hexachlorinated biphenyls (3,8,12,18,22,(31)(32)(33). Although the operon organization for the upper biphenyl degradation pathway (the upper Bph pathway) has been extensively documented, there are contradictory reports on its regulation, suggesting that there is constitutive (5,25) or biphenyl-induced expression (2,12,27). The enzymatic reactions of the upper Bph pathway result in formation of benzoate and pentadiene or their chlorinated derivatives.…”

Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

“…The transcription of the bph locus of Burkholderia sp. strain LB400, whose bph genes are very similar to those of KF707, was investigated (9). In this system, the ORF0 protein (corresponding to KF707 BphR1) mediates the activation of the bphA1 promoter.…”