New two-component regulatory system required for the constitutive expression of bph operon in Cupriavidus basilensis WS.

Cupriavidus basilensis WS degrades diphenyl ether (DE) and its lower brominated derivatives using enzymes encoded by the bph operon. However, it is not yet known under what circumstances bph genes are expressed and how they are regulated in C. basilensis WS. To answer these questions, we used transposon mutagenesis and identified a new two-component regulatory system, BphS/BphT, in C. basilensis WS, which is indispensable for the expression of the bph operon. When BphS or BphT is inactivated, C. basilensis WS no longer exhibits the ability to decompose DE. Using a β-galactosidase reporter system and RT-qPCR, we showed that bph genes are constitutively transcribed in C. basilensis WS and that deletion of bphS or bphT strongly inhibited the transcription of bph genes. We also showed that the gene ORF0, which is upstream of bphA1 and is similar to the GntR-family regulators of the bph operon, is not involved in the constitutive transcription of the bph operon in C. basilensis WS. The cis-acting elements required for the expression and regulation of bph genes in the DE degradation pathway are included in the intergenic region between ORF0 and bphA1. Our results suggest that BphS/BphT represents a new two-component regulatory system for the bph operon that is necessary for the constitutive expression of bph genes.