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The bphoperon of Pseudomonassp. KKS102 is constituted of 11 bphgenes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bphoperon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bphoperon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bphoperon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bphoperon.

The bphgenes inPseudomonassp. KKS102, which are involved in the degradation of polychlorinated biphenyl/biphenyl, are induced in the presence of biphenyl. In this study our goal was to understand the regulatory mechanisms involved in the inducible expression. Thebphgenes (bphEGF(orf4)A1A2A3BCD(orf1)A4R) constitute an operon, and its expression is strongly dependent on the pEpromoter located upstream of the bphEgene. A bphSgene, whose deduced amino acid sequence showed homology with the GntR family transcriptional repressors, was identified at the upstream region of the bphEgene. Disruption of the bphSgene resulted in constitutive expression of bphgenes, suggesting that thebphSgene product negatively regulated the pEpromoter. The gel retardation and DNase footprinting analyses demonstrated specific binding of BphS to the pEpromoter region and identified four BphS binding sites that were located within and immediately downstream of the −10 box of the pEpromoter. The four binding sites were functional in repression because their respective elimination resulted in derepression of the pEpromoter. The binding of BphS was abolished in the presence of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, an intermediate compound in the biphenyl degradation pathway. We concluded that the negative regulator BphS plays a central role in the regulation of bphgene expression through its action at the pEpromoter.