Biochemical and genetic analysis of the [gamma]-resorcylate (2,6-dihydroxybenzoate) catabolic pathway in Rhizobium sp. strain MTP-10005: identification and functional analysis of its gene cluster

We identified a gene cluster that is involved in the [gamma]-resorcylate (2,6-dihydroxybenzoate) catabolism of the aerobic bacterium Rhizobium sp. strain MTP-10005. The cluster consists of the graRDAFCBEK genes, and graA, graB, graC, and graD were heterologously expressed in Escherichia coli. Enzymological studies showed that graD, graA, graC, and graB encode the reductase (GraD) and oxygenase (GraA) components of a resorcinol hydroxylase (EC 1.14.13.x), a maleylacetate reductase (GraC) (EC 1.3.1.32), and a hydroxyquinol 1,2-dioxygenase (GraB) (EC 1.13.11.37). Bioinformatic analyses suggested that graE, graR, and graK encode a protein with an unknown function (GraE), a MarR-type transcriptional regulator (GraR), and a benzoate transporter (GraK). Quantitative reverse transcription-PCR of graF, which encodes [gamma]-resorcylate decarboxylase, revealed that the maximum relative mRNA expression level ([5.93 [+ or -] 0.82] x [10.sup.-4]) of graF was detected in the total RNA of the cells after one hour of cultivation when [gamma]-resorcylate was used as the sole carbon source. Reverse transcription-PCR of graDAFCBE showed that these genes are transcribed as a single mRNA and that the transcription of the gene cluster is induced by [gamma]-resorcylate. These results suggested that the graDAFCBE genes are responsible as an operon for the growth of Rhizobium sp. strain MTP-10005 on [gamma]-resorcylate and are probably regulated by GraR at the transcriptional level. This is the first report of the [gamma]-resorcylate catabolic pathway in an aerobic bacterium.