Recent Papers

ABSTRACT

Pseudomonas putida TW3 is able to catabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3,4-dihydroxybenzoic acid (protocatechuate) with the release of the nitro group as ammonium. The genes for the enzymes of 4NBen catabolism are separated on two regulons. One encodes 4NBen reductase (PnbA), catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate, and the second  4-hydroxylaminobenzoate lyase (PnbB), catalyzing the conversion of 4-hydroxylaminobenzoate to protocatechuate (3,4-dihydroxybenzoate) and ammonium.  Between pnbA and pnbB and transcribed in the opposite direction is  pnbR which encodes a LysR-family regulator. A frame-shift deletion within pnbR eliminated synthesis of PnbA. The sites for the start of transcription of pnbA and pnbB were mapped 35 and 27 bp upstream of the respective initiation codons by primer extension analysis. The regions upstream of both transcription starts showed significant homology and possible DNA binding sites for PnbR were identified. These had a conserved TCGA-N5-TCGA motif typical of the binding motif characteristic of LysR regulators and a third conserved TGCA closer to the transcription start. Alteration by base substitutions of two of the three TGCA repeats (R1 and R2) resulted in a major reduction of the inducibility of both PnbA and PnbB activities by 4NBen. Whereas the alteration of each R1 totally destroyed the ability of 4NBen to induce the downstream enzyme, the changes in R2 still allowed a limited degree of induction, but less than with wild type suggesting that R1 is more dominant tetrad in the PnbR-DNA interaction. These studies implicate PnbR as the regulator of both regulons of pnb genes and hence of 4-nitrobenzoate catabolism in TW3.