An additional regulator, TsaQ, is involved with TsaR in regulation of transport during the degradation of p-toluenesulfonate in Comamonas testosteroni T-2.

摘要

The degradation of p-toluenesulfonate (TSA) by Comamonas testosteroni T-2 is initiated by a transport system (TsaST) and enzymes (TsaMBCD) encoded on the tsa transposon, Tn tsa, on the TSA plasmid (pTSA). Tn tsa comprises an insert of 15 kb between two IS 1071 elements. The left-hand 6 kb and the right-hand 6 kb are nearly mirror images. The regulator of the tsaMBCD1 genes (right-hand side) is the centrally located LysR-type TsaR, which is encoded upstream of tsaMBCD1 on the reverse strand. The other centrally located genes are tsaS and tsaT, encoded downstream of tsaR and on the same strand as both tsaR and tsaMBCD2. The latter four genes are not expressed. Downstream of tsaD1 (tsaD2) is tsaQ1 (tsaQ2) and another open reading frame of unknown function. The tsaQ genes have identical sequences. Sequence analysis indicated that TsaQ could be an IclR-type regulator, whose expression during degradation of TSA was proven by data from RT-PCR. Both copies of tsaQ could be knocked-out by homologous recombination. Double mutants failed to grow with TSA but grew with p-toluenecarboxylate (TCA), which is also degraded via TsaMBCD. This showed TsaQ to be essential for the degradation of TSA but not TCA. We attributed this to regulation of the transport of TSA, especially to regulation of the expression of tsaT, which was expressed solely during growth with TSA. Seven independently isolated bacteria containing the tsa operon were available. Those six which contained tsaT on Tn tsa also contained tsaQ. The promoter region of tsaT was found to be a target of the regulator TsaR. Band-shift data indicate that TsaR is required for the expression of tsaT, which suggests that tsaR and tsaQ(1,2), together with tsaMBCD1, belong to a common regulatory unit.