The TACAN4TGCA motif upstream from the -35 region in the sigma70-sigmaS-dependent Pm promoter of the TOL plasmid is the minimum DNA segment required for transcription stimulation by XylS regulators

Abstract

Transcription from the TOL plasmid meta-cleavage pathway operon promoter Pm is dependent on the XylS regulator activated by benzoate effectors or after XylS overproduction. We have generated 5' deletions in Pm and have analyzed expression from wild-type and mutant promoters with the wild-type XylS regulator and XylS mutant regulators that stimulated transcription constitutively. We have found that the motifs T(C or A)CAN4TGCA located between -46 and -57 and -67 and -78 with respect to the main transcription initiation point are required for maximal stimulation of transcription from Pm with effector-activated wild-type XylS. Deletion of the farthest TCCA submotif decreased but did not abolish transcription mediated by the pair XylS with 3-methylbenzoate; however, removal of the motif between -67 and -78 resulted in the loss of stimulation by the wild-type regulator. XylSG44S and XylSS229I stimulated high levels of transcription in the absence of effectors from the wild-type promoter and from a mutant promoter exhibiting only the -46 to -57 motif only when an effector was present. The point mutation Pm5U (with C-47 replaced by G [C-47-->G]) and Pm4 (C-68-->G), located in each 3' TGCA submotif of each motif, resulted in a 90% decrease in transcription stimulation with wild-type XylS; however, the mutant XylSS229I stimulated high levels of transcription from the point mutation promoters both in the presence and in the absence of effectors, while mutant XylSG44S suppressed the two point mutations only with 3-methylbenzoate. Overexpression of XylS and XylSG44S allowed the two regulators to stimulate high levels of transcription from the wild-type promoter, the point mutation Pm4 and Pm5U promoters, and deltaPm promoters exhibiting at least the -46 to -57 motif. Therefore the TACAN4TGCA motif between -46 and -57 represents the minimal DNA segment required for stimulation of transcription from Pm.