THE TACAN(4)TGCA MOTIF UPSTREAM FROM THE -35 REGION IN THE SIGMA(70)-SIGMA(S)-DEPENDENT PM PROMOTER OF THE TOL PLASMID IS THE MINIMUM DNA SEGMENT REQUIRED FOR TRANSCRIPTION STIMULATION BY XYLS REGULATORS

Transcription from the TOL plasmid meta-cleavage pathway operon promot er Pm is dependent on the XylS regulator activated by benzoate effecte rs 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 stimulate d transcription constitutively. We have found that the motifs T(C or A )CAN, TGCA located between -46 and -57 and -67 and -78 with respect to the main transcription initiation point are required for maximal stim ulation of transcription from Pm,vith effector-activated wild-type Xyl S, Deletion of the farthest TCCA submotif decreased but did not abolis h transcription mediated by the pair XylS with 3-methylbenzoate; howev er, removal of the motif between -67 and -78 resulted in the loss of s timulation by the wild-type regulator, XylSG44S and XylSS229I stimulat ed high levels of transcription in the absence of effecters from the w ild-type promoter and from a mutant promoter exhibiting only the -46 t o -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 e ach 3' TGCA submotif of each motif, resulted in a 90% decrease in tran scription stimulation with wild-type XylS; however, the mutant XglSS22 9I stimulated high levels of transcription from the point mutation pro moters both in the presence and in the absence of effecters, while mut ant XylSG44S suppressed the two point mutations only with 3-methylbenz oate. Overexpression of XylS and XylSG44S allowed the two regulators t o stimulate high levels of transcription from the wild-type promoter, the point mutation Pm4 and Pm5U promoters, and Delta Pm promoters exhi biting at least the -46 to -57 motif. Therefore the TACAN(4)TGCA motif between -46 and -57 represents the minimal DNA segment required for s timulation of transcription from Pm.