Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants

The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effecters, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNA-polymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the -40 to -70 region, In vivo and in vitro methylation of Pm shows extensive methylation of T at position -42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/RNA polymerase interactions. Methylation of T-42 was highest in cells bearing XylS and in the presence of an effector. Gs in the -47 to -61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between -41 and -78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN(6)GGNCA. These motifs cover the -70 to -56 and the -49 to -35 regions. Single point mutations revealed that nucleotides located at -49 to -46 and at -59, -60, -62, and -70 are the most critical for appropriate XylS-Pm interactions.