The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, 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 TGCAN6GGNCA. 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.
This study was supported by European Commission Grant BIO-CT97-2313 and Comisión Interministerial de Ciencia y Tecnologı́a of Spain Grant BIO97-0641.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
