TraM protein of plasmid R1: In vitro selection of the target region reveals two consensus 7 bp binding motifs spaced by a 4 bp linker of defined sequence

The TraM protein encoded by plasmid R1 has three functions in conjugative transfer: (i) it positively controls transfer gene expression, (ii) it stimulates efficient site-specific ssDNA cleavage at the oriT in vivo and (iii) it couples the relaxosome to the envelope-bound transport complex. Plasmid R1 contains two binding regions for TraM, sbmA and sbmB, either of which comprises several minimal TraM targets. SELEX of a randomized minimal (18 bp) TraM binding sequence was used to search for sequences that bind TraM most efficiently. This approach resulted in a sequence with a modular structure with two 7 bp consensus motifs 5′ T1G2A3N4T5C6R7 and 5′ T1G2A3N4T5Y6R7 spaced by a 4 bp linker with the consensus sequence 5′ Y1A2/C2T3/G3A4. EMSAs revealed that evolution in vivo was for maximal binding affinity. R1 derivatives with mutated sbmA and sbmB sites based on one in vitro selected sequence were found to be either 5-fold impaired (mut sbmA) in conjugation or could not be transferred by conjugation anymore (mut sbmB). Transcriptional lacZ fusions demonstrated that the introduced sbmB mutations affected promoter activity severely and abrogated autoregulation by TraM, thereby explaining this drastic effect on conjugal transfer. The implications of these findings are discussed in the context of the role of TraM.