Relationship between <i>oriT</i> length and efficiency of RP4-mediated conjugation from <i>Escherichia coli</i> to Gram-positive bacteria

In RP4 conjugation, approximately 350 bp of the origin of transfer (oriT) is required for transfer. Within this oriT, there are binding regions for the transfer-related proteins TraI, TraK, and TraJ. We investigated the influence of deleting each protein-binding region within oriT on transfer efficiency in Escherichia coli, Streptomyces lividans, and Bacillus subtilis. The deletion of the TraI-binding region completely abolished transfer in all species. The partial deletion of the TraK-binding region had a minimal impact when targeting but affected efficiency when targeting B. subtilis. The deletion of the TraJ-binding region completely abolished transfer in E. coli and B. subtilis but only reduced efficiency in S. lividans. This is the first report to investigate the influence of each region within oriT on transfer efficiency in S. lividans and B. subtilis, suggesting that the length of oriT required for effective RP4 conjugation varies when targeting Gram-positive bacteria.