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00925njm 22002777a 4500 dc Gayetri Ramachandran, Andrés author Miguel Arribas, Andrés author Abia, David author Singh, Praveen K. author Crespo, Isidro author Gago-Córdoba, César author Hao, Jian An author Luque-Ortega, Juan Román author Alfonso, Carlos author Wu, Ling J. author Boer, Roeland author Meijer, Wilfried J. J. author 2017 Altres ajuts: MITYC/Bio2016-77883-C2-1-P Altres ajuts: MITYC/Bio2016-77883-C2-2-P Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named rel , is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the Rel shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to Rel. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, Rel constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research. Antibiotics have saved the lives of millions. However, the emergence and spread of antibiotic resistance compromises the effectiveness of antibiotics. Genes conferring antibiotic resistance are often located on genetic elements that can be transferred to other bacteria. Conjugation is the predominant route responsible for spreading antibiotic resistance genes, and depends critically on a class of proteins called relaxases. These enzymes initiate conjugation by processing the DNA of the mobile element and are therefore an Achilles' heel of the conjugation process. Although antibiotic resistance is an important health threat in both Gram-negative and Gram-positive bacteria, conjugation has so far been studied primarily in Gram-negative bacteria. Due to the extremely high concentration of bacteria, which favors conjugation, the intestinal gut is a hotspot for spreading antibiotic resistance. It is now known that a large part of the gut microbiome corresponds to Gram-positive bacteria, and many of these belong to the phylum Firmicutes. To better understand conjugation and specifically relaxases of Gram-positive conjugative elements we have identified and characterized the relaxase and the DNA region at which it acts of the conjugative plasmid pLS20 from the Gram-positive Firmicute Bacillus subtilis. We also show that the relaxase of pLS20 is the founding member of a new and large family of relaxases that is almost exclusively present in Firmicutes bacteria. This work will have important implications for the transfer of genes in Gram-positive bacteria in general and specifically for conjugation-mediated spread of antibiotic resistance in Firmicutes bacteria of the intestinal gut. Relaxases Sequence motif analysis Horizontal gene transfer Plasmid construction Sequence databases Polymerase chain reaction Sequence similarity searching Antibiotic resistance Discovery of a new family of relaxases in Firmicutes bacteria