title: Discovery of a new family of relaxases in Firmicutes bacteria.
abstract: 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 relLS20, 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 RelLS20 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 RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 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.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Gayetri
authors.initials: G
authors.lastname: Ramachandran
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Andrés
authors.identifier: 0000-0001-5679-9083
authors.initials: A
authors.lastname: Miguel-Arribas
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: David
authors.identifier: 0000-0003-0401-7590
authors.initials: D
authors.lastname: Abia
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Singh
authors.affiliation: XALOC beamline, ALBA synchrotron Light Source, Cerdanyola del Vallès, Barcelona, Spain.
authors.forename: Isidro
authors.identifier: 0000-0001-7698-1720
authors.initials: I
authors.lastname: Crespo
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: César
authors.identifier: 0000-0001-7470-4033
authors.initials: C
authors.lastname: Gago-Córdoba
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Jian An
authors.initials: JA
authors.lastname: Hao
authors.affiliation: Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
authors.forename: Juan Roman
authors.initials: JR
authors.lastname: Luque-Ortega
authors.affiliation: Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
authors.forename: Carlos
authors.identifier: 0000-0001-7165-4800
authors.initials: C
authors.lastname: Alfonso
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, United Kingdom.
authors.forename: Ling J
authors.initials: LJ
authors.lastname: Wu
authors.affiliation: XALOC beamline, ALBA synchrotron Light Source, Cerdanyola del Vallès, Barcelona, Spain.
authors.forename: D Roeland
authors.initials: DR
authors.lastname: Boer
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Wilfried J J
authors.identifier: 0000-0003-1842-0049
authors.initials: WJ
authors.lastname: Meijer
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics*; D003227:Conjugation, Genetic; D004277:DNA, Single-Stranded / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification; D000068536:Bacillota / Q000201:enzymology* / Q000235:genetics; D000069196:Gastrointestinal Microbiome / Q000235:genetics; D022761:Gene Transfer, Horizontal; D006801:Humans; D010957:Plasmids / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004706:Endodeoxyribonucleases