Relaxasas conjugativas de la familia MOBv

201 p.-51 fig.-14 tab.Conjugation is the most important mechanism involved in horizontal genetic transfer between bacteria. This process relies on the exchange of genetic material through physical contact from a donor to a recipient cell. Therefore, it is a source of diversification and adaptation of microorganisms to different niches (Gogarten & Townsend, 2005). The conjugative transfer is carried out through three specialized modules: the relaxosome (MOB) integrated by the origin of transfer (oriT) and one or more proteins involved in the DNA processing, the coupling protein (T4CP), and the type IV secretion system (T4SS). Based on this modular composition, the conjugative elements can be classified as autotransferables or mobilizables. In general, the latter contain only the relaxosome, and can be transferred only when co-residing with an autotransferable element that provides the T4CP and T4SS (Smillie et al, 2010). Based on the genomic location, the conjugative elements can be extrachromosomal (plasmids), with autonomous replication capacity; or intrachromosomal (ICEs, Integrative and Conjugative Elements), integrated within the chromosome but with excision/integration abilities. When the latter are mobilizables, they are called IMEs (Integrative and Mobilizable Elements). Reviewed in (Bellanger et al, 2014). Regarding the relaxosome, the key elements involved in the conjugative mobilization of the DNA are the oriT sequence and the relaxase protein. OriTs are DNA sequences that contain stretches of nucleotides involved in DNA-protein interactions (inverted and/or directed repeats) and the cleavage site for the relaxase. Relaxases are proteins with endonuclease/topoisomerase activity that recognize the oriT sequence where they specifically cleave the DNA to initiate the conjugation (donor cell), or close the transferred DNA strand in the termination event (recipient cell). Up to now, all relaxases described use a tyrosine-mediated transesterification by a mechanism that shares similarities with the rolling circle replication (RCR). In general, the domain involved with DNA interaction (relaxase domain) is located at the N-terminal region of the protein, whereas the C-terminal moiety can contain helicase, primase or protein-protein interactions domains. Reviewed in (Chandler et al, 2013). The aminoacid sequence of the relaxases has been used to classify the conjugative elements (MOB families) since it provides a global view on the dissemination of these elements by conjugation (Garcillán-Barcia et al, 2009). The present work is focused on the characterization of two mobilizable elements that encode relaxases belonging to the MOBV1 family: pMV158 and IMErpsI.OBJECTIVES 1. To update the MOBV1 family of plasmids, their distribution and phylogenetic relationships with emphasis on their transfer modules. 2. To characterize the relaxase domain of MobM protein, encoded by plasmid pMV158, at its structural and functional level. 3. To study the global interactions between MobM and the DNA of plasmid pMV158. 4. To characterize a newly discovered integrative element and the relaxase of the MOBV1 family it encodes. RESULTS AND CONCLUSIONS 1. PLASMIDS OF THE MOBV1 FAMILY A bioinformatic survey of plasmids belonging to the MOBV1 family (using relaxase MobMpMV158 as the prototype) allowed the identification of 97 relaxases codified by 93 different plasmids. All these plasmids are mobilizable and were isolated from G+ bacteria, mainly from phylum Firmicutes. Almost half of the plasmids (45%) encode antibiotic resistance or other determinants, and up to 80% contain RCR initiator proteins. The transfer modules of MOBV1 plasmids show that inverted repeats and putative nick sites within their oriT sequences are highly conserved. However, the relaxases encoded by these plasmids present a low overall similarity, although the three characteristic motifs of the MOBV1 family are conserved. 2. THE RELAXASE DOMAIN OF MobM Purification and characterization of a new N-terminal truncated version of MobM (that contains its relaxase domain) has shown that the DNA binding and cleavage activities are located in the first 200 residues, whereas the central region (200-243) is important in DNA positioning within the active center of the protein. The oligomerization domain is placed in the C-terminal region (243-494). Resolution of the structures of MobM with several DNA substrates together with construction of protein mutant derivatives and functional analyses revealed that the catalytic residue of MobM is the histidine at position 22 (H22). This is the first report on a relaxase that uses a residue other than tyrosine to perform the DNA nucleophilic attack.3. INTERACTION OF MobM WITH THE REPLICATION AND CONTROL MODULE OF PLASMID pMV158 Electron microscopy analyses have unveiled that MobM could bind to plasmid pMV158 in a region located within the replication-control module. This region was mapped by EMSA and DNase I footprinting assays and was located within the promoter region of gene rnaII. RNAII controls pMV158 copy number at a traductional level. In vivo experiments showed that MobM inhibits transcription of RNAII, thus reducing the intracellular levels of RNAII and, consequently, increasing plasmid copy number. We believe that our findings constitute a novel mechanism of regulation of plasmid copy number by a relaxase and demonstrate the existence of intramolecular communications between the mobilization and the replication-control gene modules of a plasmid. 4. A NEW MOBILIZABLE ELEMENT OF S. agalactiae Bioinformatic searches of MOBV1 relaxases in bacterial chromosomes has revealed the existence of a putative MobM homolog (MobSag) located within a mobilizable element (IMErpsI) of S. agalactiae. The present work has shown that IMErpsI has the ability to excise from the chromosome as well as the in vivo functionality of its transfer module (oriTIMErpsI and mobSag gene). The MobSag protein characterization revealed that its N-terminal domain encompasses the three characteristic motifs of the MOBV1 family of relaxases. MobSag has in vitro relaxase activity, since it is able to relax supercoiled plasmid DNA harbouring oriTIMErpsI. Cleavage of supercoiled DNA by MobSag depends on the protein dose, having optimal activity at 50ºC, 20 min. Relaxation of scDNA by MobSag is strand and site-specific (leader strand and 5'-GpT-3' site), corresponding with the predicted nick site for the oriTs of plasmids belonging to the MOBV1 family. Further, MobSag is active in vivo, since it mediates the conjugative transfer of a supercoiled plasmid that contains its oriTIMErpsI.Proyecto de Unión Europea (EU-CP223111, CAREPNEUMO)Proyecto Consolider (CSD2008-00013, INTERMODS) de los Ministerios de Economia y competividad (MINECO) y Ciencia e Innovación (MICINN)Proyectos BFU2008-00179-E/BMC (REDEEX-1) (MICINN) y BFU2011-14145-E (REDEEX-2) (MINECO)Peer reviewe