Deep sequencing of the cryptic plasmid mobilome in the symbiotic bacterium Sinorhizobium meliloti.

LÓPEZ, J. L.; GIUSTI, M. A.; MARTINI, C; TORRES TEJERIZO, G. A.; LOZANO, M. J.; SALAS, M, E.; SALTO, I. P.; PUHLER, A.; DEL PAPA, M.F.; SCHLÜTER, A.; PISTORIO, M.; LAGARES, A.

Santander

Congreso; International Plasmid Biology Conference; 2012

International Society for Plasmid Biology

Background and aims. Rhizobia are alfa- and beta- proteobacteria that inhabit soils, and are able to establish specific N2-fixing symbioses with legumes. Current information shows that rhizobia are significantly diverse including different bacterial genera, and that in several cases bear significant amounts of plasmid DNA ranging from few kilobases to megabases in size. Many of those plasmids are currently being characterized at the molecular level, and show to be active vectors for horizontal gene transfer (HGT) in the processes of rhizobial diversification/speciation (Cervantes et al. 2011; Torres Tejerizo et al. 2011). Methods. In a survey to investigate plasmid diversity in S. meliloti, the N2-fixing symbiont of alfalfa, we purified more than forty (mid- to high- molecular weight) cryptic plasmids which were used here to characterize, by deep sequencing, the mobilome gene content at the species level. Plasmids that sized approx. between 50-400 kb were prepared by CsCl gradients, sequenced using the Ion Torrent Technology, and the collected data analyzed via the metaSAMS software package. Results. We collected more than 0.25 Gb with 2 Mb of non-redundant plasmid sequence data. The number of read corresponding to housekeeping genes indicated that low contamination with chromosomal DNA (ca. 5-10%) was present in the purified HMW plasmid-DNA pool. Concerning functional elements of the plasmids, data mining allowed for the recovery of 6 RepA, 7 RepB, and 5 RepC complete sequences, together with 6 putative relaxases (likely due to sequence frame-shifts, 20 additional split Rep/TraA sequences were also found). The phylogenetic analysis of the replication and mobilization elements showed an intricate distribution of sequences when compared to homologous elements from other rhizobia. The diversity of relaxases found (MOBP and MOBQ-like proteins usually present in rhizobia, as well as relaxases from other families) contrasts with previous results from our laboratory where relaxases from the recently described MOBP0 subfamily were thought to be dominant. Conclusions. To our knowledge this is the first deep sequencing of a plasmid pool aiming at the characterization of a plasmid mobilome at the species level. The approach proved to be feasible, with deep penetrance at the scale of single gene analysis. We are currently investigating the kind of metabolic genes present in the rhizobial plasmid mobilome, and their relationships with the rhizobial life-style. References. Cervantes L et al. (2011) The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain. BMC Microbiol 11:149. Torres Tejerizo GA et al. (2011) First genomic analysis of the broad-host-range Rhizobium sp. LPU83 strain, a member of the low-genetic diversity Oregon-like Rhizobium sp. group. J Biotechnol 155:3-10.