Plasmid electroporation of Sinorhizobium strains: The role of the restriction gene hsdR in type strain Rm1021 Lorenzo Ferri 1 , Alessandro Gori, Emanuele G. Biondi, Alessio Mengoni, Marco Bazzicalupo * Dept. of Evolutionary Biology, University of Florence, via Romana 17-19, I-50125 Firenze, Italy article info Article history: Received 24 November 2009 Revised 13 January 2010 Available online 22 January 2010 Communicated by Manuel Espinosa Keywords: Transformation Restriction hsdR Gene transfer Sinorhizobium abstract Although horizontal gene transfer mediated by plasmids is important to the generation of the genetic variability of Sinorhizobium strains, the barriers which can reduce horizontal gene transfer between bacteria have not yet been studied in Sinorhizobium. We studied the plasmid transfer by electroporation and its restriction in strains of Sinorhizobium melil- oti and S. medicae. After conditions for electroporation were established, three S. meliloti strains (including the sequenced type strain Rm1021) and two S. medicae strains were elec- troporated with plasmid DNA extracted from strains of both species. The efficiency of transformation was found to be variable among different strains. The acquisition of plas- mid DNA was found to be donor-dependent in S. meliloti strain Rm1021 that prefers self- DNA more than the DNA from other Sinorhizobium strains. All other strains tested did not show a preference for self-DNA. In strain Rm1021, the inactivation of the hsdR gene, coding for a putative type-I restriction enzyme, increased the efficiency of transformation and conjugation with non-self DNA; the transformation capability was again reduced in hsdR mutant when the cloned hsdR gene was expressed from a lac promoter. Phylogenetic analysis of the hsdR gene clearly indicated that this gene was horizontally transferred to strain Rm1021, explaining its absence in the other strains tested. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Since horizontal gene transfer (HGT) is an important mechanism of bacterial genome evolution (Gogarten et al., 2002), barriers that limit it can induce bacterial ge- netic isolation (Matic et al., 1996). Plasmids are the main vectors of HGT in several bacterial species and in rhizobia, where they contribute to a relevant fraction of the genome and to the large genomic diversity of natural isolates (Hau- kka et al., 1998; Silva et al., 2007; Pistorio et al., 2008). Fac- tors that can reduce horizontal gene transfer between bacteria, besides geographical separation, are: surface exclusion barriers, cytoplasmic barriers such as restriction endonucleases, DNA divergence and homologous recombi- nation (Matic et al., 1996; Berndt et al., 2003; Thomas and Nielsen, 2005). Restriction–modification (R–M) systems, comprising restriction endonucleases and methyltransfer- ases, recognize and modify specific DNA sequences, pro- tecting ‘‘self” DNA from restriction while eliminating potentially harmful ‘‘foreign” DNA which lacks appropriate modification (Murray, 2000). There are three distinct well- characterized types of R–M systems, including type-I restriction enzymes, which cut DNA within specific recog- nition sequences and are therefore widely used as tools for molecular biology (Bickle and Kruger, 1993). Type-I R–M systems require the products of three genes, hsdR (restric- tion), hsdM (modification), and hsdS (sequence specificity), and cut DNA at sites remote from the recognition sequence (Murray, 2000). It has been suggested that Restriction– modification systems are related to mobile genetic ele- ments (Kobayashi et al., 1999) which can spread among different bacterial species. 0147-619X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2010.01.001 * Corresponding author. Fax: +39 0552288250. E-mail address: marco.bazzicalupo@unifi.it (M. Bazzicalupo). 1 Present address: Metabolic and Muscular Unit, Pediatric Neurology Clinic, AOU Meyer, Viale Pieraccini, 24, Firenze, Italy. Plasmid 63 (2010) 128–135 Contents lists available at ScienceDirect Plasmid journal homepage: www.elsevier.com/locate/yplas