Inverse PCR primers amplifying the rest of the plasmid molecule were designed, and after the amplification reaction, we obtained a product of about 900 bp. No ORF was found on this PCR fragment, but comparison with the GenBank database showed considerable homology (80%) to the plasmid pSRD191 on a DNA
stretch of about 450 bp downstream of the gene for replication protein. In addition to this, we detected limited homology to other plasmids from S. ruminantium, particularly to pONE429 and pONE430, pSRD192, pS23 (M86247) and pJJM1 (Z49917), which was mainly found around the location of SRSR elements of plasmids. This plasmid was designated pSRD77, and its complete nucleotide sequence was found to be 1470 bp in length with an overall GC
content of 46.5% and one open reading frame at nucleotides stretching from 260 to 790 encoding a putative replication protein belonging to RepL family Protein Tyrosine Kinase inhibitor of replication proteins. Studying plasmid selleck kinase inhibitor rep modules is a good approach to assess plasmid biodiversity and/or the evolution of these molecules (Guglielmetti et al., 2005), especially in the case of RCR plasmids that are made as interchangeable gene modules (Novick, 1989). The replication modules of RCR plasmids are made up by the gene encoding for the initiator protein (Rep) and sequences with high secondary structures containing both the binding- and nick-site for the initiator (double-strand origin, dso). Based on similarities of rep modules, RCR plasmids have been divided into several groups, but these groups usually do not correlate with similarities in plasmid single-strand origins (sso), region where replication of the lagging strand begins. High homologies between two different plasmids limited to their rep or other gene modules suggest that shuffling of modules has taken place during plasmid evolution. In this work in a PCR-based experiment, we analysed the genetic organization of putative plasmid rep modules of several S. ruminantium strains. A local collection of strains was included Clostridium perfringens alpha toxin in this study. However, it was
shown that plasmids isolated at different parts of the world shared striking similarities either in the organization of their rep modules or their whole genome (pONE-type vs. pSRD-type plasmids). pSRD-like plasmids were found to be widely distributed in our local set of strains, even though considerable structural instability of these plasmid molecules, respectively, their rep modules were observed in our experiments. While highly conserved rep genes were found among different S. ruminantium strains, in noncoding regions surrounding these genes, structural instabilities including deletions, insertions and other sequence alterations were seen. Selenomonas ruminantium Sequence Repeats (SRSR) sequence elements were found to be highly conserved and widespread among S. ruminantium plasmids originating from various ruminants and geographical locations.