Characterization, sequence, and replication of a small cryptic plasmid from Selenomonas ruminantium subspecies lactilytica.

A 2.5-kb cryptic plasmid, pJDB21, from the gram-negative ruminal anaerobe, Selenomonas ruminantium subspecies lactilytica, was mapped and sequenced. Five open reading frames (ORFs) were predicted and expression of two ORFs was demonstrated. Analysis of the predicted amino acid sequence of the ORF 1 protein indicated approximately 30% homology with the replication protein (rep) common to many gram-positive plasmids, and a highly conserved sequence representing the origin of replication in these plasmids was located upstream of ORF 1. This finding was consistent with a rolling circle form of replication for pJDB21. Transformation of Escherichia coli K-12 UB1636pol Ats with pJDB21 showed that the plasmid replicated independently of DNA polymerase I and produced a single-stranded DNA intermediate. Deletion analyses localized the E. coli replication function to a 1.4-kb sequence that was mapped to the predicted rep gene.