Structural organization of pRAM4, a cryptic plasmid from Prevotella ruminicola

A total of 530 strains of rumen bacteria were screened for the presence of plasmid DNA. The percentage of plasmid-bearing strains was found to be the highest among the Bacteroides/ Prevotella group (9.9%), while it was less than 1% in the Butyrivibrio (0.2%) and Clostridium (0.6%) genera. A small cryptic plasmid pRAM4 from Prevotella ruminicola T31 was subcloned in Escherichia coli and completely sequenced. Two open reading frames, encoding potential polypeptides of M(r) 32,322 (ORF1) and 32,122 (ORF2) with limited sequence similarity to replication initiation and mobilization proteins, respectively, could be identified within the sequence. The region upstream from ORF1 had an AT-rich (75%) region followed by four 22-bp direct repeats, a structure characteristic of replication origins. The plasmid hybridized at high stringency with plasmids from Bacteroides/Prevotella and Butyrivibrio, and with pBR322, suggesting that at least regions of the plasmid are widespread.