STRUCTURAL ORGANIZATION OF PRAM4, A CRYPTIC PLASMID FROM PREVOTELLA-RUMINICOLA

A total of 530 strains of rumen bacteria were screened for the presenc e 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 r eading frames, encoding potential polypeptides of M(r) 32,322 (ORF1) a nd 32,122 (ORF2) with limited sequence similarity to replication initi ation and mobilization proteins, respectively, could be identified wit hin the sequence. The region upstream from ORF1 had an AT-rich (75%) r egion followed by four 22-bp direct repeats, a structure characteristi c of replication origins. The plasmid hybridized at high stringency wi th plasmids from Bacteroides/Prevotella and Butyrivibrio, and with pBR 322, suggesting that at least regions of the plasmid are widespread. ( C) 1996 Academic Press, Inc.