CHARACTERIZATION, SEQUENCE AND MODE OF REPLICATION OF PLASMID PNB2 FROM THE THERMOPHILIC BACTERIUM CLOSTRIDIUM-THERMOSACCHAROLYTICUM

The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated f rom the thermophilic bacterium Clostridium thermosaccharolyticum was d etermined. pNB2 DNA has very low GC content (27%) and may serve as a m odel for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence reve aled three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were s ynthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG , and has similarities with the conserved motifs of Rep proteins encod ed by rolling circle (RC) plasmids of the pC194/pUB110 family. Souther n hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mec hanism. The most significant similarities are found between pNB2 Rep p rotein and the Rep proteins of three RC plasmids of the pC194 family ( pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparativ e analysis of these Rep proteins showed that despite the significant l evel of divergence, these Rep proteins share a high degree of similari ty in the regions of five well-known conserved domains of RC Rep prote ins and fall into two groups in accordance with the similarities found in their active sites.