Complete sequence and molecular characterization of pNB101, a rolling-circle replicating plasmid from the...

A new plasmid was isolated from the haloalkaliphilic archaeon, Natronobacterium sp. strain AS7091 and named pNB101. Sequence analysis revealed that pNB101 consists of 2,538 bp, in which three major open reading frames (ORF1, ORF2, and ORF3) were identified in the same strand. The ORF1 encodes a putative replication (Rep) protein with three typical motifs (I, II, and III) found in rolling-circle (RC) replicating plasmids. The putative double-stranded origin (DSO) and single-stranded origin (SSO) were detected within ORF3 and downstream of ORF1, respectively. S1 nuclease digestion and Southern blot analysis demonstrated the existence of the single-stranded DNA (ssDNA) intermediate from pNB101, which corresponds to the leading strand in RC replication and was further confirmed by strand-specific RNA probes. A single transcript for ORF1 ( rep) was detected by Northern blotting, and the 5' end of this transcript was determined by primer extension. Both results indicate that the three motifs (I-III) are located at the very end of the N-terminal of this Rep protein. Northern blot analysis also revealed that the ORF3 was transcribed at a very high level, which may play an important role in plasmid replication because the putative DSO is located in this gene. Together, our results indicate that pNB101, the first plasmid isolated from haloalkaliphilic Archaea, represents a novel RC-replicating plasmid.