Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

Abstract

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a transacting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 bp fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 bp DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.

Regulation of the transfer genes of Streptomyces plasmid pSN22: in vivo and in vitro study of the interaction of TraR with promoter regions

“…The regulatory gene, traR, is immediately upstream of the tra operon and is transcribed in the opposite direction (19). The promoter region also functions as an initiation site for secondstrand synthesis to convert the single-stranded intermediate from rolling-circle replication into the double-stranded form (17). Sequence analysis indicated that traR encodes a putative protein of 27 kDa.…”

Nucleotide sequence of a nicking site of the Streptomyces plasmid pSN22 replicating by the rolling circle mechanism

“…In pSN22, DSO (previously called ori1 ) exists in the Nae I‐ Bst EII region from nucleotide positions 7419 to 8195 [6]. Comparison of the nucleotide sequences of the DSO regions of pSN22, pIJ101, and pJV1 revealed three separate highly conserved sequences, from nucleotide positions 7660 to 7763, 7823 to 7869, and 7949 to 8033, respectively, of pSN22 (Fig.…”

“…The nucleotide sequence of the putative nicking site, 5′‐CTTGGGA‐3′, was not identical to the 7‐bp consensus sequence, 5′‐CTTGATA‐3′, of the pC194 group of plasmids [10]. It is proposed that conserved region I is also essential for pSN22 replication, because a plasmid molecule lacking the Nae I‐ Sma I region (from nucleotide positions 7419 to 7778) of DSO could not replicate in S. lividans [6]. Conserved region III overlaps with the putative translation start codons of rep .…”

“…With the aim of developing efficient plasmid vectors for genetic manipulation of Streptomyces , replication mechanisms of small circular plasmids in these microorganisms have been investigated. All of the plasmids so far examined have been found to replicate by the rolling circle mechanism [1–8]. Rolling circle replication starts at a site known as the double strand origin (DSO) by the introduction of a strand‐specific nick catalyzed by a plasmid‐encoded Rep protein.…”

“…pSN22 is an 11‐kbp self‐transmissible plasmid which replicates by the rolling circle mechanism [6] and is thought to belong to the pC194 group of plasmids. The nucleotide sequences of the genes in pSN22 involved in plasmid transfer are similar to those of pJV1 [11], and the nucleotide sequence of its rep region shows high homology with that of pIJ101 [12].…”