Lagging-strand origins of the promiscuous plasmid pMV158: physical and functional characterization

Abstract

The streptococcal plasmid pMVl58 replicates by a rolling circle mechanism, which involves the generation of single-stranded plasmid DNA intermediates. This plasmid has the unique feature of having two lagging-strand origins of replication. One of these origins, termed SOU, is functional in Streptococcus pneumoniae and in Bacillus subtilis in an orientation-dependent manner. The other origin, SSOA, is only functional in the former host. RNA polymerase seems to be involved in the initiation of the conversion of single-to doublestranded plasmid DNA from both ssoA and ssoU. Mutational and deletion analyses have allowed us to define ssoA as being within a highly structured, non-coding 199 bp region. Within this region, two elements which are conserved in several rolling-circle replicating plasmids are located, the recombination site RS, and a 6 base consensus sequence. Both elements may play a role in the conversion of single-to double-stranded plasmid DNA.

Plasmid Rolling-Circle Replication

“…In general, alternative plasmid-borne signals and/or bacterial mechanisms seem to exist that can partially overcome the lack of the genuine sso (12,121,139). Especially efficient appears to be the predominant sso-and RNAP-independent priming system revealed in Streptomyces lividans and proposed to result from the stabilization of RCR plasmids lacking sso in this bacterium (146).…”

“…The ssoU is the most promiscuous sso origin characterized so far, as it seems to be fully functional in many, if not all, Firmicutes (121,(139)(140)(141)(142). The ssoU origin was first identified in staphylococcal plasmid pUB110 (142), within a ∼250-bp DNA fragment with the potential to form several hairpin structures containing symmetric and asymmetric internal loops in addition to the terminal one (139).…”

“…These origins display a high degree of sequence heterogeneity, with only two well-conserved regions: the previously termed recombination site B (RS B ), which is present in the lower stem of the hairpin of all the ssoAs, and a 6-nt consensus sequence (CS-6) that is located in the terminal loop of the hairpin of most but not all of these origins (12,120,121). The RS B has been shown to be involved in binding of RNAP to the ssoA (122).…”

“…The RS B has been shown to be involved in binding of RNAP to the ssoA (122). Mutations in the RS B abolish almost completely the ssoA-dependent synthesis of the lagging strand both in vitro and in vivo, giving rise to the accumulation of very high numbers of plasmid ssDNA intermediates in the host cell (121,123). The CS-6-containing terminal loop seems to function as a terminator of the primer RNA synthesis, and changes in this region lead to a moderate increase in the intracellular number of ssDNA plasmid forms without impairing ssoA binding by RNAP (121)(122)(123).…”

“…Mutations in the RS B abolish almost completely the ssoA-dependent synthesis of the lagging strand both in vitro and in vivo, giving rise to the accumulation of very high numbers of plasmid ssDNA intermediates in the host cell (121,123). The CS-6-containing terminal loop seems to function as a terminator of the primer RNA synthesis, and changes in this region lead to a moderate increase in the intracellular number of ssDNA plasmid forms without impairing ssoA binding by RNAP (121)(122)(123). Sequences similar to CS-6 have been identified in the terminal loops of hairpins located in DNA regions that have been either proposed or shown to act as ssos in plasmids from Actinobacteria (124)(125)(126)(127)(128).…”