Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coli

Abstract

pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably maintained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30 degrees C. The optimal concentration of template DNA was 40 micrograms/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its derivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

“…Increasing the incubation temperature resulted in an augmented number of specific RepB-relaxed DNA molecules. This supports our previous findings on the need for supercoiled DNA as a substrate for RepB (8). Hairpin generation prior to RepB cleavage could explain the failure of RepB to nick linear pLS1 DNA (data not shown), unlike RepC of pT181 (16).…”

“…Supercoiled plasmid DNAs were isolated from bacterial cultures by preparation of cleared lysates followed by purification through two consecutive CsCl-ethidium bromide (EtBr) equilibrium gradients as previously described (8). The RepB protein was purified from E. coli BL21(DE3) harboring plasmid pLS19 (7) essentially as already described (6), except that the DEAE-Sephacel step was omitted.…”

Structural organization of the Corynebacterium glutamicum plasmid pCG100

“…It is tempting to assume that the 32 kDa protein is the replication protein. Its molecular mass is in agreement with that of other Gram-positive replication proteins, which vary from 25 to 40 kDa (del Solar et al, 1987;Khan et al, 1988;Byeon & Weisblum, 1990).…”

Plasmid Rolling-Circle Replication

“…The Rep nick sequence is generally located on an unpaired region within these hairpins, as exemplified by IRII of pT181 and IR-I of pMV158, which accounts for the requirement of plasmid DNA supercoiling to render the cleavage sequence a suitable ssDNA substrate for replication (66)(67)(68). The presence of secondary structures is likely to be involved in efficient recruitment and utilization of the initiator protein.…”