Mol Gen Genet (1987) 206:428-435 © Springer-Verlag 1987 Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coil Gloria del Solar, Ramon Diaz, and Manuel Espinosa Centro de Investigaciones Biol6gicas Velazquez, 144, E-28006 Madrid, Spain Summary. 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 mantained 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. Maxi- mal synthesis of streptococcal plasmid DNA occurred after 90 rain of incubation and at a temperature of 30 ° C. The optimal concentration of template DNA was 40 #g/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 ori- gins 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 strepto- coccal plasmid DNAs replicated in the E. coli in vitro sys- tem 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. Key words: In vitro replication - Streptococcal plasmids Plasmid DNA synthesis Introduction Plasmid-mediated genetic exchanges between bacteria be- longing to the same group, gram-positive or gram-negative, seem to be a general phenomenon (Bagdasarian et al. 1981 ; Ehrlich 1977). However, reports on plasmids from gram- positive hosts being able to replicate in gram-negative or- ganisms are scarce (Ehrlich 1977; Kok et al. 1984). It has been shown that hybrids between pBR322 and the staphylo- coccal plasmid pC194 replicate in thermosensitive DNA- polymerase I mutants of Escherichia coli under conditions in which pBR322 did not replicate (Goze and Ehrlich 1980). This in conjunction with other results, led to the conclusion Offprint requests to. M. Espinosa that some plasmids from Staphylococcus aureus can repli- cate in E. coli, although plasmid pC194 itself failed to trans- form E. coli. Plasmid pMV158 is a 5.4 kb multicopy streptococcal plasmid conferring constitutive resistance to tetracycline (Burdett 1980). The plasmid was originally isolated and characterized in Streptococcus agalactiae (Burdett 1980). In addition to S. pneumoniae (Saunders and Guild 1980), pMV158 is able to replicate in other streptococci (Clewell 1981) as well as in Bacillus subtilis (Espinosa et al. 1982). Furthermore, pMVI58 and several derivatives are able to efficiently transform E. coli (Lacks et al. 1986). It is likely that this very broad host range is specified by some plasmid- encoded replication functions. These features make the pMV158 replicon a very attractive system for studying gen- eral principles that apply to the initiation of plasmid replica- tion and its control. To this end, in vitro systems developed for the analysis of E. coli plasmid replication (Diaz et al. 1981; Diaz and Staudenbauer 1982a; Staudenbauer 1976) provide a useful tool. Use of these systems has revealed that plasmid ColE1 DNA replication is independent of de novo protein synthesis but requires transcription of an RNA primer (Staudenbauer 1976, 1984). In vitro replica- tion of plasmids R1 and R6K has demonstrated that these plasmids strictly need synthesis of a specific protein, in- volved in the initiation of replication (Diaz et al. 1981 ; Diaz and Ortega 1984; Inuzuka and Helinski 1978). Cell-free systems have also been developed from Pseudomonas aeru- ginosa and from S. aureus to analyse the replicative charac- teristics of plasmids RSFI010 (Diaz and Staudenbauer 1982b) and pT181 (Khan et al. 1981), respectively. These analyses have shown that both plasmids require a specific plasmid-encoded protein for their replication. While plas- mids RSF1010, R6K and pT181 replicate exclusively in ex- tracts prepared from plasmid-containing cells, in vitro repli- cation of RI and ColE1 can also occur in plasmid-free extracts (for a review, see Scott 1984). In the experiments described here, using an E. coli cell- free replication system (Staudenbauer 1976) we have stud- ied the ability of the extracts to support replication of exo- genously added DNA from pMV158 and its derivatives pLS1 and pLS5. Comparison with E. coli replicons showed that, similar to RI, the streptococcal plasmids required the host RNA polymerase and de novo protein synthesis. Anal- ysis of the reaction products demonstrated that cell-free extracts of E. coli can carry out the complete replication of the streptococcal plasmids.