Expression of temperateBacillus subtilisphage SPO2 DNA polymerase geneLinEscherichia coli

Abstract

Temperate Bacillus subtilis phage SPO2 replicates in the presence of 6-(p-hydroxyphenylazo)uracil, an inhibitor of DNA polymerase III [1,2]. Several lines of evidence suggest that replication of SPO2 DNA is dependent on a phage-specific D N A polymerase coded for by SPO2 gene L [1,3]. We have recently constructed several pC194-SPO2 gene L hybrid plasmids [4]. B. subtilis carrying one of these plasmids, pJB96 (formerly called pC19496), have substantially increased levels of in vitro D N A polymerase activity. Since all experiments concerning growth of SPO2, by necessity have to be done using B. subtilis as host, it is possible that the increased D N A polymerase activity observed is due to interaction between the SPO2 gene L product and one of the bacterial DNA polymerases. This possibility could be excluded, however, if the presence of gene L could be shown to cause an increase in D N A polymerase activity also in another bacterial species. p H V I 4 is a composite plasmid constructed by fusing Escherichia coli plasmid pBR322 with the Staphylococcus aureus plasmid pC194 [5]. It can be stably maintained in both E. coli and B. subtilis. We have inserted SPO2 gene L into pHV14. The resulting plasmid, called pLB1, was transformed into polA mutants of E. coli and B. subtilis, respectively. D N A polymerase activity was then measured in extracts from these bacteria as well as from bacteria carrying the vector pHV14. In both B. subtilis and E. coli the presence of pLB1 was accompanied by a 10-50-fold increased level of D N A polymerase activity. We can thus conclude that the increased D N A polymerase activity found in B. subtilis infected with SPO2 is not due to an interaction between the SPO2 gene L product and a bacterial DNA polymerase.