Functional analysis of pSM19035-derived replicons in Bacillus subtilis

We determined the effect of various Bacillus subtilis dna(Ts) mutations on pSM19035 replication. At non-permissive temperature, plasmid replication depends on the dnaB, dnaC, dnaG and dnaI initiation replication products, but does not require the dnaA function. DNA elongation is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in dna(Ts) mutants impaired in different subunits of the enzyme. Single-stranded plasmid replication intermediates, corresponding to the leading strand, accumulate in the dnaD strain. On the basis of electron microscopic analysis of replication intermediates, we confirmed that the plasmid replicates unidirectionally by a theta mechanism.