pBLA8, from Brevibacterium linens, belongs to a Gram-positive subfamily of ColE2-related plasmids

SUMMARY: A 3.1 kb DNA fragment from pBLA8, a Brevibacterium linens cryptic plasmid, containing all the information required for autonomous replication was cloned and sequenced. Using deletion analysis, the fragment essential and sufficient for autonomous replication was delimited to 1.5 kb. This fragment is characterized by the presence of an ori site located upstream of an operon encoding two proteins, RepA and RepB, both essential for replication. Based on structural similarities and a strong conservation of ori, RepA and RepB, pBLA8 was assigned to a new subfamily of the ColE2 plasmid family. This subfamily is distinguished by the requirement for two Rep proteins and the location of an ori site upstream of the mpAB operon. RepA is thought to encode primase activity, whereas RepB could be a DNA-binding protein. An Escherichia coli-B. linens shuttle vector, derived from pBLA8, was constructed. Its host spectrum was extended to Arthrobacter species.