The BFK20 phage replication origin confers a phage-encoded resistance phenotype to the industrial strain Brevibacterium flavum.

The phage BFK20 replication origin was identified using bioinformatics tools and a fragment with the origin nucleotide sequence was cloned into the tetracycline resistance gene of Escherichia coli vector pBR328, to make the plasmid pBOS. After transformation into the host strain Brevibacterium flavum CCM 251, pBOS was able to replicate, showing that the cloned region may function as a replication origin. The presence of the BFK20 origin sequence in a pBOS plasmid isolated from B. flavum CCM 251 was confirmed by Southern hybridisation. Monitoring pBOS stability in corynebacterial hosts showed that pBOS was stable in Corynebacterium glutamicum RM3 for 20 generations and in B. flavum CCM 251 for 10 generations. The effect of the cloned BFK20 replication origin on host resistance to BFK20 infection was tested. Growth of a B. flavum CCM 251 strain harbouring pBOS stopped after phage infection, but without complete lysis. Five hours after infection, the viability of the modified strain was about five times higher than the viability of wild-type B. flavum CCM 251. Thus, the ability of the BFK20 replication origin to confer the origin-derived phage-encoded resistance phenotype to B. flavum CCM 251 was confirmed.