The viruses are internal parasites of bacteria and affect their metabolism. The composition of replication proteins in the genome of each bacteriophage is different. According to the content and character of replication proteins are phages divided into four groups. Every group is able to exploit different host proteins for phage DNA replication and the specific interactions between replication proteins are forming in replisome. One of the methods of searching for protein-protein interactions in vivo is bacterial two-hybrid system (BACTH, Euromedex®). Bacteriophage BFK20 (EMBL AJ278322) is a lytic phage of Brevibacterium flavum CCM 251, industrial producer of L-lysine. In present study we have tested protein-protein interactions between five BFK20 replication proteins: gp40 (related with CRISPR by Cas4), gp41 (helicase from SF2 family), gp42 (replication protein), gp43 (RepA like protein) and gp44 (DNA polymerase) and three replication proteins of its host B. flavum: DnaE1 (alpha subunit of DNA polymerase III), DnaJ1 (chaperon protein) and DnaLig (DNA ligase) using a bacterial two-hybrid system. We designed the primers for PCR amplification of host replication genes according to known genome of closely relative strain Corynebacterium glutamicum ATCC 13032 (EMBL BA00036) and PCR fragments were amplified on DNA template of B. flavum CCM 251. Three positive interactions between bacterial and phage proteins were detected, all involved phage protein gp41 which interacted with bacterial proteins DnaE1, DnaJ1 and DnaLig. Measured level of beta-galactosidase activity reflects the interaction strength between proteins. The highest level was detected for the interaction of gp41 with DnaE1 followed by that between gp41 with DnaLig and gp41 with DnaJ1. We conclude proteins interaction between gp41 and DnaE1 participate in BFK20 DNA replication.

Acknowledgement: This work was supported by the VEGA Grant 2/0122/14 from Slovak Academy of Sciences.