gene as a molecular marker for genome engineering of felixounoviruses.

BACKGROUND

Bacterial viruses (bacteriophages or phages) have a lot of uncharacterized genes, which hinders the progress of their applied research. Functional characterization of these genes is often hampered by a lack of suitable methods for engineering of phage genomes.

METHODS

Phages vB_EcoM_Alf5 (Alf5) and VB_EcoM_VpaE1 (VpaE1) were used as the model phages of Felixounovirus genus. The phage-coded properties were predicted by bioinformatics analysis. The 'pull-down' assay was used for detection of protein-protein interactions. Primer extension analysis was used for the DNA polymerase (DNAP) activity testing. Bacteriophage lambda Redγβα-assisted homologous recombination was used for construction of phage mutants.

RESULTS

Bioinformatics analysis showed that felixounoviruses encode DNA polymerase, which is homologous to the T7 DNAP. We found that the Escherichia coli thioredoxin A (TrxA) in vitro interacts with the predicted DNAP of Alf5 phage (gp096) and enhances its activity. Phages Alf5 and VpaE1 do not grow on E. coli strains lacking trxA gene unless it is provided in trans. This feature was used for construction of the deletion/insertion mutants of non-essential genes of felixounoviruses.

CONCLUSION

DNA replication of phages from Felixonuvirus genus depends on the host trxA, which therefore may be used as a molecular marker for their genome engineering.

GENERAL SIGNIFICANCE

We present a proof-of-principle of a strategy for targeted engineering of bacteriophages of Felixounovirus genus. The method developed here will facilitate the basic and applied research of this unexplored phage group. Furthermore, detected functional interactions between the phage and host proteins will be significant for basic research of DNA replication.