Bad Phages in Good Bacteria: Role of the Mysterious orf63 of λ and Shiga Toxin-Converting Φ24B Bacteriophages

Abstract

Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of <i>Escherichia coli</i> strains. For example, Shiga toxin-producing <i>E. coli</i> (STEC) acquired <i>stx</i> genes, encoding Shiga toxins, via lambdoid prophages (Stx phages). The results obtained so far present the evidence for the relation between the <i>exo-xis</i> region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the <i>exo-xis</i> genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame <i>orf63</i> of the <i>exo-xis</i> region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24_B. We have demonstrated that <i>orf63</i> codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa.), is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage <i>orf63</i> does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of <i>E. coli</i> during phage lytic development. Additionally, the deletion of phage <i>orf63</i> negatively influences expression of the major phage genes and open reading frames from the <i>exo-xis</i> region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage <i>orf63</i> may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, <i>orf63</i> probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the <i>exo-xis</i> region during prophage induction.