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 Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC) acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages). The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24B. We have demonstrated that orf63 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 orf63 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 E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of the major phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo-xis region during prophage induction.

Role of orf73 in the development of lambdoid bacteriophages during infection of the Escherichia coli host

“…Survival test of host bacteria after phage infection. To determine the survival rate of E. coli bacteria after phage infection, a previously published method was used (Dydecka et al, 2017). Bacterial cells were grown in LB liquid medium at 30 o C to A 600 =0.2.…”

“…Lysogenization test of bacterial cells after phage infection. To estimate the percentage of lysogenic bacteria after bacteriophage infection, a procedure presented previously was used (Dydecka et al, 2017). Host bacteria were cultivated in LB liquid medium at 30 o C to A 600 =0.2.…”

“…Interestingly, overexpression of the ea8.5 gene was responsible for the fuzzy plaque phenotype of the λ cIb2 phage, rapid virus development after prophage induction and repression of the cII-dependent promoters (p I , p aQ , p E ) (Łoś et al, 2008a;Bloch et al, 2014). On the other hand, expression of orf60a, orf63 and orf61 promotes the lytic pathway of lambdoid phage development (Dydecka et al, 2017 andDydecka et al, 2018). Mutant phages with deletions of orf60a and orf61 influenced the lysis-vs.-lysogenization decision, and impaired prophage induction provoked by different agents, such as mitomycin C and hydrogen peroxide.…”

“…Similar results were obtained for lambdoid phages with deletion of orf63. Dydecka and others (Dydecka et al, 2017) demonstrated that Orf63 is a small-size, functional protein (63 aa.) with two alpha helices, likely intertwined to form an oligomer.…”

“…Functionally, the Orf63 protein probably participates in regulation of expression of crucial phage genes and ORFs from the exo-xis region during prophage induction. Moreover, lack of sequences of orf63 in the genomes of λ and Φ24 B resulted in the delay in phage development after prophage induction and increased survival of the host cells during lytic cycle (Dydecka et al, 2017).…”