Different Expression Patterns of Genes from the Exo-Xis Region of Bacteriophage λ and Shiga Toxin-Converting Bacteriophage Ф24B following Infection or Prophage Induction in Escherichia coli

Abstract

Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide). This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation.

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

“…Previous studies indicated that either overexpression or deletion of the whole exo-xis region or particular genes and ORFs affect the lytic cycle of lambdoid bacteriophages after infection of E. coli bacteria under standard laboratory conditions (Łoś et al, 2008a;Bloch et al, 2013 andBloch et al, 2014;Licznerska et al, 2016b;Dydecka et al, 2017 andDydecka et al, 2018). Here, we demonstrate that in one-step growth experiment both lambdoid bacteriophages with the deletion of orf73 present shorter latent period and more efficient intracellular lytic development relative to the wild-type viruses (Fig.…”

“…According to available data, this ORF remains under control of the early p L and BPROM-predicted p 1 promoters (Fig. 1) (Bloch et al, 2014). As we demonstrated in Table 2, the nucleotide sequence of orf73 is highly conserved among the group of lambdoid viruses, with the scores of similarities ≥97%.…”

“…Moreover, a few years later it was demonstrated that more efficient induction of λ and Φ24 B prophages, induced by mitomycin C and hydrogen peroxide, occurred in cultures of host cells bearing a plasmid with the exo-xis region (Bloch et al, 2013). Following prophage induction, an increase in the phage DNA amount was significantly higher in lysogenic E. coli cells containing plasmid-borne exo-xis region, while survival rate of such bacteria was lower (Bloch et al, 2013 andBloch et al, 2014). These observations were supported by a finding that deletion of the phage exoxis region resulted in a dramatic decrease in the level of phage gene expression that are crucial for the lytic development of lambdoid viruses (Licznerska et al, 2016b).…”

“…In order to explain the role of individual genes or ORFs from the exo-xis region involved in the regulation of the lysis-vs.-lysogenization decision, Kwan and others (Kwan et al, 2013) reported that Ea8.5 contains a fused homeodomain/zinc finger fold, which suggests a regulatory role of this protein. 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).…”

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages

“…Gene expression was determined by RT-qPCR essentially as described previously (Bloch et al, 2014). All experiments were performed with the LightCycler 480 Real-Time PCR System (Roche), using cDNA samples from lysogenic bacteria.…”