High diversity in the regulatory region of Shiga toxin encoding bacteriophages.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) is an emerging health challenge worldwide and outbreaks caused by this pathogen poses a serious public health concern. Shiga toxin (Stx) is the major virulence factor of EHEC, and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor which maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains. RESULTS: In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and three already described Erus, ten novel Erus (Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 are widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type. CONCLUSION: The classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain.

Ribonucleotide reductases reveal novel viral diversity and predict biological and ecological features of unknown marine viruses.

Virioplankton play a crucial role in aquatic ecosystems as top-down regulators of bacterial populations and agents of horizontal gene transfer and nutrient cycling. However, the biology and ecology of virioplankton populations in the environment remain poorly understood. Ribonucleotide reductases (RNRs) are ancient enzymes that reduce ribonucleotides to deoxyribonucleotides and thus prime DNA synthesis. Composed of three classes according to O2 reactivity, RNRs can be predictive of the physiological conditions surrounding DNA synthesis. RNRs are universal among cellular life, common within viral genomes and virioplankton shotgun metagenomes (viromes), and estimated to occur within >90% of the dsDNA virioplankton sampled in this study. RNRs occur across diverse viral groups, including all three morphological families of tailed phages, making these genes attractive for studies of viral diversity. Differing patterns in virioplankton diversity were clear from RNRs sampled across a broad oceanic transect. The most abundant RNRs belonged to novel lineages of podoviruses infecting α-proteobacteria, a bacterial class critical to oceanic carbon cycling. RNR class was predictive of phage morphology among cyanophages and RNR distribution frequencies among cyanophages were largely consistent with the predictions of the "kill the winner-cost of resistance" model. RNRs were also identified for the first time to our knowledge within ssDNA viromes. These data indicate that RNR polymorphism provides a means of connecting the biological and ecological features of virioplankton populations.

Depletion of m6A-RNA in Escherichia coli reduces the infectious potential of T5 bacteriophage.

N6-Methyladenosine (m6A) is the most abundant internal modification of mRNA in eukaryotes that plays, among other mechanisms, an essential role in virus replication. However, the understanding of m6A-RNA modification in prokaryotes, especially in relation to phage replication, is limited. To address this knowledge gap, we investigated the effects of m6A-RNA modifications on phage replication in two model organisms: Vibrio campbellii BAA-1116 (previously Vibrio harveyi BB120) and Escherichia coli MG1655. An m6A-RNA-depleted V. campbellii mutant (ΔrlmFΔrlmJ) did not differ from the wild type in the induction of lysogenic phages or in susceptibility to the lytic Virtus phage. In contrast, the infection potential of the T5 phage, but not that of other T phages or the lambda phage, was reduced in an m6A-RNA-depleted E. coli mutant (ΔrlmFΔrlmJ) compared to the wild type. This was shown by a lower plaquing efficiency and a higher percentage of surviving cells. There were no differences in the T5 phage adsorption rate, but the mutant exhibited a 5-min delay in the rise period during the one-step growth curve. This is the first report demonstrating that E. coli cells with lower m6A-RNA levels have a higher chance of surviving T5 phage infection. IMPORTANCE: The importance of RNA modifications has been thoroughly studied in the context of eukaryotic viral infections. However, their role in bacterial hosts during phage infections is largely unexplored. Our research delves into this gap by investigating the effect of host N6-methyladenosine (m6A)-RNA modifications during phage infection. We found that an Escherichia coli mutant depleted of m6A-RNA is less susceptible to T5 infection than the wild type. This finding emphasizes the need to further investigate how RNA modifications affect the fine-tuned regulation of individual bacterial survival in the presence of phages to ensure population survival.

WGS analysis of two Staphylococcus aureus bacteriophages from sewage in China provides insights into the genetic feature of highly efficient lytic phages.

The study of bacteriophages is experiencing a resurgence with the increasing development of antimicrobial resistance in Staphylococcus aureus. Nonetheless, the genetic features of highly efficient lytic S. aureus phage remain to be explored. In this study, two lytic S. aureus phages, SapYZU11 and SapYZU15, were isolated from sewage samples from Yangzhou, China. The phage morphology, one-step growth, host spectrum and lytic activity of these phages were examined, and their whole-genome sequences were analysed and compared with 280 published genomes of staphylococcal phages. The structural organisation and genetic contents of SapYZU11 and SapYZU15 were investigated. The Podoviridae phage SapYZU11 and Herelleviridae phage SapYZU15 effectively lysed all of the 53 S. aureus strains isolated from various sources. However, SapYZU15 exhibited a shorter latent period, larger burst size and stronger bactericidal ability with an anti-bacterial rate of approximately 99.9999% for 24 h. Phylogenetic analysis revealed that Herelleviridae phages formed the most ancestral clades and the S. aureus Podoviridae phages were clustered in the staphylococcal Siphoviridae phage clade. Moreover, phages in different morphology families contain distinct types of genes associated with host cell lysis, DNA packaging and lysogeny. Notably, SapYZU15 harboured 13 DNA metabolism-related genes, 5 lysin genes, 1 holin gene and 1 DNA packaging gene. The data suggest that S. aureus Podoviridae and Siphoviridae phages originated from staphylococcal Herelleviridae phages, and the module exchange of S. aureus phages occurred in the same morphology family. Moreover, the extraordinary lytic capacity of SapYZU15 was likely due to the presence of specific genes associated with DNA replication, DNA packaging and the lytic cycle.

Replication Region Analysis Reveals Non-lambdoid Shiga Toxin Converting Bacteriophages.

Shiga toxin is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the gene encoding it is carried within the genome of Shiga toxin-converting phages (Stx phages). Numerous Stx phages have been sequenced to gain a better understanding of their contribution to the virulence potential of EHEC. The Stx phages are classified into the lambdoid phage family based on similarities in lifestyle, gene arrangement, and nucleotide sequence to the lambda phages. This study explores the replication regions of non-lambdoid Stx phages that completely lack the O and P genes encoding the proteins involved in initiating replication in the lambdoid phage genome. Instead, they carry sequences encoding replication proteins that have not been described earlier, here referred to as eru genes (after EHEC phage replication unit genes). This study identified three different types of Eru-phages, where the Eru1-type is carried by the highly pathogenic EHEC strains that caused the Norwegian O103:H25 outbreak in 2006 and the O104:H4 strain that caused the large outbreak in Europe in 2011. We show that Eru1-phages exhibit a less stable lysogenic state than the classical lambdoid Stx phages. As production of phage particles is accompanied by production of Stx toxin, the Eru1-phage could be associated with a high-virulence phenotype of the host EHEC strain. This finding emphasizes the importance of classifying Stx phages according to their replication regions in addition to their Stx-type and could be used to develop a novel strategy to identify highly virulent EHEC strains for improved risk assessment and management.

High throughput sequencing provides exact genomic locations of inducible prophages and accurate phage-to-host ratios in gut microbial strains.

BACKGROUND: Temperate phages influence the density, diversity and function of bacterial populations. Historically, they have been described as carriers of toxins. More recently, they have also been recognised as direct modulators of the gut microbiome, and indirectly of host health and disease. Despite recent advances in studying prophages using non-targeted sequencing approaches, methodological challenges in identifying inducible prophages in bacterial genomes and quantifying their activity have limited our understanding of prophage-host interactions. RESULTS: We present methods for using high-throughput sequencing data to locate inducible prophages, including those previously undiscovered, to quantify prophage activity and to investigate their replication. We first used the well-established Salmonella enterica serovar Typhimurium/p22 system to validate our methods for (i) quantifying phage-to-host ratios and (ii) accurately locating inducible prophages in the reference genome based on phage-to-host ratio differences and read alignment alterations between induced and non-induced prophages. Investigating prophages in bacterial strains from a murine gut model microbiota known as Oligo-MM12 or sDMDMm2, we located five novel inducible prophages in three strains, quantified their activity and showed signatures of lateral transduction potential for two of them. Furthermore, we show that the methods were also applicable to metagenomes of induced faecal samples from Oligo-MM12 mice, including for strains with a relative abundance below 1%, illustrating its potential for the discovery of inducible prophages also in more complex metagenomes. Finally, we show that predictions of prophage locations in reference genomes of the strains we studied were variable and inconsistent for four bioinformatic tools we tested, which highlights the importance of their experimental validation. CONCLUSIONS: This study demonstrates that the integration of experimental induction and bioinformatic analysis presented here is a powerful approach to accurately locate inducible prophages using high-throughput sequencing data and to quantify their activity. The ability to generate such quantitative information will be critical in helping us to gain better insights into the factors that determine phage activity and how prophage-bacteria interactions influence our microbiome and impact human health. Video abstract.

Propagation method for persistent high yield of diverse Listeria phages on permissive hosts at refrigeration temperatures.

The efficient production of a high concentration of bacteriophage in large volumes has been a limiting factor in the exploration of the true potential of these organisms for biotechnology, agriculture and medicine. Traditional methods focus on generating small volumes of highly concentrated samples as the end product of extensive mechanical and osmotic processing. To function at an industrial scale mandates extensive investment in infrastructure and input materials not feasible for many smaller facilities. To address this, we developed a novel, scalable, generic method for producing significantly higher titer psychrophilic phage (P < 2.0 × 10(-6)), 2- to 4-fold faster than traditional methods. We generate renewable high yields from single source cultures by propagating phage under refrigeration conditions in which Listeria, Yersinia and their phages grow in equilibrium. Diverse Yersinia and Listeria phages tested yielded averages of 3.49 × 10(8) to 3.36 × 10(12) PFU/ml/day compared to averages of 1.28 × 10(5) to 1.30 × 10(10) PFU/ml/day by traditional methods. Host growth and death kinetics made this method ineffective for extended propagation of mesophilic phages.