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Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. We cultivated temperate phages produced by 900 E. coli strains isolated from 648 fecal samples of children 1-year-old. Next, we isolated coliphages directly from the viral fraction of the same fecal samples. We found that 63% of strains hosted phages, while 24% of the viromes contained phages targeting E. coli. 150 of these phages, half recovered from strain supernatants, half from virome (73% temperate and 27% virulent) were tested for their host range on 75 E. coli strains isolated from the same cohort. Temperate phages barely infected the gut strains, whereas virulent phages killed up to 68% of them. We conclude that in fecal samples from children, temperate coliphages dominate, while virulent ones have greater infectivity and broader host range, likely playing a role in gut microbiota dynamics.

Rapid turnover of mobile elements drives the plasticity of bacterial genomes. Integrated bacteriophages (prophages) encode host-adaptive traits and represent a sizable fraction of bacterial chromosomes. We hypothesized that natural selection shapes prophage integration patterns relative to the host genome organization. We tested this idea by detecting and studying 500 prophages of 69 strains of Escherichia and Salmonella. Phage integrases often target not only conserved genes but also intergenic positions, suggesting purifying selection for integration sites. Furthermore, most integration hotspots are conserved between the two host genera. Integration sites seem also selected at the large chromosomal scale, as they are nonrandomly organized in terms of the origin–terminus axis and the macrodomain structure. The genes of lambdoid prophages are systematically co-oriented with the bacterial replication fork and display the host high frequency of polarized FtsK-orienting polar sequences motifs required for chromosome segregation. matS motifs are strongly avoided by prophages suggesting counter selection of motifs disrupting macrodomains. These results show how natural selection for seamless integration of prophages in the chromosome shapes the evolution of the bacterium and the phage. First, integration sites are highly conserved for many millions of years favoring lysogeny over the lytic cycle for temperate phages. Second, the global distribution of prophages is intimately associated with the chromosome structure and the patterns of gene expression. Third, the phage endures selection for DNA motifs that pertain exclusively to the biology of the prophage in the bacterial chromosome. Understanding prophage genetic adaptation sheds new lights on the coexistence of horizontal transfer and organized bacterial genomes.

Phages drive bacterial diversity, profoundly influencing microbial communities, from microbiomes to the drivers of global biogeochemical cycling. Aiming to broaden our understanding of Escherichia coli (MG1655, K-12) phages, we screened 188 Danish wastewater samples and isolated 136 phages. Ninety-two of these have genomic sequences with less than 95% similarity to known phages, while most map to existing genera several represent novel lineages. The isolated phages are highly diverse, estimated to represent roughly one-third of the true diversity of culturable virulent dsDNA Escherichia phages in Danish wastewater, yet almost half (40%) are not represented in metagenomic databases, emphasising the importance of isolating phages to uncover diversity. Seven viral families, Myoviridae, Siphoviridae, Podoviridae, Drexlerviridae, Chaseviridae, Autographviridae, and Microviridae, are represented in the dataset. Their genomes vary drastically in length from 5.3 kb to 170.8 kb, with a guanine and cytosine (GC) content ranging from 35.3% to 60.0%. Hence, even for a model host bacterium, substantial diversity remains to be uncovered. These results expand and underline the range of coliphage diversity and demonstrate how far we are from fully disclosing phage diversity and ecology.

Background Amidst rising antimicrobial resistance, bacteriophage (phage) therapy has re-emerged as a pivotal weapon against multidrug-resistant pathogens. Jumbo phages, distinguished by large genomes, show particular therapeutic promise. Yet current understanding of jumbo phages is still lacking. Methods Phage was isolated from domestic sewage. The biological properties of JP4 was characterized via transmission electron microscopy, stability tests, one-step growth curve. The genome of JP4 were elucidated by sequencing and bioinformatics tools. Structural proteins were identified via mass spectrometry. Bactericidal and biofilm eradication activities were evaluated using bacterial turbidity measurements and crystal violet assays, respectively. Statistical significance was determined by using one-way ANOVA in GraphPad Prism. Results Phage JP4 has an icosahedral head (approximately 110 nm in diameter) and a contractile tail (about 120 nm in length). JP4 possesses a linear dsDNA genome of 370,741 bp, encoding 738 proteins and 8 tRNAs. Phylogenetic analysis revealed that JP4 is a new member of the Asteriusvirus genus, and shares close evolutionary relationships with Escherichia phage UB. Additionally, mass spectrometry identified four novel structural protein encoding genes of JP4. Phage JP4 exhibited rapid infection cycle, high stability, potent in vitro bactericidal activity, and strong inhibitory effect on E. coli biofilms. Conclusions Phage JP4 is a new member of the Asteriusvirus genus. As a lytic jumbo phage with rapid bactericidal activity and strong biofilm degradation capacity, JP4 is a promising therapeutic candidate against E. coli O157:H7 infections. This study provides insights into the diversity and clinical potential of jumbo phages in combating pathogens.

Background Urinary tract infections (UTIs) caused by antibiotic-resistant bacteria have become a significant public health concern. The increasing ineffectiveness of antibiotics has led to a renewed focus on investigating other strategies, such as bacteriophages, to target specific pathogenic bacteria and prevent future resistance. Results This study reports the isolation and characterization of bacteriophage vB_Eco_ZCEC08 targeting uropathogenic Escherichia coli (UPEC). Phage vB_Eco_ZCEC08 is morphologically a non-contractile tailed phage that exhibits strong lytic activity against UPEC with a short latent period of less than 15 min and a lysis time of 20 min to produce a high burst of around 900 phage particles per host cell. vB_Eco_ZCEC08 phage activity demonstrated exceptional stability against temperature [-80–60 ̊C], pH [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 – 11 ], UV exposure and incubation in artificial human urine. The phage effectively reduced UPEC counts over a range of infection rates, with MOI 1 the most effective, and which resulted in the limited emergence of phage-insensitive bacteria. A whole-genome study of the 47.926 bp vB_Eco_ZCEC08 phage identified one tRNA gene and 84 predicted genes. Comparative genomics and phylogenetic analysis suggest that the vB_Eco_ZCEC08 phage belongs to the same genus as the Salmonella phage vB_SenS_ST1 but represents a new species. Phage vB_Eco_ZCEC08 showed minimal cytotoxicity against human urinary bladder cancer and skin fibroblast cell lines. Conclusion vB_Eco_ZCEC08 phage demonstrates strong selective lytic activity against UPEC in the absence of any lysogenic behavior. These properties coupled with inherent physiochemical stability and low cytotoxicity support the development of vB_Eco_ZCEC08 as an alternative treatment for multidrug-resistant UPEC.

Research on bacteriophages, the viruses infecting bacteria, has fueled the development of modern molecular biology and inspired their therapeutic application to combat bacterial multidrug resistance. However, most work has so far focused on a few model phages which impedes direct applications of these findings in clinics and suggests that a vast potential of powerful molecular biology has remained untapped. We have therefore recently composed the BASEL collection of Escherichia coli phages (BActeriophage SElection for your Laboratory), which made a relevant diversity of phages infecting the E. coli K-12 laboratory strain accessible to the community. These phages are widely used, but their assorted diversity has remained limited by the E. coli K-12 host. We have therefore now genetically overcome the two major limitations of E. coli K-12, its lack of O-antigen glycans and the presence of resident bacterial immunity. Restoring O-antigen expression resulted in the isolation of diverse additional viral groups like Kagunavirus , Nonanavirus , Gordonclarkvirinae , and Gamaleyavirus , while eliminating all known antiviral defenses of E. coli K-12 additionally enabled us to isolate phages of Wifcevirus genus. Even though some of these viral groups appear to be common in nature, no phages from any of them had previously been isolated using E. coli laboratory strains, and they had thus remained largely understudied. Overall, 37 new phage isolates have been added to complete the BASEL collection. These phages were deeply characterized genomically and phenotypically with regard to host receptors, sensitivity to antiviral defense systems, and host range. Our results highlighted dominant roles of the O-antigen barrier for viral host recognition and of restriction-modification systems in bacterial immunity. We anticipate that the completed BASEL collection will propel research on phage–host interactions and their molecular mechanisms, deepening our understanding of viral ecology and fostering innovations in biotechnology and antimicrobial therapy.

The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ₀) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2′-deoxy-preQ₀ and 2′-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ₀ derivative, 2′-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.

The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution. The directed evolution of antibodies yields important tools for research and therapy. Here the authors develop a periplasmic phage-assisted continuous evolution platform for improvement of protein-protein interactions in the disulfidecompatible E. coli periplasm.

Background Bacteriophages are the most genetically diverse biological entities in nature. Our current understanding of phage biology primarily stems from studies on a limited number of model bacteriophages. Jumbo phages, characterized by their exceptionally large genomes, are less frequently isolated and studied. Some jumbo phages exhibit remarkable genetic diversity, unique infection mechanisms, and therapeutic potential. Methods In this study, we describe the isolation of Sharanji, a novel Escherichia coli jumbo phage, isolated from chicken feces. The phage genome was sequenced and analyzed extensively through gene annotation and phylogenetic analysis. The jumbo phage was phenotypically characterized through electron microscopy, host range analysis, and survival at different pH and temperatures, and one-step growth curve assay. Finally, Sharanji mediated infection of E. coli is studied through fluorescence microscopy, to analyze its mechanism of infection compared to well-studied nucleus-forming jumbo phages. Results Whole genome sequencing reveals that Sharanji has a genome size of 350,079 bp and is a phage encompassing 593 ORFs. Genomic analysis indicates that the phage belongs to the Asteriusvirus genus and is related to E. coli jumbo phages PBECO4 and 121Q. Phenotypic analysis of isolated phage Sharanji, indicates that the phage size is 245.3 nm, and it is a narrow-spectrum phage infecting E. coli K12 strains, but not other bacteria including avian pathogenic E. coli . Infection analysis using microscopy shows that Sharanji infection causes cell filamentation. Furthermore, intracellular phage nucleus-like structures were not observed in Sharanji-infected cells, in contrast to infection by ΦKZ-like jumbo phages. Conclusions Our study reports the isolation and characterization of Sharanji, one of the large E. coli jumbo phages. Both genotypic and phenotypic analyses suggest that Sharanji serves as a unique model system for studying phage-bacteria interactions, particularly within the context of non-nucleus-forming jumbo phages. Further exploration of jumbo phages holds promise for uncovering new paradigms in the study of microbial viruses.

Five lytic bacteriophages specific for Salmonella enterica and Escherichia coli were isolated from wastewater in Minnesota. These phages, designated vB_Sal_EH1, vB_Sal_EH2, vB_Sal_EH3, vB_Sal_EH4, and vB_Sal_EH7, were characterized, and their genomes were sequenced. Phylogenetic analysis showed that they grouped within the genus Epseptimavirus, with genome sizes ranging from 108,554 to 115,218 bp. All five phages exhibited lytic activity against both S. enterica and Shiga-toxin-producing E. coli O157:H7. Transposon mutagenesis of the host genome identified the outer membrane protein BtuB as essential for phage infection, suggesting that it is a putative receptor. Genome sequence comparisons revealed genetic loci that are variable among the isolated phages and potentially influence their host specificity and virulence.