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Background: N4-like viruses, with specific genomic features and propagation signatures, comprise a unique viral clade within the Podoviridae family. N4-like viruses are commonly characterized by the N4-like major capsid protein (MCP) and a giant virion-encapsulated RNA polymerase (N4-like RNAP) with a size of approximately 3,500-aa, which is the largest viral protein so far described. To date, our understanding of N4-like viruses is largely derived from 80 viral isolates that infect bacteria. Thus, it is necessary to expand the diversity of N4-like viruses in culturing-independent methods.Methods: A Hidden-Markov-Module based method was designed based on two characterized N4-specific marker genes, major capsid protein and N4-like virion-encapsulated RNA polymerase. Viral sub-clades were classified based on the monophyly presented in phylogenic tree and the results of pangenome analysis. Further analysis assessed different distribution patterns, genomic properties, hosts’ metabolism reprogramming potentialities, significance of viral tRNA and horizontal gene transfer landscape.Results: We identified 1,000 N4-like virus sequences from genomes and metagenomes representing diverse habitats from around the world. N4-like viruses have been classified into 27 sub-clades and detected in almost all habitats from pole to pole, including some novel habitats, such as oral mucosa and Antarctica. Virulent factors might be crucial for some human-associated N4-like viruses to reprogram the metabolism of host cells and mediate their pathogenic ability through horizontal gene transfer. From the pangenome analysis, the protein diversity was expended over 7-fold and 17 conserved house-keeping genes were identified. Transcriptional compensation of tRNA indicates that producing progeny virion might be the main significance of viral tRNAs. From the horizontal gene transfer network, some N4-like viral sub-clades were observed that potentially infect some important human pathogens, such as Campylobacteria and Veillonella , which have not been considered as potential hosts of N4-like virus or even any virus.Conclusion: This study expands the knowledge of N4-like viruses via global metagenomic datasets, reveals the novel ecological and genomic signatures of these viruses and will provide the backbone for further N4-like virus studies.

In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen P. aeruginosa in sewage and water bodies in India is well documented., Multidrug-resistant community-acquired infections caused by the opportunistic human pathogen Pseudomonas aeruginosa are increasingly reported in India and other locations globally. Since this organism is ubiquitous in the environment, samples such as sewage and wastewater are rich reservoirs of P. aeruginosa bacteriophages. In this study, we report the isolation and characterization of a novel P. aeruginosa N4-like lytic bacteriophage, vB_Pae_AM.P2 (AM.P2), from wastewater in Kerala, India. AM.P2 is a double-stranded DNA podovirus that efficiently lyses the model strain, PAO1, at a multiplicity of infection as low as 0.1 phage per bacterium and resistance frequency of 6.59 × 10−4. Synergy in bactericidal activity was observed between AM.P2 and subinhibitory concentrations of the antibiotic ciprofloxacin. Genome sequencing of AM.P2 revealed features similar to those of the N4-like P. aeruginosa phages LUZ7 and KPP21. As judged by two independent assay methods, spot tests and growth inhibition, AM.P2 successfully inhibited the growth of almost 30% of strains from a contemporary collection of multidrug-resistant P. aeruginosa clinical isolates from South India. Thus, AM.P2 may represent an intriguing candidate for inclusion in bacteriophage cocktails developed for various applications, including water decontamination and clinical bacteriophage therapy. IMPORTANCE In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen P. aeruginosa in sewage and water bodies in India is well documented. The isolation and characterization of bacteriophages that could target emerging P. aeruginosa strains, representing possible epicenters for community-acquired infections, could serve as a useful alternative tool for various applications, such as phage therapy and environmental treatment. Continuing to supplement the repertoire of broad-spectrum bacteriophages is an essential tool in confronting this problem.