Genomic and proteomic analyses of Nus-dependent non-lambdoid phages reveal a novel coliphage group prevalent in gut: mEpimmI

Honorio Negrete-Méndez ¹ Guadalupe Valencia-Toxqui ² Omar Alejandro Sepúlveda-Robles ³ Emmanuel Ríos-Castro ⁴ Jairo César Hurtado-Cortés ¹ Victor Flores ⁵ Adrián Adrián Cázares ⁶ Luis Kameyama ¹ Eva Martínez-Peñafiel ^1* Fernando Fernández-Ramírez ^7*

• ¹ Department of Genetics and Molecular Biology, Center for Research and Advanced Studies (CINVESTAV), Mexico City, México, Mexico
• ² Department of Biology, College of Science, Texas A&M University College Station, College Station, Texas, United States
• ³ Unidad de Investigación Médica en Genética Humana, UMAE Hospital de Pediatría, Centro Médico Nacional “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), México City, Mexico
• ⁴ Genomics, Proteomics and Metabolomic core facility (UGPM), LaNSE, CINVESTAV-IPN, México City, Mexico
• ⁵ Department of Biochemistry, University of Cambridge, Cambridge, England, United Kingdom
• ⁶ Parasites and Microbes Programme, Welcome Sanger Institute, Cambridge, United Kingdom
• ⁷ Unidad de Genética, Hospital General de México. Dr. Balmis 148, C.P. 06726, México City, Mexico

Nus-dependent Mexican Escherichia coli phages (mEp) were previously isolated from clinical samples of human feces. Approximately 50 % corresponded to non-lambdoid temperate phages integrating a single immunity group, namely immunity I (mEpimmI), and these were as prevalent in as the lambdoid phages identified in such collection. In this work, we present the structural and functional characterization of six representative mEpimmI phages (mEp010, mEp013, mEp021, mEp044, mEp515, and mEp554). We searched for homologous phages and prophages in the GenBank sequence database, and found 42 complete genomes corresponding to 38 prophages from E. coli strains and 4 phages from metagenomes, displaying a wide geographical distribution. We performed several phylogenetic analyses on these 48 genomes. Intergenomic distance analyses revealed that these phages differ from previously established phage clades, and whole-proteome similarity analyses yielded a cohesive and monophyletic branch, when compared to >5,600 phages with dsDNA genomes. According to current taxonomic criteria, our results are consistent with a novel family demarcation, and the studied genomes correspond to 9 genera and 45 distinct species. Further, we identified 50 core genes displaying high synteny among the mEpimmI genomes, and these genes were found arranged in functional clusters. Furthermore, a biological feature-based characterization of these phages was carried out, with experiments focusing on proteins relevant to phage biological activities, including mass spectrometry analysis of virion structural proteins and a series of infection assays to characterize the main repressor protein, the lipoproteins associated with superinfection-exclusion, the main host receptor proteins recognized by these phages, the prophage insertion sites within the host genome and the specific integrase sequence-types involved. Finally, the antiterminator N-like protein (Gp17) was found to be preserved in all these genomes, as well as the three nut sites previously described for mEp021. With the integration of all these experimental and bioinformatics findings, our results indicate that the mEpimmI phages constitute a novel branch of Caudoviricetes distinct to other known siphovirus, contributing to the current knowledge on the diversity of phages infecting Escherichia coli.