Phage Genome Research Articles

Construction and characterization of DNA libraries from cultured phages and environmental viromes.

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (ΦT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology.IMPORTANCEFunctional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes.

A Vibrio cholerae Type IV restriction system targets glucosylated 5-hydroxymethylcytosine to protect against phage infection.

A major challenge faced by Vibrio cholerae is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, V. cholerae has acquired and/or evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized that more were encoded in V. cholerae given the low diversity of phages that have been isolated, which infect this species. Using a V. cholerae genomic library, we identified a Type IV restriction system consisting of two genes within a 16-kB region of the Vibrio pathogenicity island-2, which we name TgvA and TgvB (Type I-embedded gmrSD-like system of VPI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modifications are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff, becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We also show that the Type I restriction-modification system that embeds the tgvAB genes protects against phage T3, secΦ18, secΦ27, and λ, suggesting that this region is a phage defense island. Our study uncovers a novel Type IV restriction system in V. cholerae, increasing our understanding of the evolution and ecology of V. cholerae, while highlighting the evolutionary interplay between restriction systems and phage genome modification.IMPORTANCEBacteria are constantly being predated by bacteriophage (phage). To counteract this predation, bacteria have evolved a myriad of defense systems. Some of these systems specifically digest infecting phage by recognizing unique base modifications present on the phage DNA. In this study, we discover a Type IV restriction system encoded in V. cholerae, which we name TgvAB, and demonstrate it recognizes and restricts phage that have 5-hydroxymethylcytosine glucosylated DNA. Moreover, the evolution of resistance to TgvAB render phage susceptible to other Type IV restriction systems, demonstrating a significant evolutionary tradeoff. These results enhance our understanding of the evolution of V. cholerae and more broadly how bacteria evade phage predation.

Nine Rhizobium phage genomes recovered from wastewater in Tempe, AZ, October 2019-March 2020.

We describe nine Rhizobium microvirus genomes identified in wastewater in Tempe, AZ, USA, between October 2019 and March 2020. The major capsid protein (MCP) encoded in these genomes phylogenetically cluster together and are distinct from the MCPs of Rhizobium microviruses identified in Mexico and Argentina.

Functional and practical insights into three lactococcal antiphage systems.

The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities.

PhageScanner: a reconfigurable machine learning framework for bacteriophage genomic and metagenomic feature annotation.

Bacteriophages are the most prolific organisms on Earth, yet many of their genomes and assemblies from metagenomic sources lack protein sequences with identified functions. While most bacteriophage proteins are structural proteins, categorized as Phage Virion Proteins (PVPs), a considerable number remain unclassified. Complicating matters further, traditional lab-based methods for PVP identification can be tedious. To expedite the process of identifying PVPs, machine-learning models are increasingly being employed. Existing tools have developed models for predicting PVPs from protein sequences as input. However, none of these efforts have built software allowing for both genomic and metagenomic data as input. In addition, there is currently no framework available for easily curating data and creating new types of machine learning models. In response, we introduce PhageScanner, an open-source platform that streamlines data collection for genomic and metagenomic datasets, model training and testing, and includes a prediction pipeline for annotating genomic and metagenomic data. PhageScanner also features a graphical user interface (GUI) for visualizing annotations on genomic and metagenomic data. We further introduce a BLAST-based classifier that outperforms ML-based models and an efficient Long Short-Term Memory (LSTM) classifier. We then showcase the capabilities of PhageScanner by predicting PVPs in six previously uncharacterized bacteriophage genomes. In addition, we create a new model that predicts phage-encoded toxins within bacteriophage genomes, thus displaying the utility of the framework.

An experimental census of retrons for DNA production and genome editing.

Retrons are bacterial immune systems that use reverse-transcribed DNA (RT-DNA) to detect phage infection. They are also deployed for genome editing, where they are modified so that the RT-DNA encodes an editing donor. Retrons are common in bacterial genomes, and thousands of unique retrons have been predicted bioinformatically. However, few have been characterized experimentally. We add to the corpus of experimentally studied retrons, finding 62 empirically determined, natural RT-DNAs that are not predictable from the retron sequence alone. We synthesize >100 previously untested retrons to identify the natural sequence of RT-DNA they produce, quantify their RT-DNA production and test the relative efficacy of editing using retron-derived donors to edit bacterial, phage and human genomes. We observe large diversity in RT-DNA production and editing rates across retrons, finding that top-performing editors are drawn from a subset of the retron phylogeny and outperform those used in previous studies, reaching precise editing rates of up to 40% in human cells.

PSIX-18 Multiomic analysis to identify host and microbiome contributions to digestibility in beef cattle

Abstract This study evaluated beef heifers selected for high (efficient) or low (inefficient) digestible fiber intake (DFI). Initial analysis showed that high DFI animals had reduced methane production versus low-DFI under a high forage diet. Using the same cohort of animals maintained on a further 4 diets of varying forage:concentrate ratios, we employed multi-kingdom amplicon sequencing and metagenome shotgun sequencing of rumen digesta and feces alongside RNA sequencing of rumen epimural samples to evaluate the compositional and functional interplay between different microbial groups, and their relationship with host gene expression in cattle divergent for DFI. Samples were collected from 16 cattle during 2 metabolism trials, comprising 5 diets. Amplicon sequencing analysis was conducted using QIIME2; 16S rRNA and 18S rRNA reads were analyzed using the SILVA database, while LSU (fungal) sequences were analyzed using a custom D1/D2 database. Additional analysis of archaeal 16S rRNA sequences was conducted using the Rumen and Intestinal Methanogens (RIM) database. Metagenome shotgun reads underwent a two-pass classification with Kraken2 using a database of prokaryotic genomes derived from the GTDB taxonomy, with the unclassified output undergoing classification using a custom database containing all NCBI protozoa, fungi, and phage genomes, enriched with selected rumen-specific ciliate and fungal genomes. Downstream analysis of taxonomic data from all microbiome work was conducted in R, and differentially abundant taxa were identified using ANCOM-BC and Aldex2. Functional analysis of metagenome contigs using the CAZY database implemented in dbCAN3 is ongoing. RNA-seq data were analyzed using the ARS-UCD reference genome, with identification of DE genes conducted using DeSeq2. Preliminary results indicate no major effect of DFI ranking on host gene expression, bacterial 16S rRNA, or metagenome compositional profiles. Several bacterial genera were differentially abundant between digestibility groups (P < 0.05), but these were all minor (< 0.01%) members of the microbiome. Fungal and methanogen communities different significantly (P < 0.05) according to DFI group, with efficient (high DFI) containing and more diverse communities under high-grain diet (P < 0.05). The same difference showed a tendency toward significance for the 18S rRNA protozoa data (P < 0.1). These preliminary data indicate that the microbial factors underpinning divergence in efficiency measured by DFI vary according to diet and may be more prominent in the non-bacterial fraction of the microbiome. Ongoing functional analysis of metagenome data as well as integration of multiomic data will provide deeper insight into these relationships and how they contribute to feed digestibility and efficiency in cattle.

Biological and genomic characterization of the novel bacteriophage vB_VpM-pA2SJ1, which infects Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease.

Vibrio parahaemolyticus is a major seafood-borne zoonotic pathogen that causes gastroenteritis in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, we isolated and characterized Vibrio phage vB_VpM-pA2SJ1, which infects clinical and AHPND-associated strains of V. parahaemolyticus. The phage genome is a linear dsDNA 51,054 bp in length with a G + C content of 43.7%, and it contains 89 open reading frames. Genome comparisons revealed basal similarity to other Vibrio phages, particularly Vibrio phage vB_VpP_1, with 84.2% identity and 46% coverage. Phylogenetic analysis based on the whole genome, the terminase large subunit, and the major capsid protein revealed that phage vB_VpM-pA2SJ1 did not cluster with other known phage families, thus indicating its uniqueness.

Complete genome sequence of E. coli lytic bacteriophage BAU.Micro_ELP-22, isolated from sewage water, Bangladesh.

Escherichia coli lytic bacteriophage BAU.Micro_ELP-22 was isolated from sewage wastewater as a therapeutic agent alternative to antibiotics. The phage genome is 373,488 bp in length, encoding 744 protein-coding sequences and 7 tRNAs, and contains no antibiotic resistance, virulence, or temperate marker genes, which specifies its potentiality as a compatible phage therapy candidate.

Potential of training of anti-Staphylococcus aureus therapeutic phages against Staphylococcus epidermidis multidrug-resistant isolates is restricted by inter- and intra-sequence type specificity.

Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus, while a limited number of studies investigated phage activity against S. epidermidis. We studied the potential of phage training to extend the host range of two types of anti-S. aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S. epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti-S. aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.ImportanceS. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

Phage genome engineering with retrons

Phage genome engineering with retrons

Giant viruses as reservoirs of antibiotic resistance genes

Nucleocytoplasmic large DNA viruses (NCLDVs; also called giant viruses), constituting the phylum Nucleocytoviricota, can infect a wide range of eukaryotes and exchange genetic material with not only their hosts but also prokaryotes and phages. A few NCLDVs were reported to encode genes conferring resistance to beta‑lactam, trimethoprim, or pyrimethamine, suggesting that they are potential vehicles for the transmission of antibiotic resistance genes (ARGs) in the biome. However, the incidence of ARGs across the phylum Nucleocytoviricota, their evolutionary characteristics, their dissemination potential, and their association with virulence factors remain unexplored. Here, we systematically investigated ARGs of 1416 NCLDV genomes including those of almost all currently available cultured isolates and high-quality metagenome-assembled genomes from diverse habitats across the globe. We reveal that 39.5% of them carry ARGs, which is approximately 37 times higher than that for phage genomes. A total of 12 ARG types are encoded by NCLDVs. Phylogenies of the three most abundant NCLDV-encoded ARGs hint that NCLDVs acquire ARGs from not only eukaryotes but also prokaryotes and phages. Two NCLDV-encoded trimethoprim resistance genes are demonstrated to confer trimethoprim resistance in Escherichia coli. The presence of ARGs in NCLDV genomes is significantly correlated with mobile genetic elements and virulence factors.

New evidence supports the prophage origin of RcGTA.

Gene transfer agents (GTAs) are phage-like entities that package and transfer random host genome fragments between prokaryotes. RcGTA, produced by Rhodobacter capsulatus, is hypothesized to originate from a prophage ancestor. Most of the evidence supporting this hypothesis came from the finding of RcGTA-like genes in phages. More than 75% of the RcGTA genes have a phage homolog. However, only a few RcGTA homologs have been identified in a (pro)phage genome, leaving the hypothesis that GTAs evolved from prophages through gene loss with only weak evidence. We herein report the discovery of an inducible prophage (vB_MseS-P1) from a Mesorhizobium sediminum strain that contains the largest number (12) of RcGTA homologs found in a phage genome to date. We also identified three putative prophages and two prophage remnants harboring 12-14 RcGTA homologs in a Methylobacterium nodulans strain. The protein remote homology detection also revealed more RcGTA homologs from other phages than we previously thought. Moreover, the head-tail gene architecture of these newly discovered prophage-related elements closely resembles that of RcGTA. Furthermore, vB_MseS-P1 virions have structural proteins similar to RcGTA particles. Close phylogenetic relationships between certain prophage genes and RcGTA-like genes in Alphaproteobacteria further support the shared ancestry between RcGTA and prophages. Our findings provide new relatively direct evidence of the origin of RcGTA from a prophage progenitor.IMPORTANCEGTAs are important genetic elements in certain groups of bacteria and contribute to the genetic diversification, evolution, and ecological adaptation of bacteria. RcGTA, a common type of GTA, is known to package and transfer random fragments of the bacterial genome to recipient cells. However, the origin of RcGTA is still elusive. It has been hypothesized that RcGTA evolved from a prophage ancestor through gene loss. However, the few RcGTA homologs identified in a (pro)phage genome leave the hypothesis lacking direct evidence. This study uncovers the presence of a large number of RcGTA homologs in an inducible prophage and several putative prophages. The similar head-tail gene architecture and structural protein compositions of these newly discovered prophage-related elements and RcGTA further demonstrate an unprecedentedly observed close evolutionary relationship between prophages and RcGTA. Together, our findings provide more direct evidence supporting the origin of RcGTA from prophage.

DNA polymerase swapping in Caudoviricetes bacteriophages

BackgroundViruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families.MethodsGroups of phages in which DNAP swapping likely occurred were identified as subtrees of a defined depth in a comprehensive evolutionary tree of tailed bacteriophages that included phages with DNAPs of different families. The DNAP swaps were validated by constrained tree analysis that was performed on phylogenetic tree of large terminase subunits, and the phage genomes encoding swapped DNAPs were aligned using Mauve. The structures of the discovered unusual DNAPs were predicted using AlphaFold2.ResultsWe identified four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs “in situ”, without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage genome replication. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication.ConclusionsReplacement of the DNAP gene by one encoding a DNAP of a different family occurred on many independent occasions during the evolution of different families of tailed phages, in some cases, resulting in very closely related phages encoding unrelated DNAPs. DNAP swapping was likely driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility.

Cell-free synthesis of infective phages from in vitro assembled phage genomes for efficient phage engineering and production of large phage libraries.

Bacteriophages are promising alternatives to traditional antimicrobial treatment of bacterial infections. To further increase the potential of phages, efficient engineering methods are needed. This study investigates an approach to phage engineering based on phage rebooting and compares selected methods of assembly and rebooting of phage genomes. GG assembly of phage genomes and subsequent rebooting by cell-free transcription-translation reactions yielded the most efficient phage engineering and allowed production of a proof-of-concept T7 phage library of 1.8 × 107 phages. We obtained 7.5 × 106 different phages, demonstrating generation of large and diverse libraries suitable for high-throughput screening of mutant phenotypes. Implementing versatile and high-throughput phage engineering methods allows vastly accelerated and improved phage engineering, bringing us closer to applying effective phages to treat infections in the clinic.

Phage Immunoprecipitation and Sequencing—a Versatile Technique for Mapping the Antibody Reactome

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

A Bioinformatic Ecosystem for Bacteriophage Genomics: PhaMMSeqs, Phamerator, pdm_utils, PhagesDB, DEPhT, and PhamClust.

The last thirty years have seen a meteoric rise in the number of sequenced bacteriophage genomes, spurred on by both the rise and success of groups working to isolate and characterize phages, and the rapid and significant technological improvements and reduced costs associated with sequencing their genomes. Over the course of these decades, the tools used to glean evolutionary insights from these sequences have grown more complex and sophisticated, and we describe here the suite of computational and bioinformatic tools used extensively by the integrated research-education communities such as SEA-PHAGES and PHIRE, which are jointly responsible for 25% of all complete phage genomes in the RefSeq database. These tools are used to integrate and analyze phage genome data from different sources, for identification and precise extraction of prophages from bacterial genomes, computing "phamilies" of related genes, and displaying the complex nucleotide and amino acid level mosaicism of these genomes. While over 50,000 SEA-PHAGES students have primarily benefitted from these tools, they are freely available for the phage community at large.

Diversity of Shiga toxin transducing phages in Escherichia coli O145:H28 and the different Shiga toxin 2 production levels associated with short- or long-tailed phages.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes serious gastrointestinal illness, including hemorrhagic colitis and hemolytic uremic syndrome. Two types of Stxs (Stx1 and Stx2) are known and both are encoded by bacteriophages (Stx phages), but the production of Stx2 is known to be a major risk factor for severe STEC infections. The production of Stx2, but not Stx1, is tightly coupled with the induction of Stx phages, and Stx2 production levels vary between STEC strains even within the same serotype. Here, we analyzed the genomic diversity of all Stx phages in 71 strains representing the entire O145:H28 lineage, one of the often highly pathogenic STECs, and the relationship between the variations in Stx phage genomes and the levels of Stx2 production by host strains. Our analysis reveals highly dynamic natures of Stx phages in O145:H28, including the independent acquisition of similar Stx phages by different sublineages, the recent transfer of Stx phage between different sublineages, and the frequent gain and loss of Stx phages in some sublineages. We also show the association of the Stx2 phage types with the Stx2 production levels of host strains: strains carrying short-tailed Stx2 phages exhibited significantly higher Stx2 production levels than those carrying long-tailed Stx2 phages. Detailed analyses of the Stx2 phage genomes revealed that both of short- and long-tailed phages exhibited sequence diversification and they were divided into two groups, respectively, based on the sequence similarity of the phage early region encoding genes responsible for phage induction, short-tailed phages contained early regions clearly different in genetic organization from those in long-tailed phages. Therefore, the variations in the early regions between short-and long-tailed Stx2 phages appeared to be linked to a striking difference in Stx2 production levels in their host strains. These results broaden our understanding of the diversification and dynamism of Stx phages in O145:H28 and the association of Stx2 phage types with the Stx2 production level in this STEC lineage.

DepoScope: Accurate phage depolymerase annotation and domain delineation using large language models.

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to identify depolymerase sequences and their enzymatic domains precisely. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which is subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can greatly enhance our understanding of phage-host interactions at the level of depolymerases.

Bacteriophage-encoded protein utilization in bacterial ghost production: a mini-review.

Bacterial ghosts (BGs) are described as bacterial cell envelopes that retain their structure but lack cytoplasmic contents. The study of BGs spans multiple disciplinary domains, and the development of BG production techniques to obtain ample and stable BG samples holds significant implications for probing the biological characteristics of BGs, devising novel disease treatment strategies, and leveraging their industrial applications. Numerous products encoded within bacteriophage (phage) genomes possess the capability to lyse bacteria, thereby inducing BG formation primarily via disruption of bacterial cell wall integrity. This review comprehensively surveys the utilization of phage-encoded proteins in BG production techniques, encompassing methodologies such as phage E protein-mediated lysis, perforin protein-induced lysis, and strategies combining E protein with holin-endolysin systems. Additionally, discussions and summaries are provided on the current applications, challenges, and modification strategies associated with different techniques. Through a focused exploration of BG production techniques, with an emphasis on precise manipulation of BG formation using phage-encoded protein technologies, this study aims to furnish robust tools and methodologies for delving into the mechanisms underlying BG formation, as well as for the development of novel therapeutic strategies and applications based on BGs.