Phables: from fragmented assemblies to high-quality bacteriophage genomes.

MOTIVATION: Microbial communities have a profound impact on both human health and various environments. Viruses infecting bacteria, known as bacteriophages or phages, play a key role in modulating bacterial communities within environments. High-quality phage genome sequences are essential for advancing our understanding of phage biology, enabling comparative genomics studies and developing phage-based diagnostic tools. Most available viral identification tools consider individual sequences to determine whether they are of viral origin. As a result of challenges in viral assembly, fragmentation of genomes can occur, and existing tools may recover incomplete genome fragments. Therefore, the identification and characterization of novel phage genomes remain a challenge, leading to the need of improved approaches for phage genome recovery. RESULTS: We introduce Phables, a new computational method to resolve phage genomes from fragmented viral metagenome assemblies. Phables identifies phage-like components in the assembly graph, models each component as a flow network, and uses graph algorithms and flow decomposition techniques to identify genomic paths. Experimental results of viral metagenomic samples obtained from different environments show that Phables recovers on average over 49% more high-quality phage genomes compared to existing viral identification tools. Furthermore, Phables can resolve variant phage genomes with over 99% average nucleotide identity, a distinction that existing tools are unable to make. AVAILABILITY AND IMPLEMENTATION: Phables is available on GitHub at https://github.com/Vini2/phables.

Marginal lands and fungi - linking the type of soil contamination with fungal community composition.

Fungi can be found in almost all ecosystems. Some of them can even survive in harsh, anthropogenically transformed environments, such as post-industrial soils. In order to verify how the soil fungal diversity may be changed by pollution, two soil samples from each of the 28 post-industrial sites were collected. Each soil sample was characterized in terms of concentration of heavy metals and petroleum derivatives. To identify soil fungal communities, fungal internal transcribed spacer 2 (ITS2) amplicon was sequenced for each sample using Illumina MiSeq platform. There were significant differences in the community structure and taxonomic diversity among the analysed samples. The highest taxon richness and evenness were observed in the non-polluted sites, and lower numbers of taxa were identified in multi-polluted soils. The presence of monocyclic aromatic hydrocarbons, gasoline and mineral oil was determined as the factors driving the differences in the mycobiome. Furthermore, in the culture-based selection experiment, two main groups of fungi growing on polluted media were identified - generalists able to live in the presence of pollution, and specialists adapted to the usage of BTEX as a sole source of energy. Our selection experiment proved that it is long-term soil contamination that shapes the community, rather than temporary addition of pollutant.

Heterologous production and characterization of a pyomelanin of Antarctic Pseudomonas sp. ANT_H4: a metabolite protecting against UV and free radicals, interacting with iron from minerals and exhibiting priming properties toward plant hairy roots.

BACKGROUND: Antarctica has one of the most extreme environments in the world. This region is inhabited by specifically adapted microorganisms that produce various unique secondary metabolites (e.g. pigments) enabling their survival under the harsh environmental conditions. It was already shown that these natural, biologically active molecules may find application in various fields of biotechnology. RESULTS: In this study, a cold-active brown-pigment-producing Pseudomonas sp. ANT_H4 strain was characterized. In-depth genomic analysis combined with the application of a fosmid expression system revealed two different pathways of melanin-like compounds biosynthesis by the ANT_H4 strain. The chromatographic behavior and Fourier-transform infrared spectroscopic analyses allowed for the identification of the extracted melanin-like compound as a pyomelanin. Furthermore, optimization of the production and thorough functional analyses of the pyomelanin were performed to test its usability in biotechnology. It was confirmed that ANT_H4-derived pyomelanin increases the sun protection factor, enables scavenging of free radicals, and interacts with the iron from minerals. Moreover, it was shown for the first time that pyomelanin exhibits priming properties toward Calendula officinalis hairy roots in in vitro cultures. CONCLUSIONS: Results of the study indicate the significant biotechnological potential of ANT_H4-derived pyomelanin and open opportunities for future applications. Taking into account protective features of analyzed pyomelanin it may be potentially used in medical biotechnology and cosmetology. Especially interesting was showing that pyomelanin exhibits priming properties toward hairy roots, which creates a perspective for its usage for the development of novel and sustainable agrotechnical solutions.

Characteristics and Comparative Genomic Analysis of a Novel Virus, VarioGold, the First Bacteriophage of Variovorax.

Variovorax represents a widespread and ecologically significant genus of soil bacteria. Despite the ecological importance of these bacteria, our knowledge about the viruses infecting Variovorax spp. is quite poor. This study describes the isolation and characterization of the mitomycin-induced phage, named VarioGold. To the best of our knowledge, VarioGold represents the first characterized virus for this genus. Comparative genomic analyses suggested that VarioGold is distinct from currently known bacteriophages at both the nucleotide and protein levels; thus, it could be considered a new virus genus. In addition, another 37 prophages were distinguished in silico within the complete genomic sequences of Variovorax spp. that are available in public databases. The similarity networking analysis highlighted their general high diversity, which, despite clustering with previously described phages, shows their unique genetic load. Therefore, the novelty of Variovorax phages warrants the great enrichment of databases, which could, in turn, improve bioinformatic strategies for finding (pro)phages.

Genome Study of a Novel Virulent Phage vB_SspS_KASIA and Mu-like Prophages of Shewanella sp. M16 Provides Insights into the Genetic Diversity of the Shewanella Virome.

Shewanella is a ubiquitous bacterial genus of aquatic ecosystems, and its bacteriophages are also isolated from aquatic environments (oceans, lakes, ice, and wastewater). In this study, the isolation and characterization of a novel virulent Shewanella phage vB_SspS_KASIA and the identification of three prophages of its host, Shewanella sp. M16, including a mitomycin-inducible Mu-like siphovirus, vB_SspS_MuM16-1, became the starting point for comparative analyses of phages infecting Shewanella spp. and the determination of their position among the known bacterial viruses. A similarity networking analysis revealed the high diversity of Shewanella phages in general, with vB_SspS_KASIA clustering exclusively with Colwellia phage 9A, with which it forms a single viral cluster composed of two separate viral subclusters. Furthermore, vB_SspS_MuM16-1 presented itself as being significantly different from the phages deposited in public databases, expanding the diversity of the known Mu-like phages and giving potential molecular markers for the identification of Mu-like prophages in bacterial genomes. Moreover, the functional analysis performed for vB_SspS_KASIA suggested that, despite the KASIA host, the M16 strain grows better in a rich medium and at 30 °C the phage replication cycle seems to be optimal in restrictive culture conditions mimicking their natural environment, the Zloty Stok gold and arsenic mine.

Plasmidome of Listeria spp.-The repA-Family Business.

Bacteria of the genus Listeria (phylum Firmicutes) include both human and animal pathogens, as well as saprophytic strains. A common component of Listeria spp. genomes are plasmids, i.e., extrachromosomal replicons that contribute to gene flux in bacteria. This study provides an in-depth insight into the structure, diversity and evolution of plasmids occurring in Listeria strains inhabiting various environments under different anthropogenic pressures. Apart from the components of the conserved plasmid backbone (providing replication, stable maintenance and conjugational transfer functions), these replicons contain numerous adaptive genes possibly involved in: (i) resistance to antibiotics, heavy metals, metalloids and sanitizers, and (ii) responses to heat, oxidative, acid and high salinity stressors. Their genomes are also enriched by numerous transposable elements, which have influenced the plasmid architecture. The plasmidome of Listeria is dominated by a group of related replicons encoding the RepA replication initiation protein. Detailed comparative analyses provide valuable data on the level of conservation of these replicons and their role in shaping the structure of the Listeria pangenome, as well as their relationship to plasmids of other genera of Firmicutes, which demonstrates the range and direction of flow of genetic information in this important group of bacteria.

Identification, Characterization, and Genomic Analysis of Novel Serratia Temperate Phages from a Gold Mine.

Bacteria of the genus Serratia inhabit a variety of ecological niches like water, soil, and the bodies of animals, and have a wide range of lifestyles. Currently, the complete genome sequences of 25 Serratia phages are available in the NCBI database. All of them were isolated from nutrient-rich environments like sewage, with the use of clinical Serratia strains as hosts. In this study, we identified a novel Serratia myovirus named vB_SspM_BZS1. Both the phage and its host Serratia sp. OS31 were isolated from the same oligotrophic environment, namely, an abandoned gold mine (Zloty Stok, Poland). The BZS1 phage was thoroughly characterized here in terms of its genomics, morphology, and infection kinetics. We also demonstrated that Serratia sp. OS31 was lysogenized by mitomycin-inducible siphovirus vB_SspS_OS31. Comparative analyses revealed that vB_SspM_BZS1 and vB_SspS_OS31 were remote from the known Serratia phages. Moreover, vB_SspM_BZS1 was only distantly related to other viruses. However, we discovered similar prophage sequences in genomes of various bacteria here. Additionally, a protein-based similarity network showed a high diversity of Serratia phages in general, as they were scattered across nineteen different clusters. In summary, this work broadened our knowledge on the diverse relationships of Serratia phages.

Identification and Characterization of the First Virulent Phages, Including a Novel Jumbo Virus, Infecting Ochrobactrum spp.

The Ochrobactrum genus consists of an extensive repertoire of biotechnologically valuable bacterial strains but also opportunistic pathogens. In our previous study, a novel strain, Ochrobactrum sp. POC9, which enhances biogas production in wastewater treatment plants (WWTPs) was identified and thoroughly characterized. Despite an insightful analysis of that bacterium, its susceptibility to bacteriophages present in WWTPs has not been evaluated. Using raw sewage sample from WWTP and applying the enrichment method, two virulent phages, vB_OspM_OC and vB_OspP_OH, which infect the POC9 strain, were isolated. These are the first virulent phages infecting Ochrobactrum spp. identified so far. Both phages were subjected to thorough functional and genomic analyses, which allowed classification of the vB_OspM_OC virus as a novel jumbo phage, with a genome size of over 227 kb. This phage encodes DNA methyltransferase, which mimics the specificity of cell cycle regulated CcrM methylase, a component of the epigenetic regulatory circuits in Alphaproteobacteria. In this study, an analysis of the overall diversity of Ochrobactrum-specific (pro)phages retrieved from databases and extracted in silico from bacterial genomes was also performed. Complex genome mining allowed us to build similarity networks to compare 281 Ochrobactrum-specific viruses. Analyses of the obtained networks revealed a high diversity of Ochrobactrum phages and their dissimilarity to the viruses infecting other bacteria.

Characterization of a Unique Bordetella bronchiseptica vB_BbrP_BB8 Bacteriophage and Its Application as an Antibacterial Agent.

Bordetella bronchiseptica, an emerging zoonotic pathogen, infects a broad range of mammalian hosts. B. bronchiseptica-associated atrophic rhinitis incurs substantial losses to the pig breeding industry. The true burden of human disease caused by B. bronchiseptica is unknown, but it has been postulated that some hypervirulent B. bronchiseptica isolates may be responsible for undiagnosed respiratory infections in humans. B. bronchiseptica was shown to acquire antibiotic resistance genes from other bacterial genera, especially Escherichia coli. Here, we present a new B. bronchiseptica lytic bacteriophage-vB_BbrP_BB8-of the Podoviridae family, which offers a safe alternative to antibiotic treatment of B. bronchiseptica infections. We explored the phage at the level of genome, physiology, morphology, and infection kinetics. Its therapeutic potential was investigated in biofilms and in an in vivo Galleria mellonella model, both of which mimic the natural environment of infection. The BB8 is a unique phage with a genome structure resembling that of T7-like phages. Its latent period is 75 ± 5 min and its burst size is 88 ± 10 phages. The BB8 infection causes complete lysis of B. bronchiseptica cultures irrespective of the MOI used. The phage efficiently removes bacterial biofilm and prevents the lethality induced by B. bronchiseptica in G. mellonella honeycomb moth larvae.

Genome-Wide and Functional View of Proteolytic and Lipolytic Bacteria for Efficient Biogas Production through Enhanced Sewage Sludge Hydrolysis.

In this study, we used a multifaceted approach to select robust bioaugmentation candidates for enhancing biogas production and to demonstrate the usefulness of a genome-centric approach for strain selection for specific bioaugmentation purposes. We also investigated the influence of the isolation source of bacterial strains on their metabolic potential and their efficiency in enhancing anaerobic digestion. Whole genome sequencing, metabolic pathway reconstruction, and physiological analyses, including phenomics, of phylogenetically diverse strains, Rummeliibacillus sp. POC4, Ochrobactrum sp. POC9 (both isolated from sewage sludge) and Brevundimonas sp. LPMIX5 (isolated from an agricultural biogas plant) showed their diverse enzymatic activities, metabolic versatility and ability to survive under varied growth conditions. All tested strains display proteolytic, lipolytic, cellulolytic, amylolytic, and xylanolytic activities and are able to utilize a wide array of single carbon and energy sources, as well as more complex industrial by-products, such as dairy waste and molasses. The specific enzymatic activity expressed by the three strains studied was related to the type of substrate present in the original isolation source. Bioaugmentation with sewage sludge isolates-POC4 and POC9-was more effective for enhancing biogas production from sewage sludge (22% and 28%, respectively) than an approach based on LPMIX5 strain (biogas production boosted by 7%) that had been isolated from an agricultural biogas plant, where other type of substrate is used.

cytb as a New Genetic Marker for Differentiation of Prototheca Species.

Achlorophyllous unicellular microalgae of the genus Prototheca (Trebouxiophyceae, Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused by Protothecawickerhamii, followed by Protothecazopfii and, sporadically, by Protothecacutis and Protothecamiyajii In veterinary medicine, however, P. zopfii is a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification of Prototheca spp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among all Prototheca spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all Prototheca spp. We propose the mitochondrial cytb gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae. The cytb gene displayed important advantages over the rDNA markers. Not only did the cytb gene have the highest discriminatory capacity for resolving all Prototheca species, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partial cytb gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all Prototheca species described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.

Molecular characterization of the pA3J1 plasmid from the psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_J3.

The knowledge on plasmids of cold-active bacteria is highly limited. In this study, the molecular characterization of the pA3J1 plasmid of Antarctic psychrotolerant bacterium Pseudomonas sp. ANT_J3 was performed. Within this plasmid, thirteen putative open reading frames were identified. Nine of them encoded proteins involved in replication, partitioning, postsegregational elimination of plasmid-less cells (via a toxin-antitoxin system activity), multimer resolution and mobilization by conjugal transfer. These genes constitute the plasmid backbone. The functional analysis of the pA3J1 maintenance region revealed that it is a narrow host range replicon, stably maintained in the host cells by the combined activities of the partitioning and relBE-type toxin-antitoxin systems. It was also suggested that the replication system of the pA3J1 plasmid may be temperature-sensitive. Comparative analyses revealed the presence of 16 Pseudomonas plasmids encoding homologous replication proteins and 5 plasmids carrying mobA genes homologous to the corresponding gene of pA3J1. The relaxase (MobA) of the pA3J1 plasmid was classified into MOBQ family, and the phylogenetic analysis suggested that this may be a representative of a novel group (or subgroup) within this family. The structural and comparative analyses revealed that the arrangement of genetic modules in the pA3J1 plasmid is unique.

Application of Metagenomic Analyses in Dentistry as a Novel Strategy Enabling Complex Insight into Microbial Diversity of the Oral Cavity.

The composition of the oral microbiome in healthy individuals is complex and dynamic, and depends on many factors, such as anatomical location in the oral cavity, diet, oral hygiene habits or host immune responses. It is estimated at present that worldwide about 2 billion people suffer from diseases of the oral cavity, mainly periodontal disease and dental caries. Importantly, the oral microflora involved in local infections may spread and cause systemic, even life-threatening infections. In search for etiological agents of infections in dentistry, traditional approaches are not sufficient, as about 50% of oral bacteria are not cultivable. Instead, metagenomic analyses are particularly useful for studies of the complex oral microbiome - both in healthy individuals, and in patients with oral and dental diseases. In this paper we review the current and future applications of metagenomic studies in evaluation of both the composition of the oral microbiome as well as its potential pathogenic role in infections in dentistry.

Structure and functions of a multireplicon genome of Antarctic Psychrobacter sp. ANT_H3: characterization of the genetic modules suitable for the construction of the plasmid-vectors for cold-active bacteria.

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT_H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT_H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT_H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations.

Revealing the diversity of bacteria and fungi in the active layer of permafrost at Spitsbergen island (Arctic) - Combining classical microbiology and metabarcoding for ecological and bioprospecting exploration.

Arctic soils are constantly subjected to extreme environmental conditions such as low humidity, strong winds, high salinity, freeze-thaw cycles, UV exposition, and low nutrient availability, therefore, they have developed unique microbial ecosystems. These environments provide excellent opportunities to study microbial ecology and evolution within pristine (i.e. with limited anthropogenic influence) regions since the High Arctic is still considered one of the wildest and least explored environments on the planet. This environment is also of interest for the screening and recovery of unique microbial strains suitable for various biotechnological applications. In this study, a combination of culture-depended and culture-independent approaches was used to determine the cultivation bias in studies of the diversity of cold-active microorganisms. Cultivation bias is a reduction in recovered diversity, introduced when applying a classical culturing technique. Six different soil types, collected in the vicinity of the Polish Polar Station Hornsund (Spitsbergen, Norway), were tested. It was revealed that the used media allowed recovery of only 6.37 % of bacterial and 20 % of fungal genera when compared with a culture-independent approach. Moreover, it was shown that a combination of R2A and Marine Broth media recovered as much as 93.6 % of all cultivable bacterial genera detected in this study. Based on these results, a novel protocol for genome-guided bioprospecting, combining a culture-dependent approach, metabarcoding, next-generation sequencing, and genomic data reuse was developed. With this methodology, 14 psychrotolerant, multi-metal-resistant strains, including the highly promising Rhodococcus spp., were obtained. These strains, besides increased metal tolerance, have a petroleum hydrocarbon utilization capacity, and thus may be good candidates for future bioremediation technologies, also suited to permanently cold regions.

Spoligotyping of Mycobacterium tuberculosis - Comparing in vitro and in silico approaches.

Spoligotyping is one of the molecular typing methods widely used for exploring the genetic variety of Mycobacterium tuberculosis. The aim of this study was to compare the spoligoprofiles of M. tuberculosis clinical isolates, obtained using in vitro and in silico approaches. The study included 230 M. tuberculosis isolates, recovered from Poland and Lithuania between 2018 and 2021. Spoligotyping in vitro was performed with a commercially available kit. Whole genome sequencing (WGS) was done with Illumina NovaSeq 6000 sequencer. Spoligotype International Types (SITs) were assigned according to the SITVIT2 database or using three different in silico tools, and based on WGS data, namely SpoTyping, SpolPred, and lorikeet. Upon in vitro spoligotyping, the isolates produced 65 different spoligotypes. Spoligotypes inferred from the WGS data were congruent with in vitro generated patterns in 81.7% (188/230) for lorikeet and 81.3% (187/230) for SpolPred and SpoTyping. Spacers 18 and 31 produced the highest ratio of discrepant results between in vitro and in silico approaches, with their signals discordantly assigned for 15 (6.5%) and 9 (3.9%) isolates, respectively. All three in silico approaches used were similarly efficient for M. tuberculosis spoligotype prediction. However, only SpoTyping could predict spoligotypes without a need for manual curation. Thus, we consider it as the most accurate tool. Its use is further advocated by the shortest time of analysis. A relatively high (ca. 20%) discordance between in vitro and in silico spoligotyping results was observed. While we discourage comparing conventional spoligotyping with in silico equivalents, we advise the use of the latter, as it improves the accuracy of spoligopatterns, and thus depicts the relatedness between the isolates more reliably.

The Promise and Pitfalls of Prophages.

Phages dominate every ecosystem on the planet. While virulent phages sculpt the microbiome by killing their bacterial hosts, temperate phages provide unique growth advantages to their hosts through lysogenic conversion. Many prophages benefit their host, and prophages are responsible for genotypic and phenotypic differences that separate individual microbial strains. However, the microbes also endure a cost to maintain those phages: additional DNA to replicate and proteins to transcribe and translate. We have never quantified those benefits and costs. Here, we analysed over two and a half million prophages from over half a million bacterial genome assemblies. Analysis of the whole dataset and a representative subset of taxonomically diverse bacterial genomes demonstrated that the normalised prophage density was uniform across all bacterial genomes above 2 Mbp. We identified a constant carrying capacity of phage DNA per bacterial DNA. We estimated that each prophage provides cellular services equivalent to approximately 2.4 % of the cell's energy or 0.9 ATP per bp per hour. We demonstrate analytical, taxonomic, geographic, and temporal disparities in identifying prophages in bacterial genomes that provide novel targets for identifying new phages. We anticipate that the benefits bacteria accrue from the presence of prophages balance the energetics involved in supporting prophages. Furthermore, our data will provide a new framework for identifying phages in environmental datasets, diverse bacterial phyla, and from different locations.

Phables: from fragmented assemblies to high-quality bacteriophage genomes.

Microbial communities influence both human health and different environments. Viruses infecting bacteria, known as bacteriophages or phages, play a key role in modulating bacterial communities within environments. High-quality phage genome sequences are essential for advancing our understanding of phage biology, enabling comparative genomics studies, and developing phage-based diagnostic tools. Most available viral identification tools consider individual sequences to determine whether they are of viral origin. As a result of the challenges in viral assembly, fragmentation of genomes can occur, leading to the need for new approaches in viral identification. Therefore, the identification and characterisation of novel phages remain a challenge. We introduce Phables, a new computational method to resolve phage genomes from fragmented viral metagenome assemblies. Phables identifies phage-like components in the assembly graph, models each component as a flow network, and uses graph algorithms and flow decomposition techniques to identify genomic paths. Experimental results of viral metagenomic samples obtained from different environments show that Phables recovers on average over 49% more high-quality phage genomes compared to existing viral identification tools. Furthermore, Phables can resolve variant phage genomes with over 99% average nucleotide identity, a distinction that existing tools are unable to make. Phables is available on GitHub at https://github.com/Vini2/phables.

Host interactions of novel Crassvirales species belonging to multiple families infecting bacterial host, Bacteroides cellulosilyticus WH2.

Bacteroides, the prominent bacteria in the human gut, play a crucial role in degrading complex polysaccharides. Their abundance is influenced by phages belonging to the Crassvirales order. Despite identifying over 600 Crassvirales genomes computationally, only few have been successfully isolated. Continued efforts in isolation of more Crassvirales genomes can provide insights into phage-host-evolution and infection mechanisms. We focused on wastewater samples, as potential sources of phages infecting various Bacteroides hosts. Sequencing, assembly, and characterization of isolated phages revealed 14 complete genomes belonging to three novel Crassvirales species infecting Bacteroides cellulosilyticus WH2. These species, Kehishuvirus sp. 'tikkala' strain Bc01, Kolpuevirus sp. 'frurule' strain Bc03, and 'Rudgehvirus jaberico' strain Bc11, spanned two families, and three genera, displaying a broad range of virion productions. Upon testing all successfully cultured Crassvirales species and their respective bacterial hosts, we discovered that they do not exhibit co-evolutionary patterns with their bacterial hosts. Furthermore, we observed variations in gene similarity, with greater shared similarity observed within genera. However, despite belonging to different genera, the three novel species shared a unique structural gene that encodes the tail spike protein. When investigating the relationship between this gene and host interaction, we discovered evidence of purifying selection, indicating its functional importance. Moreover, our analysis demonstrated that this tail spike protein binds to the TonB-dependent receptors present on the bacterial host surface. Combining these observations, our findings provide insights into phage-host interactions and present three Crassvirales species as an ideal system for controlled infectivity experiments on one of the most dominant members of the human enteric virome. Impact statement: Bacteriophages play a crucial role in shaping microbial communities within the human gut. Among the most dominant bacteriophages in the human gut microbiome are Crassvirales phages, which infect Bacteroides. Despite being widely distributed, only a few Crassvirales genomes have been isolated, leading to a limited understanding of their biology, ecology, and evolution. This study isolated and characterized three novel Crassvirales genomes belonging to two different families, and three genera, but infecting one bacterial host, Bacteroides cellulosilyticus WH2. Notably, the observation confirmed the phages are not co-evolving with their bacterial hosts, rather have a shared ability to exploit similar features in their bacterial host. Additionally, the identification of a critical viral protein undergoing purifying selection and interacting with the bacterial receptors opens doors to targeted therapies against bacterial infections. Given Bacteroides role in polysaccharide degradation in the human gut, our findings advance our understanding of the phage-host interactions and could have important implications for the development of phage-based therapies. These discoveries may hold implications for improving gut health and metabolism to support overall well-being. Data summary: The genomes used in this research are available on Sequence Read Archive (SRA) within the project, PRJNA737576. Bacteroides cellulosilyticus WH2, Kehishuvirus sp. 'tikkala' strain Bc01, Kolpuevirus sp. ' frurule' strain Bc03, and 'Rudgehvirus jaberico' strain Bc11 are all available on GenBank with accessions NZ_CP072251.1 ( B. cellulosilyticus WH2), QQ198717 (Bc01), QQ198718 (Bc03), and QQ198719 (Bc11), and we are working on making the strains available through ATCC. The 3D protein structures for the three Crassvirales genomes are available to download at doi.org/10.25451/flinders.21946034.

Host interactions of novel Crassvirales species belonging to multiple families infecting bacterial host, Bacteroides cellulosilyticus WH2.

Bacteroides, the prominent bacteria in the human gut, play a crucial role in degrading complex polysaccharides. Their abundance is influenced by phages belonging to the Crassvirales order. Despite identifying over 600 Crassvirales genomes computationally, only few have been successfully isolated. Continued efforts in isolation of more Crassvirales genomes can provide insights into phage-host-evolution and infection mechanisms. We focused on wastewater samples, as potential sources of phages infecting various Bacteroides hosts. Sequencing, assembly, and characterization of isolated phages revealed 14 complete genomes belonging to three novel Crassvirales species infecting Bacteroides cellulosilyticus WH2. These species, Kehishuvirus sp. 'tikkala' strain Bc01, Kolpuevirus sp. 'frurule' strain Bc03, and 'Rudgehvirus jaberico' strain Bc11, spanned two families, and three genera, displaying a broad range of virion productions. Upon testing all successfully cultured Crassvirales species and their respective bacterial hosts, we discovered that they do not exhibit co-evolutionary patterns with their bacterial hosts. Furthermore, we observed variations in gene similarity, with greater shared similarity observed within genera. However, despite belonging to different genera, the three novel species shared a unique structural gene that encodes the tail spike protein. When investigating the relationship between this gene and host interaction, we discovered evidence of purifying selection, indicating its functional importance. Moreover, our analysis demonstrated that this tail spike protein binds to the TonB-dependent receptors present on the bacterial host surface. Combining these observations, our findings provide insights into phage-host interactions and present three Crassvirales species as an ideal system for controlled infectivity experiments on one of the most dominant members of the human enteric virome.