PHACTS, a computational approach to classifying the lifestyle of phages

Motivation: Bacteriophages have two distinct lifestyles: virulent and temperate. The virulent lifestyle has many implications for phage therapy, genomics and microbiology. Determining which lifestyle a newly sequenced phage falls into is currently determined using standard culturing techniques. Such laboratory work is not only costly and time consuming, but also cannot be used on phage genomes constructed from environmental sequencing. Therefore, a computational method that utilizes the sequence data of phage genomes is needed

Inference of the life cycle of environmental phages from genomic signature distances to their hosts

The environmental impact of uncultured phages is shaped by their preferred life cycle (lytic or lysogenic). However, our ability to predict it is very limited. We aimed to discriminate between lytic and lysogenic phages by comparing the similarity of their genomic signatures to those of their hosts, reflecting their co-evolution. We tested two approaches: (1) similarities of tetramer relative frequencies, (2) alignment-free comparisons based on exact k = 14 oligonucleotide matches. First, we explored 5126 reference bacterial host strains and 284 associated phages and found an approximate threshold for distinguishing lysogenic and lytic phages using both oligonucleotide-based methods. The analysis of 6482 plasmids revealed the potential for horizontal gene transfer between different host genera and, in some cases, distant bacterial taxa. Subsequently, we experimentally analyzed combinations of 138 Klebsiella pneumoniae strains and their 41 phages and found that the phages with the largest number of interactions with these strains in the laboratory had the shortest genomic distances to K. pneumoniae. We then applied our methods to 24 single-cells from a hot spring biofilm containing 41 uncultured phage-host pairs, and the results were compatible with the lysogenic life cycle of phages detected in this environment. In conclusion, oligonucleotide-based genome analysis methods can be used for predictions of (1) life cycles of environmental phages, (2) phages with the broadest host range in culture collections, and (3) potential horizontal gene transfer by plasmids

ZOMBIES IN BACTERIAL GENOMES: IDENTIFICATION AND ANALYSIS OF PREVIOUSLY VIRULENT PHAGE

Bacteriophage (or ‘phage’) are viruses that infect and reproduce within their bacterial hosts. They have a major global impact on bacterial evolution and ecology, and might influence the pathogenicity of their host bacterium by providing virulence factors. Phage can either be described as “virulent” or “temperate”; the distinguishing feature between the two is their method of replication. This study sought to identify phage sequences within bacterial host genomes and determine the life cycle of the phage, exploring whether there is a connection between defective phage and previously virulent phage. It would normally be expected that any phage sequences identified within a bacterial host would have a temperate life cycle, since only temperate phage enter the lysogenic cycle and insert their DNA into the host as a ‘prophage,’ while virulent phage replicate via the lytic cycle, in which phage DNA replicates separately from that of the host’s and infected cells are lysed. Defective phage–‘zombies’ in bacterial genomes–are dormant phage that have become inactive through mutational decay or some other process. It is possible that some of these defective phage are in fact previously virulent phage that have become accidentally inserted within the host genome. This study detected phage within bacterial genomes using the prophage identification tools PHAge Search Tool (PHAST) and Prophage Finder. Identified sequences were categorized as ‘intact,’ ‘questionable,’ or ‘incomplete’; questionable and incomplete phage were classified as defective. The lifestyles of the uncovered phage sequences were then determined using PHACTS; six phage were identified as possibly virulent. The life cycles of the phage were further analyzed by assessing the genomic signature distances (GSD) and codon adaptation indexes (CAI) for each phage. Three phage were shown to have a GSD consistent with a virulent life cycle, and the CAI values of four phage corresponded with that of virulent phage. Although previous studies have indicated that some virulent phage may have a temperate lineage, identifying prophage as previously virulent is a novel finding. This has implications for our understanding of phage life cycles and the infection process, as it challenges the idea that only temperate phage insert their DNA into the host genome

Complete genome sequence of new bacteriophage phiE142, which causes simultaneously lysis of multidrug-resistant Escherichia coli O157:H7 and Salmonella enterica

Bacterial strains used in the host range spectrum of the bacteriophage phiE142. Phage was assessed for host range by spot testing. (+) indicate positive sensitivity to phage lysis, and (-) indicate negative sensitivity to phage lysis. (DOCX 41 kb

High-level diversity of tailed phages, eukaryote-associated viruses, and virophage-like elements in the metaviromes of Antarctic soils

The metaviromes of two distinct Antarctic hyperarid desert soil communities have been characterized. Hypolithic communities, cyanobacterium-dominated assemblages situated on the ventral surfaces of quartz pebbles embedded in the desert pavement, showed higher virus diversity than surface soils, which correlated with previous bacterial community studies. Prokaryotic viruses (i.e., phages) represented the largest viral component (particularly Mycobacterium phages) in both habitats, with an identical hierarchical sequence abundance of families of tailed phages (Siphoviridae>Myoviridae>Podoviridae). No archaeal viruses were found. Unexpectedly, cyanophages were poorly represented in both metaviromes and were phylogenetically distant from currently characterized cyanophages. Putative phage genomes were assembled and showed a high level of unaffiliated genes, mostly from hypolithic viruses. Moreover, unusual gene arrangements in which eukaryotic and prokaryotic virus-derived genes were found within identical genome segments were observed. Phycodnaviridae and Mimiviridae viruses were the second-mostabundant taxa and more numerous within open soil. Novel virophage-like sequences (within the Sputnik clade) were identified. These findings highlight high-level virus diversity and novel species discovery potential within Antarctic hyperarid soils and may serve as a starting point for future studies targeting specific viral groups.IS

Phage Genome Diversity in a Biogas-Producing Microbiome Analyzed by Illumina and Nanopore GridION Sequencing

The microbial biogas network is complex and intertwined, and therefore relatively stable in its overall functionality. However, if key functional groups of microorganisms are affected by biotic or abiotic factors, the entire efficacy may be impaired. Bacteriophages are hypothesized to alter the steering process of the microbial network. In this study, an enriched fraction of virus-like particles was extracted from a mesophilic biogas reactor and sequenced on the Illumina MiSeq and Nanopore GridION sequencing platforms. Metagenome data analysis resulted in identifying 375 metagenome-assembled viral genomes (MAVGs). Two-thirds of the classified sequences were only assigned to the superkingdom Viruses and the remaining third to the family Siphoviridae, followed by Myoviridae, Podoviridae, Tectiviridae, and Inoviridae. The metavirome showed a close relationship to the phage genomes that infect members of the classes Clostridia and Bacilli. Using publicly available biogas metagenomic data, a fragment recruitment approach showed the widespread distribution of the MAVGs studied in other biogas microbiomes. In particular, phage sequences from mesophilic microbiomes were highly similar to the phage sequences of this study. Accordingly, the virus particle enrichment approach and metavirome sequencing provided additional genome sequence information for novel virome members, thus expanding the current knowledge of viral genetic diversity in biogas reactors.BMBF, 031L0103, de.NBI - Partner - MetaProtServBMBF, 031A532B, de.NBI - Etablierungsphase - Koordinierungs- und Administrationseinheit - CAU - GeschäftsstelleBMBF, 031A533A, de.NBI - Etablierungsphase - Leistungszentrum - BiGi - Bioinformatisches Resourcenzentrum für mikrobielle Genomforschung in Biotechnologie und MedizinBMBF, 031A533B, de.NBI - Etablierungsphase - Leistungszentrum - BiGi - Bioinformatisches Resourcenzentrum für mikrobielle Genomforschung in Biotechnologie und MedizinBMBF, 031A534A, de.NBI - Etablierungsphase - Leistungszentrum - BioInfra.Prot - Bioinformatik der ProteomikBMBF, 031A535A, de.NBI - Etablierungsphase - Leistungszentrum CiBi - Zentrum für integrative BioinformatikBMBF, 031A537A, de.NBI - Etablierungsphase - Heidelberg Center for Human Bioinformatics - HD-HuB - Humane Genetik und Genomik, Humane Mikrobiomik, systematische Phänotypisierung humaner ZellenBMBF, 031A537B, de.NBI - Etablierungsphase-European Molecular Biology Laboratory-HD-HUBBMBF, 031A537C, de.NBI - Etablierungsphase - Heidelberg Center for Human Bioinformatics - HD-HuBBMBF, 031A537D, de.NBI - Etablierungsphase - Heidelberg Center for Human Bioinformatics - HD-HuB - Humane Genetik und Genomik, Humane Metagenomik, systematische Phänotypisierung humaner Zellen, Epigenetik und de.NBI Cloud Standort Heidelberg/BerlinBMBF, 031A538A, de.NBI - Etablierungsphase - Leistungszentrum: RBC - RNA Bioinformati

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae

Characterization and diversity of phages infecting Aeromonas salmonicida subsp. salmonicida

Phages infecting Aeromonas salmonicida subsp. salmonicida, the causative agent of the fsh disease furunculosis, have been isolated for decades but very few of them have been characterized. Here, the host range of 12 virulent phages, including three isolated in the present study, was evaluated against a panel of 65A. salmonicida isolates, including representatives of the psychrophilic subspecies salmonicida, smithia, masoucida, and the mesophilic subspecies pectinolytica. This bacterial set also included three isolates from India suspected of being members of a new subspecies. Our results allowed to elucidate a lytic dichotomy based on the lifestyle of A. salmonicida (mesophilic or psychrophilic) and more generally, on phage types (lysotypes) for the subspecies salmonicida. The genomic analyses of the 12 phages from this study with those available in GenBank led us to propose an A. salmonicida phage pan-virome. Our comparative genomic analyses also suggest that some phage genes were under positive selection and A. salmonicida phage genomes having a discrepancy in GC% compared to the host genome encode tRNA genes to likely overpass the bias in codon usage. Finally, we propose a new classifcation scheme for A. salmonicida phages

Genomic Analysis of Prophages from Klebsiella pneumoniae Clinical Isolates

Klebsiella pneumoniae is an increasing threat to public health and represents one of the most concerning pathogens involved in life-threatening infections. The resistant and virulence determinants are coded by mobile genetic elements which can easily spread between bacteria populations and co-evolve with its genomic host. In this study, we present the full genomic sequences, insertion sites and phylogenetic analysis of 150 prophages found in 40 K. pneumoniae clinical isolates obtained from an outbreak in a Portuguese hospital. All strains harbored at least one prophage and we identified 104 intact prophages (69.3%). The prophage size ranges from 29.7 to 50.6 kbp, coding between 32 and 78 putative genes. The prophage GC content is 51.2%, lower than the average GC content of 57.1% in K. pneumoniae. Complete prophages were classified into three families in the order Caudolovirales: Myoviridae (59.6%), Siphoviridae (38.5%) and Podoviridae (1.9%). In addition, an alignment and phylogenetic analysis revealed nine distinct clusters. Evidence of recombination was detected within the genome of some prophages but, in most cases, proteins involved in viral structure, transcription, replication and regulation (lysogenic/lysis) were maintained. These results support the knowledge that prophages are diverse and widely disseminated in K. pneumoniae genomes, contributing to the evolution of this species and conferring additional phenotypes. Moreover, we identified K. pneumoniae prophages in a set of endolysin genes, which were found to code for proteins with lysozyme activity, cleaving the β-1,4 linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan network and thus representing genes with the potential for lysin phage therapy.F.F.V. is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (PTDC/BTM-SAL/28978/2017) that supported this work. The work is partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020.info:eu-repo/semantics/publishedVersio