Genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences

Bacterial viruses (bacteriophages) have a key role in shaping the development and functional outputs of host microbiomes. Although metagenomic approaches have greatly expanded our understanding of the prokaryotic virosphere, additional tools are required for the phage-oriented dissection of metagenomic data sets, and host-range affiliation of recovered sequences. Here we demonstrate the application of a genome signature-based approach to interrogate conventional whole-community metagenomes and access subliminal, phylogenetically targeted, phage sequences present within. We describe a portion of the biological dark matter extant in the human gut virome, and bring to light a population of potentially gut-specific Bacteroidales-like phage, poorly represented in existing virus like particle-derived viral metagenomes. These predominantly temperate phage were shown to encode functions of direct relevance to human health in the form of antibiotic resistance genes, and provided evidence for the existence of putative ‘viral-enterotypes’ among this fraction of the human gut virome

Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome

Characterisation and UV inactivation of bacteriophages infecting human-specific bacteroides strain GB-124

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterisation and UV inactivation of bacteriophages infecting human-specific bacteroides strain GB-124

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Resolution of habitat-associated ecogenomic signatures in bacteriophage genomes and application to microbial source tracking

Just as the expansion in genome sequencing has revealed and permitted the exploitation of phylogenetic signals embedded in bacterial genomes, the application of metagenomics has begun to provide similar insights at the ecosystem-level for microbial communities. However, little is known regarding this aspect of bacteriophage associated with microbial ecosystems, and if phage encode discernible habitat-associated signals diagnostic of underlying microbiomes. Here we demonstrate that individual phage can encode clear habitat-related “ecogenomic signatures”, based on relative representation of phage encoded gene homologues in metagenomic datasets. Furthermore, we show the  ecogenomic signature encoded by the gut-associated ɸB124-14 can be used to segregate metagenomes according to environmental origin, and distinguish “contaminated” environmental metagenomes (subject to simulated in silico human faecal pollution) from uncontaminated datasets. This indicates phage encoded ecological signals likely possess sufficient discriminatory power for use in biotechnological applications, such as development of microbial source tracking tools for monitoring water quality

Analysis of the mature φB124-14 proteome.

<p>Spectra of φB124-14 proteins identified by tandem mass spectrometry. Example peptide spectra for each of the three proteins identified are shown. Table provides protein coverage and associated number of unique peptides matched and the sequence of the top four matches (ranked by by XCorr score).</p

Incidence of sequences homologous to ΦB124-14 and ΦB40-8 human gut metagenomes.

<p>Percentage of individual metagenomes in which sequences homologous to φB124-14 or φB40-8 were identified (≥80% identity over ≥100 nucleotides, 1e⁻⁵ or lower). The microbial metagenomes examined were derived from individuals of European (MetaHit) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Qin1" target="_blank">[28]</a>, Japanese <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Kurokawa1" target="_blank">[8]</a> and American <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Gill1" target="_blank">[60]</a> origin, alongside the combined viromes from 12 individuals of American descent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Reyes1" target="_blank">[6]</a>. <b>MH MetaHit</b>– All individuals represented in the MetaHit dataset; <b>Jap</b> – All individuals of Japanese origin; <b>AM</b> – All individuals of American descent; <b>Virome</b> – All viromes from individuals of American origin. <b>B.</b> Scatter plots illustrating the relationship between size of individual metagenomes searched and detection of sequences homologous to φB124-14. r² =  Pearson correlation co-efficient. **<i>P</i><0.0001.</p

Comparative genomic analysis of ΦB124-14 and ΦB40-8 (ATCC 51477-B1).

<p><b>A.</b> Nucleotide sequences of φB124-14 and φB40-8 were compared using the Artemis Comparison Tool (ACT). Shaded areas between linear phage genome maps represent areas of high nucleotide identity (90% or greater). Colour scale represents level of nucleotide identity at each region of homology. The ORF map for φB40-8 corresponds to the annotations available in the GenBank submission (FJ008913.1). For the purposes of this analysis, the φB124-14 genome was linearised between ORFs 29 and 30 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone-0035053-g002" target="_blank">Figure 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053.s004" target="_blank">Table S2</a>), in order to compare the circular φB124-14 genome with that of φB40-8. Colours of ORFs correspond to functional assignments as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone-0035053-g002" target="_blank">Figure 2</a>. <b>B.</b> Comparison of amino acid sequences from φB124-14 ORFs with those annotated in the φB40-8 genome. Shading between arrows indicates those sharing high amino acid sequence identity. Colour scale indicates level of amino acid identity between each homologous ORF.</p

Physical structure and host range of ΦB124-14.

<p><b>A.</b> Transmission electron micrograph of ΦB124-14 showing phage capsid composed of an icosahedral head and a non-contractile tail. Magnification 50,000×. Scale Bar, 20 nm. <b>B.</b> Phylogenetic characterisation of <i>B. fragilis</i> φB124-14 host strains. Consensus maximum likelihood trees were constructed from 16S rRNA gene sequences, with 1000 bootstrap resamplings using MEGA v5. Bootstrap values of 40 or greater are shown adjacent to respective nodes. Accession numbers for bacterial 16S sequences are given in brackets following species names on the tree. The ability of φB124-14 to replicate in a particular host species was tested in standard agar overlay assays, in which replication of φB124-14 in a particular host was indicated by production of plaques in bacterial lawns. Species tested in host range assays are denoted by open or filled circles. Filled red circles indicate strains which support φB124-14 replication, and open grey circles indicate strains in which φB124 did not replicate.</p

Physical structure of φB124-14 genome. Left and middle panels

<p>show <i>in silico</i> digest and electrophoresis to visualise restriction fragment profiles of φB124-14 expected for each permutation of the genome (linear or circular) generated by pDRAW32. <b>Right panel</b> shows results obtained from digestion of 1.5 µg of φB124-14 DNA (3 h at 37°C) with restriction enzymes used in <i>in silico</i> analysis. Restriction enzymes tested are indicated above each lane. MW  = 1 kb Molecular Weight marker (Promega). UC  =  uncut φB124-14 DNA.</p