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

Occurrence of bacteriophages infecting Bacteroides host strains (ARABA 84 and GB-124) in fecal samples of human and animal origin

Bacteriophage-based microbial source-tracking studies are an economical and simple way of identifying fecal sources in polluted water systems. Recently isolated Bacteroides spp. strains ARABA 84, and GB-124 have been shown to detect bacteriophages exclusively in aquatic systems impacted by human fecal material. To date, limited examination of the occurrence or concentration of phages capable of infecting Bacteroides fragilis strain GB-124 or B. thetaiotaomicron strain ARABA 84 in human and animal feces has been carried out. This study reports the prevalence rates and concentrations of phages infecting ARABA 84 and GB-124 host strains in human and a range of animal feces. Discrete human fecal samples (n = 55) and pooled animal samples (n = 46, representing the feces of over 230 animals) were examined for phages infecting the host strains ARABA 84, GB-124, and E. coli strain WG5. Both human Bacteroides host strains were highly specific (95% and 100% for ARABA 84 and GB-124, respectively), challenging results from previous studies. This study supports the use of Bacteroides strains GB-124 and ARABA 84 in fecal source tracking studies for the detection of human fecal contamination.</jats:p

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

Phylogeny of ΦB124-14 large subunit terminase.

<p>Amino acid sequences homologous to φB124-14 terminase (ORF43), based on bit-score, were retrieved from GenBank and metagenomic datasets, including human gut microbiomes and viromes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Reyes1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Kurokawa1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Qin1" target="_blank">[28]</a> and marine microbial metagenomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Yooseph1" target="_blank">[92]</a>, and aligned using ClustalW. The unrooted concensus neighbour joining tree (1000 bootstrap resamplings) was produced using MEGA v5. Bootstrap values ≥40 are shown adjacent to respective tree nodes. Scale indicates amino acid substitutions. Colours indicate phylum level grouping or origin of metagenomic sequences. Black triangles indicate φB124-14 or φB40-8 terminase sequences; white triangles represent other phage sequences; white circles represent sequences originating from human gut metagenomes.</p

Comparison of tetranucleotide repeat frequency patterns in bacteriophage genomes and ecological profiling of φB124-14 and φB40-8.

<p>The tetranucleotide repeat frequency (TRF) correlation scores for φB124-14, φB40-8 and <i>Burkholderia</i> φKS10, were compared using scatter plots and correlation of data examined using the Pearson coefficient. A complete list of genomes and sequences utilised in this analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053.s005" target="_blank">Table S3</a>. <b>A.</b> Comparison of TRF scores for φB124-14 (x-axis) vs φB40-8 (y-axis). <b>B.</b> Comparison of TRF scores for φB124-14 (x-axis) vs <i>Burkholderia</i> φKS10 (y-axis). <b>A, B. <u>Upper charts</u></b> plot scores for all phage genomes, viral metagenome fragments, and <i>Bacteroides</i> genomes. <b>Phage</b>  =  TRF scores from comparisons to 611 phage and prophage genomes. <b>Virome</b>  =  TRF scores from comparisons to 188 large fragments (>10 Kb) from human gut viral metagenomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Reyes1" target="_blank">[6]</a>. <b>Chromosome</b>  =  TRF scores from comparison to 48 <i>Bacteroides spp</i>. genome sequences. Each sequence type is represented by a different colour and symbol as indicated in the figure legends on each chart. The intensity of shading of data points reflects the number of data points represented in a given area with a greater intensity indicating more overlapping data points. Values in parentheses provide Pearson correlation scores for each sequence type. <b><u>Lower charts</u></b> plot TRF scores for sequences assigned to one of three categories based on their relation to the human gut microbiome: <b>Gut </b><b> = </b> comprises bacteriophage infecting bacterial genera commonly forming part of the normal human gut microbiota. <b>Gut Associated  = </b> comprises bacteriophage genomes infecting bacterial genera whose member species are associated with the gut but not generally considered to be members of the normal gut microbiota (such as primary invasive gut pathogens), or where member species are more commonly associated with environmental habitats. <b>Non-Gut  = </b> contains bacteriophage infecting bacterial genera with member species not considered to be part of the human gut microbiota or typically associated with this community, and primarily encompasses bacteriophage infecting genera of environmental origin. <b>Virome  = </b> All large fragments (n = 188, >10 Kb) assembled using CAMERA workflows (per individual) from human gut viral metagenomic libraries <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Reyes1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Sun1" target="_blank">[91]</a>. Each sequence category is represented by a different colour and symbol as indicated in the figure legends on each chart. For the purposes of this analysis phage infecting a particular host bacterial genus were only utilised if four or more representative phage genomes were available (540 complete phage genomes, representing 31 bacterial genera). The intensity of shading of data points reflects the number of data points represented in a given area with a greater intensity indicating more overlapping data points. Values in parentheses provide Pearson correlation scores for each sequence type.</p