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

Abstract

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.

Keywords: bacteriophages; fragment recruitment; phage enrichment; phage particle extraction protocol; virome; virome structure.

Figure 1

Morphological diversity of the phage-like particles observed in a sample originating from a laboratory-scale biogas reactor: (A) Myoviridae, (B) icosahedral phage-like particles, (C) Podoviridae, (D) Rudiviridae, and (E) Myoviridae.

Figure 2

Taxonomic profiling of the biogas metavirome applying the “What the Phage” (WtP) pipeline [44]. The colors represent different families of phages as well as unclassified viruses. (A) Number of identified and classified sequences of metagenome-assembled viral genomes (MAVGs) that could be assigned to a certain family. (B) Number of identified and classified sequences and taxonomic classification of MAVGs assigned in Figure 2A as ‘others’.

Figure 3

Protein-based phage similarity network constructed applying the vConTact 2.0 pipeline [49]. The vConTact 2.0 network generated by using the ProkaryoticViralRefSeq v94 virus database in conjunction with the phage dataset originating from a laboratory-scale biogas reactor (375 sequences) analyzed in this study using the perfused forced directed layout. Related bacteriophages are represented as triangles in pink with the edges between them representing shared protein clusters. Node colors were used to indicate phage hosts, whose taxonomic affiliations were positioned right next to the respective cluster. Ellipses around the groups indicate the biogas phages of this study. Network visualization was generated using Cytoscape 3.8.2.

Figure 4

(A) Classification of the identified ORFs in selected MAVGs. ORFs were grouped into the categories: hypothetical proteins, AMGs, DNA replication, packaging, host modification, putative functions, DNA modification, host lysis, structural proteins, and lysis-related proteins. (B) Genomic map of MAVG 6 created with the Snapgene viewer tool. ORFs are represented by arrows and those encoding hypothetical proteins are shown in grey. The ADP–ribosyltransferase (ADPRT) gene, highlighted in pink, represents a genetic determinant involved in the modification of host proteins. (C) Representation of the viral class, lifestyle prediction, host prediction, and host prediction confidence generated by PHACTS for 40 MAVG genomes.

Figure 5

The 50-most-abundant MAVGs found in different microbial communities derived from publicly available metagenomes. Selected samples (the top) showing metagenome fragment mapping results of greater than 0.5% were visualized. Abbreviation: BGP, biogas plant.