Diversity and comparative genomics of Microviridae in Sphagnum- dominated peatlands

Abstract

Microviridae, a family of bacteria-infecting ssDNA viruses, is one of the still poorly characterized bacteriophage groups, even though it includes phage PhiX174, one of the main models in virology for genomic and capsid structure studies. Recent studies suggest that they are diverse and well represented in marine and freshwater virioplankton as well as in human microbiomes. However, their diversity, abundance, and ecological role are completely unknown in soil ecosystems. Here we present the comparative analysis of 17 completely assembled Microviridae genomes from 12 viromes of a Sphagnum-dominated peatland. Phylogenetic analysis of the conserved major capsid protein sequences revealed the affiliation to Gokushovirinae and Pichovirinae as well as to two newly defined subfamilies, the Aravirinae and Stokavirinae. Additionally, two new distinct prophages were identified in the genomes of Parabacteroides merdae and Parabacteroides distasonis representing a potential new subfamily of Microviridae. The differentiation of the subfamilies was confirmed by gene order and similarity analysis. Relative abundance analysis using the affiliation of the major capsid protein (VP1) revealed that Gokushovirinae, followed by Aravirinae, are the most abundant Microviridae in 11 out of 12 peat viromes. Sequences matching the Gokushovirinae and Aravirinae VP1 matching sequences, respectively, accounted for up to 4.19 and 0.65% of the total number of sequences in the corresponding virome, respectively. In this study we provide new genome information of Microviridae and pave the way toward quantitative estimations of Microviridae subfamilies.

Keywords: Aravirinae; Gokushovirinae; Microviridae; Stokavirinae; ssDNA phage; viral metagenomics; virus diversity; virus ecology.

FIGURE 1

Maximum likelihood phylogenetic analysis of full-length major capsid protein sequences present in the Microviridae genomes from Sphagnum-dominated peat viromes. A total of 253 unambiguously aligned positions from 76 sequences were used in the analysis. Bootstrap values are indicated at the nodes. The scale bar indicates the number of substitutions per position for a unit branch length. Chlamydiaphages: Chp 1–4, CPAR39, phiCPG1; Bdellovibriophage: phiMH2K.

FIGURE 2

Modeling of major capsid proteins. (A) Three-dimensional models of Microviridae major capsid protein. Different subfamilies are depicted with, from left to right, the reference model from Spiroplasma Phage 4 (Pdb Id: 1KVP), representatives from Stokavirinae and Aravirinae. (B) Hypervariable regions of Aravirinae major capsid protein. The sequence conservation across the seven Aravirinae sequences was mapped on the three-dimensional model obtained for the Bog5275_51 genome. The three hypervariable regions were numbered (left panel). This model was mapped on the capsid structure of Spiroplasma phage 4 to estimate the position of these hypervariable regions relative to the whole virion (right panel).

FIGURE 3

Major capsid protein phylogeny and genome structure of major subfamilies of Microviridae bacteriophages. The affiliation of the peat genomes to Microviridae is shown by major capsid protein phylogeny (Maximum likelihood, 252aa positions, 1000 iterations, JTT+G model). Bootstrap values above 50% are indicated at the nodes. The genome structures of the assembled viral peat genomes (blue) were compared to other Microviridae genomes. Pairwise comparison (TBLASTX) was visualized with ACT (Carver et al., 2005). Gray shading indicates the level of similarities. Homologous genes specific for some subfamilies are color-coded as indicated in the legend. P. multiformis, Prevotella multiformis (WP_007368517); P. nigrescens, Prevotella nigrescens (WP_023924568); Pa. distasonis, Parabacteroides distasonis (ZP_17317332); Pa. merdae, Parabacteroides merdae (WP_022322420). Dragonfly, Dragonfly associated phage (YP_006908226); GOS_10803 (ECU79166); GM1 (444297902); GOM (530695360); CPAR39 (NP_063895); Chp4 (YP_338238); SI2 (KC131023); Metagenome: genome assembled from the metagenome.

FIGURE 4

Whole genome phylogeny and conserved genome structure of peat Gokushovirinae. The affiliation of the peat genomes to Gokushovirinae was shown by whole genome phylogeny (Maximum likelihood, 2052 positions, 1000 iterations, GTR3 model). Maximum likelihood bootstrap values above 50% are indicated at the nodes. The genome structure of the assembled viral peat genomes (virome blue, metagenome red) was compared to Gokushovirinae. Pairwise comparisons (TBLASTX) were visualized by ACT (Carver et al., 2005). Gray shading indicates the level of similarities.

FIGURE 5

Relative proportions of Microviridae subfamilies based on their content of major capsid protein sequences. (A) VP1 match counts in 69 viromes from public databases; (B) in the 12 peat viromes. Matches to major capsid proteins were normalized to the number of total sequences in the viromes. Best matches were determined by BLASTX with an e-value 10^-10 cut off.