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. 2018 Jun 13:9:1223.
doi: 10.3389/fmicb.2018.01223. eCollection 2018.

Viral Communities of Shark Bay Modern Stromatolites

Richard Allen White Iii  1   2   3   4   5 , Hon L Wong  4   6 , Rendy Ruvindy  4   6 , Brett A Neilan  4   6 , Brendan P Burns  4   6
Affiliations

Viral Communities of Shark Bay Modern Stromatolites

Richard Allen White Iii et al. Front Microbiol. .

Abstract

Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients.

Keywords: BREX; CRISPR-Cas; Shark Bay; ssDNA viruses; stromatolites; viral defense; viral metagenomics.

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Figures

FIGURE 1
FIGURE 1
Shark Bay viral and cellular fraction comparison. MG-RAST Functional annotations using KEGG (KO) and SEED where based on BLAT based comparison (e-value ≤ 10-5) against respective database. (A) Viral taxonomic classification characterization by nucleic acid state in MetaVir2. (B) Viral taxonomic classification characterization by viral family in MetaVir2. (C) Viral taxonomic classification characterization by viral genus in MetaVir2 by cellular or viral fraction metagenome. (D) GAAS predictive viral genome size estimations in MetaVir2. (E) SEED subsystem functional annotations in MG-RAST. (F) KEGG (KO) level 1 functional annotations in MG-RAST. MetaVir2 viral (BLAST) based comparison (e-value ≤ 10-5) against NCBI viral refseq database and normalized to genome length using the built-in Genome-relative Abundance and Average Size (GAAS) normalization tool (Angly et al., 2006).
FIGURE 2
FIGURE 2
Viral fraction comparison Shark Bay against Highbourne Cay, Rios Mesquites and Pozas Azules II microbialite sites. (A) Viral taxonomic classification characterization by nucleic acid state in MetaVir2. (B) Viral taxonomic classification characterization by viral family in MetaVir2. (C) Viral taxonomic classification characterization by viral genus in MetaVir2. (D) Principal coordinate analyses (PCA) comparing the viral diversity in disparate stromatolite locations. PCA were constructed from similarity matrices utilizing protein coding sequence recruitment using NCBI viral refseq database (refseq update 2017-1-11) and normalized to genome length using the built-in GAAS. Proportion variance (PC) was explained by each component printed next to the PC1/PC2 axes labels. MetaVir2 viral (BLAST) based comparison (e-value ≤ 10-5) against NCBI viral refseq database and normalized to genome length using the built-in Genome-relative Abundance and Average Size (GAAS) normalization tool (Angly et al., 2006). MG-RAST Functional annotations using KEGG (KO) and SEED where based on BLAT based comparison (e-value ≤ 10-5) against respective database. SB, Shark Bay; HBC, Highbourne Cay; PAII, Pozas Azule II; RM, Rio Mesquites.
FIGURE 3
FIGURE 3
Antiviral resistance genes amongst Shark Bay viral and cellular fraction. (A) CRISPR-Cas associated genes (B) BREX associated genes (C) DISARM associated genes. The relative abundance is the number of hits from GhostKoala and the Pfam database using InterProScan 5.
FIGURE 4
FIGURE 4
Phylogenetic analysis of microbial and viral fractions from Shark Bay stromatolites for the Replication-associated protein (Rep) in CRESS viruses. Maximum likelihood phylogenetic tree of Rep protein sequences found in CRESS viruses in Shark Bay stromatolites. Protein sequence alignments were performed using MUSCLE and gaps in alignment were removed with UGENE. The tree was constructed with IQ-TREE v. 1.6.1 with 1000 bootstrap replicates and was visualized with iTOL. Number indicates bootstrap values, nodes with bootstrap values lower than 70 were not shown and represented by the collapsed branch. The collapsed branches in this figure represent reference sequences from Rosario et al. (2009a) for the replication (rep) protein that have lower than 70 bootstrap values.
FIGURE 5
FIGURE 5
Phylogenetic analysis of viral fractions from Shark Bay stromatolites for the major capsid protein (VP1) in Microviridae viruses. Maximum likelihood phylogenetic tree of VP1 protein sequences obtained from Microviridae viruses in Shark Bay stromatolites. Reference sequences were retrieved from the Uniprot database. Short sequences (<160 amino acid) were removed prior to alignment. Alignments were performed using MUSCLE and gaps in alignment were removed with UGENE. The tree was constructed with IQ-TREE v. 1.6.1 with 1000 bootstrap replicates and was visualized with iTOL. Number indicates bootstrap values, nodes with bootstrap values lower than 70 were not shown and represented by the collapsed branch. The collapsed branches in this figure represent reference sequences from Desnues et al. (2008) for major capsid protein (VP1) that have lower than 70 bootstrap values.
FIGURE 6
FIGURE 6
Phylogenetic analysis of viral fractions from Shark Bay stromatolites for phoH (of the pho regulon) in dsDNA viruses. Maximum-likelihood tree of phoH protein sequences obtained from dsDNA viruses. Reference sequences were retrieved from Uniprot database. Alignments were performed using MUSCLE and gaps in alignment were removed with UGENE. The tree was constructed with IQ-TREE v. 1.6.1 with 1000 bootstrap replicates and was visualized with iTOL. Number indicates bootstrap values, nodes with bootstrap values lower than 70 were not shown and represented by the collapsed branch. The collapsed branches in this figure represent reference sequences from Goldsmith et al. (2011) for the phosphate starvation inducible protein (phoH) that have lower than 70 bootstrap values.
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