Expanding our understanding of marine viral diversity through metagenomic analyses of biofilms

Abstract

Recent metagenomics surveys have provided insights into the marine virosphere. However, these surveys have focused solely on viruses in seawater, neglecting those associated with biofilms. By analyzing 1.75 terabases of biofilm metagenomic data, 3974 viral sequences were identified from eight locations around the world. Over 90% of these viral sequences were not found in previously reported datasets. Comparisons between biofilm and seawater metagenomes identified viruses that are endemic to the biofilm niche. Analysis of viral sequences integrated within biofilm-derived microbial genomes revealed potential functional genes for trimeric autotransporter adhesin and polysaccharide metabolism, which may contribute to biofilm formation by the bacterial hosts. However, more than 70% of the genes could not be annotated. These findings show marine biofilms to be a reservoir of novel viruses and have enhanced our understanding of natural virus-bacteria ecosystems.

Keywords: Biofilm; Metagenomics; Ocean; Virus.

Fig. 1

Sampling locations of the 101 biofilms. The eight locations include (1) South Atlantic, (2) Red Sea, (3) Hong Kong Water, (4) Yung Shue O Bay, (5) East China Sea, (6) South China Sea 1, (7) South China Sea 2, and (8) South China Sea 3. Tara surface seawater samples used for comparison are also shown

Fig. 2

Similarity between viral sequences identified from marine biofilms and those documented in the IMG/VR database. The biofilm-derived viral sequences were BLASTn searched against the IMG/VR database. The BLASTn hits with over 90% similarity for more than 1000 bp alignments were considered to be known viruses (dots in red), while the other hits (dots in blue) and those with no significant similarity were considered to be novel viruses

Fig. 3

Phylogenetic tree of the terminase large subunit gene VOG9355 identified from the biofilm phage sequences. Closely related terminase large subunit gene sequences documented in the VOG database were revealed by hmmscan and then used as a reference. The protein sequences that could be aligned by ClustalW were used to construct a maximum likelihood tree with 1000 replicates. Bootstrap values (> 50) are shown on the branches. All the gene sequences from biofilms are shown in blue, and branches that represent potentially novel viral lineages are shown in red

Fig. 4

The endemism of biofilm-derived viruses. Metagenomic reads of 101 biofilm and 91 seawater samples were mapped to the biofilm-derived viral contigs to compare their abundance in biofilms and seawater. Metagenomic reads (101 bp and ten million reads per sample) were mapped to gene sequences. Viral sequences with coverage > 1 in biofilms and coverage = 0 in seawater samples are presented

Fig. 5

Identification of viral sequences from microbial genome bins and viral gene functional annotation. A Viral sequences were identified from 100 microbial genomes distributed across 11 bacterial phyla (Proteobacteria were divided into Alpha- and Gamma-proteobacteria). B The number of viral genes annotated by BLASTp searching against the COG database for functional classification

Fig. 6

Potential auxiliary metabolic genes of phage genes extracted from the bacterial bins. The gene functions were predicted by BLASTp searching against the KEGG database. The top 18 (gene number > 10) KEGGs are shown