Exploring Viral Diversity in a Gypsum Karst Lake Ecosystem Using Targeted Single-Cell Genomics

Abstract

Little is known about the diversity and distribution of viruses infecting green sulfur bacteria (GSB) thriving in euxinic (sulfuric and anoxic) habitats, including gypsum karst lake ecosystems. In this study, we used targeted cell sorting combined with single-cell sequencing to gain insights into the gene content and genomic potential of viruses infecting sulfur-oxidizing bacteria Chlorobium clathratiforme, obtained from water samples collected during summer stratification in gypsum karst Lake Kirkilai (Lithuania). In total, 82 viral contigs were bioinformatically identified in 62 single amplified genomes (SAGs) of C. clathratiforme. The majority of viral gene and protein sequences showed little to no similarity with phage sequences in public databases, uncovering the vast diversity of previously undescribed GSB viruses. We observed a high level of lysogenization in the C. clathratiforme population, as 87% SAGs contained intact prophages. Among the thirty identified auxiliary metabolic genes (AMGs), two, thiosulfate sulfurtransferase (TST) and thioredoxin-dependent phosphoadenosine phosphosulfate (PAPS) reductase (cysH), were found to be involved in the oxidation of inorganic sulfur compounds, suggesting that viruses can influence the metabolism and cycling of this essential element. Finally, the analysis of CRISPR spacers retrieved from the consensus C. clathratiforme genome imply persistent and active virus-host interactions for several putative phages prevalent among C. clathratiforme SAGs. Overall, this study provides a glimpse into the diversity of phages associated with naturally occurring and highly abundant sulfur-oxidizing bacteria.

Keywords: Kirkilai; Lithuania; Microviridae; Natura 2000; Single Cell Genomics; chlorobium phages; green sulfur bacteria; gypsum karst ecosystem; rare ecosystems; virus diversity.

Figure 1

Bacterial community composition of the most abundant (>1% from total reads) taxa throughout the water column of the gypsum karst Lake Kirkilai (Lithuania).

Figure 2

Taxonomic (Kaiju) classification (a) and distribution (b) of viral contigs recovered from 62 C. clathratiforme single amplified genomes (SAGs). Phage contigs were ordered by detection frequency among 62 C. clathratiforme SAGs.

Figure 3

Phylogenetic tree reconstruction of Microviridae family bacteriophages (RefSeq DB; accessed on March 2021) and representation of C. clathratiforme SAGs-associated Microviridae genome (NODE_396). Members of Bullavirinae are marked in blue, members of Gokushovirinae are marked in green and unclassified microviruses are marked in red. The C. clathratiforme SAGs-associated Microviridae phage genome (NODE_396) is shown in black. The tree was calculated using a neighbor joining algorithm of the conserved sites in the genomes, with the bootstrap values higher than 75% given at the nodes.