Novel viruses of Haloquadratum walsbyi expand the known archaeal virosphere of hypersaline environments

Abstract

Solar salterns represent unique systems with low diversity microbial communities that serve as an excellent model for studying the evolution and ecology of archaeal viruses and the interactions with their hosts. This is particularly relevant for the extremely abundant "square" archaeon Haloquadratum walsbyi, for which isolated viruses have remained elusive despite the fact that this microbe governs the salt-saturated ponds of most solar salterns worldwide. In this work, we have used cutting-edge imaging techniques, based on virus fluorescence in situ hybridization (virusFISH), and a combination of -omic techniques, at both population and single-cell levels, to provide an in-depth characterization of the Hqr. walsbyi virosphere. Our analyses have led to the identification of a new subfamily of tailed low-GC dsDNA viruses, which we propose to name "Haloquadravirinae", with host assignment confirmed by virusFISH in natural samples. Haloquadraviruses can represent more than 50% of the viral community in solar saltern viromes and infect nearly 40% of square cells in natural environments. The genetic imprint of these viruses, which are globally distributed in hypersaline environments, has provided insights into the structure of their virions and their potential life strategy. Along with the identification of other virus-like elements associated with Hqr. walsbyi through single-cell genomics, this work expands our current understanding of the archaeal virosphere.

Keywords: Haloquadratum; archaeal virus; halophile; halovirus; hypersaline; virome.

Figure 1

Alignment of the 25 “Haloquadravirinae” complete genomes, retrieved from the CR30 crystallizer in the period 2007–2014, and grouped by genus (“Polavirus”, “Squarevirus” and “Walsbyivirus”). Predicted ORFs are indicated by horizontal arrows. Core genes are coloured according to their amino acid identities (grey, pink, red), whereas genus-specific genes and singletons are coloured in purple and black, respectively. Vertical arrows indicate the genome position of predicted proto-spacers against the Hqr. walsbyi C23^T CRISPR systems (nomenclature as in 21 and 25). Representation is based on Easyfig (blue intensities are related to the identity percentages in the alignment as shown in the bottom right gradient key) [58].

Figure 2

Confocal laser scanning microscopy images of the virusFISH for haloquadraviruses. A. DAPI staining of a CR30apr17 water sample, where square cells are clearly observed. B. Fluorescence of the hybridized AlexaFluor 594-labelled probes for “Haloquadravirinae”. C. Combined DAPI-virusFISH image. White arrows indicate some infected square cells. Scale bar: 5 µm.

Figure 3

A. Relative abundance of “Haloquadravirinae” in a set of 28 viral metagenomes from Bras del Port salterns, spanning the period 2014–2019. Red bars represent the percentage of viromic nucleotides recruited by five “Haloquadravirinae” core genes, whereas pink bars refer to the estimated percentage of nucleotides recruited after normalization of the core genome sequencing depth by the average genome length of haloquadraviruses. The dynamics of VLP, total cells, Archaea and Bacteria per ml of brine sample are also indicated. B. Estimated percentages of “Haloquadravirinae” genera compared with other haloviruses in the studied system.

Figure 4

Experimental design (adapted from [26]) used to link uncultured viruses to individual cells. Step 1 shows the isolation of SAGs from a CR30 sample collected in June 2011, alongside the parallel construction of a virochip. Step 2 depicts the separate hybridization of five Haloquadratum SAGs against the virochip. Fosmid 4G12 exhibited positive hybridization with SAG AB577-A23 (hereafter referred to as HqrSAG). In Step 3, both fosmid 4G12 and HqrSAG were sequenced, and subsequent analyses confirmed that the fosmid 4G12 insert shared a high sequence identity with one of the contigs from HqrSAG (contig017). Predicted ORFs in both 4G12 and contig017 are marked by arrows and color-coded according to their putative functions. Genome visualizations were generated using Easyfig, where the intensity of blue shading corresponds to the percentage identity in the alignment [58].