Cellular and Genomic Properties of Haloferax gibbonsii LR2-5, the Host of Euryarchaeal Virus HFTV1

Abstract

Hypersaline environments are the source of many viruses infecting different species of halophilic euryarchaea. Information on infection mechanisms of archaeal viruses is scarce, due to the lack of genetically accessible virus-host models. Recently, a new archaeal siphovirus, Haloferax tailed virus 1 (HFTV1), was isolated together with its host belonging to the genus Haloferax, but it is not infectious on the widely used model euryarcheon Haloferax volcanii. To gain more insight into the biology of HFTV1 host strain LR2-5, we studied characteristics that might play a role in its virus susceptibility: growth-dependent motility, surface layer, filamentous surface structures, and cell shape. Its genome sequence showed that LR2-5 is a new strain of Haloferax gibbonsii. LR2-5 lacks obvious viral defense systems, such as CRISPR-Cas, and the composition of its cell surface is different from Hfx. volcanii, which might explain the different viral host range. This work provides first deep insights into the relationship between the host of halovirus HFTV1 and other members of the genus Haloferax. Given the close relationship to the genetically accessible Hfx. volcanii, LR2-5 has high potential as a new model for virus-host studies in euryarchaea.

Keywords: N-glycosylation; S-layer; archaeal virus; archaellum; haloarchaea; type IV pili.

FIGURE 1

Cell shape change and motility of Hfx. gibbonsii LR2-5. (A) Left side: typical growth curve of Hfx. gibbonsii LR2-5 in CA medium with 18% (wt/vol) SW at 42°C. Average optical density at 600 nm (OD₆₀₀) was calculated from three independent technical replicates; error bars represent the standard deviation. Right side: phase contrast images show typical cell shapes correlating with three different growth phases: early exponential (I), mid-exponential (II), and stationary (III) growth phases. (B) Representative example of motility rings from Hfx. gibbonsii LR2-5 on semi-solid agar plates with different media after 3 days of growth at 45°C. (C) Quantification of the diameter of motility rings formed on semi-solid agar plates with different media in 18% SW. Calculations were made using more than three independent experiments including three biological replicates each. Middle line indicates the mean, and lower and upper lines the standard deviation. (D) Cells from early exponential growth phase were negatively stained with 2% (wt/vol) uranyl acetate. Top: Hfx. gibbonsii LR2-5 cell with typical rod-shaped morphology and archaella at the cell pole. Scale bar, 1 μm. Bottom: Close up of a bundle of archaella filaments at the cell surface. Scale bar, 0.2 μm.

FIGURE 2

HFTV1 susceptibility of Haloferax strains by spot-on-lawn assay. Spot-on-lawn assay conducted with lawns of (A) Hfx. gibbonsii LR2-5, (B) Hfx. gibbonsii Ma2.38, and (C) Haloferax volcanii H26. Different dilutions of HFTV1 lysate (undiluted 5 × 10¹¹ PFU/mL) were spotted on the host lawns and incubated for 3 days. Control spots were prepared with medium.

FIGURE 3

Comparison of the Hfx. gibbonsii LR2-5 chromosome and plasmids to those of three close relatives. (A) Hfx. gibbonsii LR2-5 chromosome map showing similarity to replicons of closely related strains. The two outermost rings depict the annotated genes (CDS, tRNA, and rRNA) of strain LR2-5, for the forward and reverse DNA strands (color key, upper right). Rings 3–5 depict BLASTn similarities between strain LR2-5 and the three strains listed in the color key (upper left). Colored bars represent regions of similarity (Expect value ≤ 10^{– 20}, cutoff = 90% nucleotide identity), while uncolored (white) regions represent no significant similarity. Ring 6 (gray blocks) are predicted genomic islands (IslandViewer 4). The two innermost rings represent plots of GC content (black) and GC-skew (green/purple) for strain LR2-5, and the color key for these plots is given in the lower left. The GC content ring plots differences from the average GC%, with outward pointing peaks indicating higher than average, and inward pointing peaks indicating lower than average GC%. IGE1-3, integrative genetic elements (see text). rRNA1 and rRNA2 are the two ribosomal RNA operons. Tick marks around the inner-most and outer-most rings show DNA size in kb. The maps and plots were made using the CGView Server (http://stothard.afns.ualberta.ca/cgview_server). (B) Hfx. gibbonsii LR2-5 plasmid maps showing similarity to replicons of closely related strains. The color key for LR2-5 genes (outer two rings) and the inner GC content plots (black) are as described in (A). The comparison strains used to produce the BLASTn similarity rings are indicated by the colored boxes lower left. The actual replicons used are as follows. Plasmid pHGLR1 is compared to Hfx. gibbonsii ARA6 plasmid pHG1 (488,062 bp) (red), Hfx. gibbonsii Ma2.38 (green), and to Hfx. volcanii DS2^T plasmid pHV3 (437,906 bp) (orange). Plasmid pHGLR2 is compared to Hfx. gibbonsii ARA6 plasmid pHG2 (335,881 bp) (red), contig 28 (AOLJ01000028) of Hfx. gibbonsii Ma2.38 (green), and to Hfx. volcanii DS2^T plasmid pHV4 (635,786 bp) (orange). Plasmid pHGLR3 is compared to Hfx. volcanii DS2^T plasmid pHV1 (85,092 bp) (orange).

FIGURE 4

Surface protein and pili of Hfx. gibbonsii LR2-5. (A) Coomassie stained SDS-PAGE gel of Hfx. volcanii, Hfx. gibbonsii Ma2.38, and Hfx. gibbonsii LR2-5 whole cell lysates. The prominent bands at around 245 kDa are considered to run at the height of the S-layer glycoproteins (red boxes). The LR2-5 band was subjected to mass spectrometry. (B) Comparison of the genetic loci of Hfx. gibbonsii LR2-5 PilAs with those of Hfx. gibbonsii ARA6 and Hfx. volcanii DS2^T. Homologs to all six Hfx. volcanii pilins are present in Hfx. gibbonsii LR2-5; however, particularly PilA1, PilA2, and PilA5 are more distant homologs (protein sequence identity 51–68%). PilA3 and PilA4 are organized in an operon in all three strains and the proteins have high protein sequence identity (≥85%).

FIGURE 5

Hfx. gibbonsii LR2-5 N-glycosylation genes with comparison to their closest homologs. Genes are located in two clusters (marked as I and II in A,B) with some genes being located apart, e.g., on a plasmid (C). Close homologs in Hfx. volcanii are indicated with the corresponding agl name. Predicted protein function is indicated by color code; blue: glycosyltransferase, red: flippase, white: rfbX family protein (potential flippase), orange: oligosaccharyltransferase, green: sugar modification enzyme, gray: not directly related to N-glycosylation, yellow: hypothetical protein/pseudogene not apparently related to glycosylation. Protein homology and sequence identity are indicated by colored bars. Bar color indicates the level of amino acid sequence identity (see the color coding on the right). Background coloring indicates additional close homologs, red: Hfx. gibbonsii ARA6, green: Hfx. sp. Atlit-16N and Atlit-10N. Gene sizes are arbitrary.