Complete Genome Sequence of the Model Halovirus PhiH1 (ΦH1)

Abstract

The halophilic myohalovirus Halobacterium virus phiH (ΦH) was first described in 1982 and was isolated from a spontaneously lysed culture of Halobacterium salinarum strain R1. Until 1994, it was used extensively as a model to study the molecular genetics of haloarchaea, but only parts of the viral genome were sequenced during this period. Using Sanger sequencing combined with high-coverage Illumina sequencing, the full genome sequence of the major variant (phiH1) of this halovirus has been determined. The dsDNA genome is 58,072 bp in length and carries 97 protein-coding genes. We have integrated this information with the previously described transcription mapping data. PhiH could be classified into Myoviridae Type1, Cluster 4 based on capsid assembly and structural proteins (VIRFAM). The closest relative was Natrialba virus phiCh1 (φCh1), which shared 63% nucleotide identity and displayed a high level of gene synteny. This close relationship was supported by phylogenetic tree reconstructions. The complete sequence of this historically important virus will allow its inclusion in studies of comparative genomics and virus diversity.

Keywords: Archaea; Halobacterium salinarum; genome inversion; haloarchaea; halobacteria; halophage; halovirus; virus.

Figure 1

Diagram of the phiH1 genome with lines below showing regions previously sequenced (red) along with their database accessions. The blue lines (NEW) indicate regions sequenced in the present study by Sanger sequencing. Tick marks (dark green) below the blue lines show the positions of oligonucleotide primers used for PCR and primer-walking. Dots at the right and left contig ends indicate sequence continuity between them. Scale bar at top shows position in bp.

Figure 2

PhiH1 GC-profile, genetic map, and corresponding transcription program (adapted from [16,42]). (a) GC-profile of the phiH1 genome. (b) Genetic map of the phiH1 genome, showing coding sequences as red, blue or grey arrows. Dotted lines above indicate gene clusters involved in particular functions. Some CDS are labelled above the map, e.g., TerL, terminase large subunit; Portal, portal protein; Tape, tape-measure protein; RepH, replicase (label within CDS arrow); Mt, DNA methylases; TerS, terminase small subunit. Some genes are shown below the map, such as hp32, encoding the major capsid protein HP32. Panels c, d and e summarise transcription data from previously published studies, and above them is a colour key that indicates the time of appearance of early (0–1 h, blue), middle (1–2 h, green) and late (>2 h, pink) transcripts. (c) Precise mapping of viral transcripts, including start and termination sites [16,39]. (d) Summary transcription program of lytic infection based on hybridisation of labelled infected-cell transcripts to restriction fragments of virus DNA [42]. Thin coloured lines indicate whether continuing transcription persists over time. (e) The transcription map data of [42] are shown, projected onto the in silico restriction map of phiH1, as determined from the complete genome sequence (this study). Enzymes are indicated at the left. Numbers on the restriction map refer to those of the original publication of [42] (see also Figure S1). Coloured shading follows that of panels c and d. Dotted pattern shown beyond the right-hand pac site indicates terminal redundancy of virus DNA. Scale bars (in kb) are shown below panels a and e.

Figure 3

Circos plot of amino acid similarity (tBLASTx) between phiH1 and the haloviruses phiCh1, BJ1, CGphi46 and HSTV-1. The threshold for connecting lines was E-value ≤ 10⁻⁴⁰, with line colours reflecting the ratio of actual tBLASTx bitscore to the maximal score (using ‘score/max’ ratio colouring with blue ≤ 0.25, green ≤ 0.50, orange ≤ 0.75, red > 0.75). The outer histogram counts how many times each colour has hit the specific part of the sequence and uses an equivalent colouring scheme. The distance between successive tick marks shown along each virus genome represents 0.1 of the full genome length. Protein names shown along the phiH1 genome indicate the positions of the corresponding genes.

Figure 4

Phylogenetic tree reconstruction (NJ method) of major capsid proteins (MCP) of phiH1, other haloviruses and related proteins of haloarchaea. Species names of haloarchaeal species are shown, with accession numbers given at the right side. Bootstrap confidence values (100 repetitions) are shown at branch points. The pink shading highlights taxa belonging to the class Halobacteria. Scale bar (expected changes per site) is shown at top. The outgroup (not shown) consisted of distantly related MCP sequences of Bacillus spp. (WP_001060157.1, WP_098773561.1, WP_001064748.1 and WP_000178926.1).