Genomic characterization of three novel Basilisk-like phages infecting Bacillus anthracis

Abstract

Background: In the present study, we sequenced the complete genomes of three novel bacteriophages v_B-Bak1, v_B-Bak6, v_B-Bak10 previously isolated from historical anthrax burial sites in the South Caucasus country of Georgia. We report here major trends in the molecular evolution of these phages, which we designate as "Basilisk-Like-Phages" (BLPs), and illustrate patterns in their evolution, genomic plasticity and core genome architecture.

Results: Comparative whole genome sequence analysis revealed a close evolutionary relationship between our phages and two unclassified Bacillus cereus group phages, phage Basilisk, a broad host range phage (Grose JH et al., J Vir. 2014;88(20):11846-11860) and phage PBC4, a highly host-restricted phage and close relative of Basilisk (Na H. et al. FEMS Microbiol. letters. 2016;363(12)). Genome comparisons of phages v_B-Bak1, v_B-Bak6, and v_B-Bak10 revealed significant similarity in sequence, gene content, and synteny with both Basilisk and PBC4. Transmission electron microscopy (TEM) confirmed the three phages belong to the Siphoviridae family. In contrast to the broad host range of phage Basilisk and the single-strain specificity of PBC4, our three phages displayed host specificity for Bacillus anthracis. Bacillus species including Bacillus cereus, Bacillus subtilis, Bacillus anthracoides, and Bacillus megaterium were refractory to infection.

Conclusions: Data reported here provide further insight into the shared genomic architecture, host range specificity, and molecular evolution of these rare B. cereus group phages. To date, the three phages represent the only known close relatives of the Basilisk and PBC4 phages and their shared genetic attributes and unique host specificity for B. anthracis provides additional insight into candidate host range determinants.

Keywords: Bacillus anthracis; Phage evolution; Phage genome.

Fig. 1

Transmission electron microscopy of the three phages. TEM images of phages v_B-Bak1 (a), v_B-Bak6 (b), and v_B-Bak10 (c) illustrate their morphology consistent with members of the Siphoviridae family

Fig. 2

Whole genome open reading frame alignments of phage v_B-Bak1, v_B-Bak6, v_B-Bak10 and the Basilisk and PBC4 reference genomes. Color designations are according to putative function: teal, packaging; blue, structural genes; green, host lysis; red, DNA manipulation; pink tRNA genes, purple, additional functions; and yellow, hypothetical genes

Fig. 3

Major genomic diversity regions in the genomes of the Basilisk-Like-Phages. a) left terminal region InDels, b) structural module, c) nucleic acid metabolism genes, d) putative virulence genes, and e) right terminal region. Boxed Diversity Regions (DRs) in dotted orange highlight illustrate major variations among phages v_B-BaK1, v_B-BaK6, v_B-BaK10, and Basilisk

Fig. 4

Whole genome map of phage v_B-BaK10. A circular representation of the linear phage genome was used here for illustration purposes

Fig. 5

Phylogenetic relationships among Basilisk-like phages. Unrooted maximum-likelihood dendrogram derived from amino acid sequences of the phage Terminase (a), Tape measure protein (b), DNA primase (c), and DNA helicase (d) proteins