A Novel Genus of Actinobacterial Tectiviridae

Abstract

Streptomyces phages WheeHeim and Forthebois are two novel members of the Tectiviridae family. These phages were isolated on cultures of the plant pathogen Streptomyces scabiei, known for its worldwide economic impact on potato crops. Transmission electron microscopy showed viral particles with double-layered icosahedral capsids, and frequent instances of protruding nanotubes harboring a collar-like structure. Mass-spectrometry confirmed the presence of lipids in the virion, and serial purification of colonies from turbid plaques and immunity testing revealed that both phages are temperate. Streptomycesphages WheeHeim and Forthebois have linear dsDNA chromosomes (18,266 bp and 18,251 bp long, respectively) with the characteristic two-segment architecture of the Tectiviridae. Both genomes encode homologs of the canonical tectiviral proteins (major capsid protein, packaging ATPase and DNA polymerase), as well as PRD1-type virion-associated transglycosylase and membrane DNA delivery proteins. Comparative genomics and phylogenetic analyses firmly establish that these two phages, together with Rhodococcusphage Toil, form a new genus within the Tectiviridae, which we have tentatively named Deltatectivirus. The identification of a cohesive clade of Actinobacteria-infecting tectiviruses with conserved genome structure but with scant sequence similarity to members of other tectiviral genera confirms that the Tectiviridae are an ancient lineage infecting a broad range of bacterial hosts.

Keywords: Streptomyces; actinobacteria; capsid protein; lipid membrane; pathogen; plant; potato scab; tectivirus.

Figure 1

Representative TEM images of (a) CsCl-purified WheeHeim; (b) WheeHeim with nanotube from crude lysate; (c) Forthebois from crude lysate; (d) Forthebois with nanotube from crude lysate. Black arrowheads indicate lipid membrane. White arrowheads indicate collar-like structure. Scale bar = 100 nm for all panels.

Figure 2

MALDI-TOF spectra for (A) Mycobacterium phage Rosebush and (B) Streptomyces phage WheeHeim. Asterisks denote DHB (2,5-Dihydroxybenzoic acid) matrix peaks. m/z denotes mass (m) to charge (z) ratio.

Figure 3

Lysogen isolation and testing. (a) Liquid phage release from WheeHeim lysogens. Serially diluted stock Streptomyces phage WheeHeim (row 1), streak purified S. scabiei (WheeHeim) lysogen supernatant (row 2), and stock Streptomyces phage Forthebois (row 3) spotted on NA⁺ overlayed with S. scabiei RL-34 after 48 h at 30 °C. (b) Superinfection immunity test of S. scabiei (Forthebois) lysogen. Serially diluted crude lysate stocks of Streptomyces phage Scap1 (row 1) Forthebois (row 2), WheeHeim (row 3), and S. scabiei (Forthebois) lysogen supernatant (row 4) spotted on NA⁺ overlayed with S. scabiei (Forthebois) lysogen after 48 h at 30 °C.

Figure 4

Genome maps of Rhodococcus phage Toil, Streptomyces phages WheeHeim and Forthebois, Gluconobacter phage GC1, Pseudomonas virus PRD1 and Bacillus virus Bam35. Colored arrows indicate genes. Vertical bars indicate percent amino acid identity from pairwise tBLASTX comparisons on a grey scale. For genes with assigned function in Table 1, gene numbers for Streptomyces phages WheeHeim and Forthebois are superimposed on the corresponding colored arrows.

Figure 5

Phylogenetic trees resulting from Bayesian inference on multiple sequence alignments of the packaging ATPase, the DNA polymerase and the major capsid protein sequences, and from minimal evolution inference on BLAST-derived inter-genomic distances. Only support values above 0.5 posterior probability or 50% bootstrap support are shown. Trees were rooted arbitrarily for display purposes. Colors denote the previously described Tectiviridae genera (Alphatectivirus, blue; Betatectivirus, red; Gammatectivirus, brown) as well as the proposed novel genus Deltatectivirus (green).