Siphophage 0105phi7-2 of Bacillus thuringiensis: Novel Propagation, DNA, and Genome-Implied Assembly

Abstract

Diversity of phage propagation, physical properties, and assembly promotes the use of phages in ecological studies and biomedicine. However, observed phage diversity is incomplete. Bacillus thuringiensis siphophage, 0105phi-7-2, first described here, significantly expands known phage diversity, as seen via in-plaque propagation, electron microscopy, whole genome sequencing/annotation, protein mass spectrometry, and native gel electrophoresis (AGE). Average plaque diameter vs. plaque-supporting agarose gel concentration plots reveal unusually steep conversion to large plaques as agarose concentration decreases below 0.2%. These large plaques sometimes have small satellites and are made larger by orthovanadate, an ATPase inhibitor. Phage head–host-cell binding is observed by electron microscopy. We hypothesize that this binding causes plaque size-increase via biofilm evolved, ATP stimulated ride-hitching on motile host cells by temporarily inactive phages. Phage 0105phi7-2 does not propagate in liquid culture. Genomic sequencing/annotation reveals history as temperate phage and distant similarity, in a virion-assembly gene cluster, to prototypical siphophage SPP1 of Bacillus subtilis. Phage 0105phi7-2 is distinct in (1) absence of head-assembly scaffolding via either separate protein or classically sized, head protein-embedded peptide, (2) producing partially condensed, head-expelled DNA, and (3) having a surface relatively poor in AGE-detected net negative charges, which is possibly correlated with observed low murine blood persistence.

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“…That being said, we already know that persistence is dependent on more than σ N . Specifically, phage G, a myophage, is a low-persistence phage [43]. The value of σ N , however, is 1.20 (plot indicated by G in Figure 1; the T3 plot is indicated by T3 in Figure 1).…”

“…All three phages were analyzed in the same 18-track multigel. The preparation of phages has been previously described: T3 [35], T4 [33], and G [43]. Clear-plaque phage G has a genomic sequence that differs from both the reference GenBank sequence (JN638751) and its parent at three single nucleotide locations.…”

“…We accidentally discovered a possible way to do this. We (primarily MA) subjected low-persistence phage G [43] to three serial passages through murine blood, with each passage interspersed with propagation of phage G, although propagation was in non-mutagenic conditions. High persistence was indeed achieved for partially purified (Legend to Figure 2) phages.…”

“…The initial phage G was a clear-plaque mutant, which had been selected to improve the accuracy of phage titers (Legend to Figure 1). To increase phage G persistence, the clear-plaque mutant of phage G was passed through mouse blood three times, as previously described [43]. After each passage, a sample of blood was taken at 45 min after IP inoculation.…”

“…Phage 0105phi7-2 was obtained as described in [43]. Phages and phage mixtures indicated in the text were made and injected intraperitoneally into a 11-12-week-old female C57/BL6 mouse, as previously described [35,43]. The total PFU inoculated were: 0105phi7-2, 1.0 × 10 10 ; G, 1.0 × 10 10 in 180 µL final volume.…”