Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.
Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages. Listeria monocytogenes is a Gram-positive food-borne pathogen that causes the rare but severe disease listeriosis, which is characterized by a high mortality rate (6, 28). Although many Listeria-specific bacteriophages have been described, only a limited number have been fully sequenced (2,5,15,21,29). Most Listeria phages are temperate siphoviruses, with B054, A511, and P100 being the only described myoviruses (2,5,15). Listeria can be efficiently detected and controlled by the use of highly virulent and specific bacteriophages (2, 22, 23). Bacteriophage-based biocontrol of food-borne pathogens has received increasing interest in recent years, due to its practicability, efficacy, safety, and low cost (8, 10-12). In our search for additional virulent phages useful for biocontrol, we discovered phage P70, which turned out to be a novel type of Listeriaspecific bacterial virus.Phage P70 was isolated from a grass silage sample from a dairy farm in Homburg, Switzerland. Samples taken were amplified using L. monocytogenes WSLC1001 and WSLC1042 as phage hosts, and potential phage in the resulting lysates was assessed by the soft agar overlay method (16,27). Phage P70 was propagated on semiconfluent soft agar plates of Listeria ivanovii WSLC3009 (16). Purified phage stocks were obtained by polyethylene glycol (PEG) precipitation and density gradient centrifugation (15,24). Hostrange analysis was performed as described elsewhere (18,19), and P70 was found to feature an unusually broad host range within the genus Listeria, infecting serovars 1/2a, 1/2b, 1/2c, 4a, 4c, 4d, 4e, 5, 6a, and 6b with similar efficiencies. A total of 18 of 29 Listeria strains tested were lysed by P70, including food isolates, reference strains, and the novel species L. marthii (9) but not L. rocourtiae
The Bacillus ACT group includes three important pathogenic species of Bacillus: anthracis, cereus and thuringiensis. We characterized three virulent bacteriophages, Bastille, W.Ph. and CP-51, that infect various strains of these three species. We have determined the complete genome sequences of CP-51, W.Ph. and Bastille, and their physical genome structures. The CP-51 genome sequence could only be obtained using a combination of conventional and second and third next generation sequencing technologies - illustrating the problems associated with sequencing highly modified DNA. We present evidence that the generalized transduction facilitated by CP-51 is independent of a specific genome structure, but likely due to sporadic packaging errors of the terminase. There is clear correlation of the genetic and morphological features of these phages validating their placement in the Spounavirinae subfamily (SPO1-related phages) of the Myoviridae. This study also provides tools for the development of phage-based diagnostics/therapeutics for this group of pathogens.
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