Bacteriophage P70: Unique Morphology and Unrelatedness to Other Listeria Bacteriophages

Abstract

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

Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments

“…Comparative genomics revealed five conserved orthoclusters of Listeria phages and one additional orthocluster of Enterococcus phages, which is closely related to Listeria phage orthocluster V. With the high level of similarity between previously reported complete phage genomes (13)(14)(15) and genome sequences reported here (including those with multiple contigs [LP-032 and LP-083-1]), it is extremely unlikely that assembly gaps resulted in a failure to identify genes affecting the orthoclustering of phages sequenced in this study. Two orthoclusters (orthoclusters III and IV) contain phages previously characterized as temperate phages, e.g., A118 and PSA (41,42); as expected, genomes of these phages encode an integrase, which enables the phage chromosome to integrate into the host chromosome through site-specific recombination.…”

“…Orthocluster V, which, like the others, is supported by a bootstrap value of 100, contains Listeria-infecting bacteriophages LP-026, LP-032, LP-037, LP-110, and LP-114 as well as previously described Listeria phage P70 (15). The phages in this orthocluster have genome lengths ranging from 65 to 67 kb (with an average GC content of 33.3 to 33.6%).…”

“…For example, phages isolated in North America (sequenced in this study) clustered with phages isolated in Europe (A511, P100, and P70) (15,29,30) and in East Asia (SAP6 and BC-611) (38,39). Three orthoclusters (orthoclusters I, V, and VI, all lytic) each contained phages isolated on different continents.…”

“…Molecular and in silico analyses by Dorscht et al (14) showed that Listeria phages sequenced to date belong to several phylogenetic clades and display a conserved genome organization. Recently, the unique Listeria phage P70 was described by Schmuki et al (15). P70 has a medium-sized genome (ϳ67 kb), similar in size to several phages isolated by our group from dairy farms in the state of New York (16).…”

Phylogenomic Network and Comparative Genomics Reveal a Diverged Member of the ϕKZ-Related Group, Marine Vibrio Phage ϕJM-2012

“…In HPR IV, JM-2012 was found to harbor a site-specific recombinase (HJR)-related gene. Although future studies are warranted to functionally characterize this gene, its presence in or absence from the six phage genomes indicated that HJR, which is known to be associated with a temperate life cycle (79), could play an important role in the rearrangement of the JM-2012 genome (53). Another notable feature that we identified was the localization of prophage-related genes (with the ACLAME database) in HPRs IV and IX (the latter of which is likely to be a hot spot, as it contains two moron-like elements).…”