The Terminally Redundant, Nonpermuted Genome ofListeriaBacteriophage A511: a Model for the SPO1-Like Myoviruses of Gram-Positive Bacteria

Abstract

Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G؉C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (ϳ6 kb) and K (ϳ20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G؉C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.Listeria monocytogenes is a gram-positive, opportunistic pathogen that can cause a wide spectrum of diseases, such as meningitis, septicemia, abortion, and gastroenteritis, associated with mortality rates as high as 25 to 30% (17, 62). Listeria bacteria are ubiquitously found in nature and can enter the food chain at many points (26, 66). They are able to proliferate over a wide range of external conditions, including low temperatures and high salt concentrations, thus posing severe problems to the food industry (2,22,15,48).Almost all of the Listeria bacteriophages described to date are temperate (38); only very little is known about virulent phages infecting this host. Listeria phages are valuable sources of biological information and useful tools for the study, differentiation, manipulation, and control of these bacteria (6,33,41). Phage A511 and some of its components have been particularly useful in phage typing schemes (35, 61), as reporter phage (39), with respect to their endolysins (16), and also for the control of Listeria contamination of foods (7; S. Guenther, D. Huwyler, S. Richard, and M. J. Loessner, submitted for publication). In contrast to temperate Listeria phages A118 and PSA (36, 71), A511 is a virulent (i.e., s...

Romulus and Remus, Two Phage Isolates Representing a Distinct Clade within the Twortlikevirus Genus, Display Suitable Properties for Phage Therapy Applications

“…Three major modules encoding virion proteins, DNA metabolism functions, and proteins necessary for cell lysis can be distinguished. Predicted structural proteins of Romulus, Remus, and SA11 show approximately 70 to 80% sequence identity to proteins of the other twortlikeviruses infecting S. aureus and approximately 60% identity to proteins of the Listeria phages A511 and P100 (5,50,51). In general, other proteins encoded by the Romulus and Remus genomes displayed an average 50% homology with proteins of K, G1, ISP, and Twort.…”

Bacteriophage P70: Unique Morphology and Unrelatedness to Other Listeria Bacteriophages

“…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).…”

“…Transmission electron micrographs of P70 were obtained as described previously (15) and revealed a particle morphology previously not observed for Listeria phages, featuring a long, flexible tail connected to an elongated head (B3 morphotype [1]) with a head measuring 128 nm in length and 57 nm in diameter and a tail length of 141 nm (Fig. 1A).…”

“…The closest homologues are found in the PBSX-defective prophage of Bacillus subtilis (25), which supposedly employs a P22-like headful packaging mechanism. We investigated the physical genome structure of P70 by restriction profiles, Bal31 nuclease digestion (15,21), pulsed-field gel electrophoresis (PFGE), and Sanger runoff sequencing (Fig. 2B).…”

“…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.…”