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Bacteriophage P70: Unique morphology and unrelatedness to 2

other Listeria bacteriophages 3

4 5 6

Martina M. Schmuki, Doris Erne, Martin J. Loessner, and Jochen Klumpp* 7 8

Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, 9 Switzerland 10

11 12 13 14 15 16 17 18 19 20 21 22 23 24 Running title: Listeria phage P70 genome 25 26 27 28 29 30 Keywords: Listeria, bacteriophage, PacBio, hybrid sequence assembly 31 32 33 34 35 36 37 * Corresponding author mailing address: 38

Schmelzbergstrasse 7, 8092 Zurich, Switzerland 39

Phone: +41 44 632 5378, e-mail: jklumpp@ethz.ch 40

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.02350-12 JVI Accepts, published online ahead of print on 19 September 2012

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Abstract 41

42

Listeria monocytogenes is an important foodborne pathogen, and its bacteriophages find many 43

uses in detection and biocontrol of its host. The novel broad host-range virulent phage P70 has 44

a unique morphology with an elongated capsid. Its genome sequence was determined by a 45

hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome 46

contains 67,170 bp and 119 ORFs. Our analyses suggest that P70 represents an archetype of 47

virus unrelated to other known Listeria bacteriophages. 48

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Main Text 49

Listeria monocytogenes is a Gram-positive foodborne pathogen causing the rare but 50

severe disease Listeriosis, which is characterized by a high mortality rate (6, 29). Although many 51

Listeria-specific bacteriophages have been described, only a limited number have been fully 52

sequenced (2, 5, 15, 22, 30). Most Listeria phages are temperate siphoviruses, with B054, A511 53

and P100 being the only described myoviruses (2, 5, 15). Listeria can be efficiently detected and 54

controlled by the use of highly virulent and specific bacteriophages (2, 23, 24). Bacteriophage-55

based biocontrol of foodborne pathogens has received increasing interest in recent years, due to 56

its practicability, efficacy, safety, and low cost (8, 10-12). In our search of additional virulent 57

phages useful for biocontrol, we discovered phage P70, which turned out to be a novel type of 58

Listeria-specific bacterial virus. 59

Phage P70 was isolated from a grass silage sample from a dairy farm in Homburg, 60

Switzerland. Samples taken were amplified using L. monocytogenes WSLC1001 and 61

WSLC1042 as phage hosts, and potential phage in the resulting lysates assessed by soft-agar 62

overlay method (17, 28). Phage P70 was propagated on semi-confluent soft-agar plates of L. 63

ivanovii WSLC3009 (17). Purified phage stocks were obtained by PEG-precipitation and density 64

gradient centrifugation (15, 25). Host-range analysis was performed as described elsewhere (19, 65

20), and P70 was found to feature an unusually broad host range within the genus Listeria, 66

infecting serovars 1/2a, 1/2b, 1/2c, 4a, 4c, 4d, 4e, 5, 6a and 6b with similar efficiencies. A total of 67

18 of 29 Listeria strains tested were lysed by P70, including food isolates, reference strains and 68

the novel species L. marthii (9) but not L. rocourtiae strain (18). Consistent with other Listeria 69

phages isolated so far, P70 does not bind to or lyse serovar 3 strains that lack the rhamnose 70

residue in the polyribitol phosphate backbone of wall teichoic acids (7, 27). 71

Transmission electron micrographs of P70 were obtained as described (15) and revealed 72

a particle morphology previously not observed for Listeria phages, featuring a long, flexible tail 73

connected to an elongated head (B3 morphotype, (1)) with a head measuring 128 nm in length 74

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and 57 nm in diameter, and a tail length of 141 nm (Figure 1A). P70 is a member of the 75

Siphoviridae family in the order Caudovirales. 76

The P70 genome sequence was obtained from DNA purified by CsCl density gradient 77

purification as previously described (15), employing a novel Sanger/Pacbio hybrid sequencing 78

strategy. A total of 643 Sanger reads (average length 884 nt) were obtained from three shotgun 79

libraries (insert size 2 kb, 1.5 kb and 0.5 kb), and assembly of seven contigs, however, also 80

resulting in a very high cloning bias (16). Then, an alignment scaffold was created by 81

sequencing P70 DNA on a Pacific Biosciences RS device (3 SMRT cells, 2 kb library). A total of 82

354.960.481 pre-filter bases were reduced to 70.378.003 post-filter bases (33.848 reads), with 83

an average read length of 1881 nt and an accuracy of 0.871. A single contig of approximately 68 84

kb could be assembled from these data, revealing a missing sequence of approximately 4 kb in 85

the Sanger assembly. The frequent features of PacBio assemblies are false indels of several 86

bases in length, which could be corrected by high sequencing coverage and combination with 87

the Sanger reads, using the software CLC Genomics Workbench 5.1 (CLC Bio, Denmark). A 88

hybrid Sanger/PacBio assembly produced a unit genome sequence of 67,170 bp, with a 752-fold 89

average coverage. Ambiguities, conflicts and few regions of lower coverage were addressed by 90

direct Sanger sequencing using P70 DNA as the template. The P70 genome features 117 open 91

reading frames, and putative functions could be assigned to 39 gene products based on 92

sequence homology and amino acid motif searches (Figure 1B). The sequence has been 93

deposited at GenBank (accession JX442241). Nucleotide dotplot analyses against the genomes 94

of Listeria phages 01761, A006, A118, A500, A511, B025, B053, B054, P35 and P40 (results not 95

shown) revealed that P70 possess a unique genome, about 50% larger than other Listeria 96

siphoviruses, and half the size of the largest known Listeria phage genomes (5, 16, 21). The 97

genome is divided in several functional modules corresponding to lifestyle (5), but highly mosaic 98

in nature and composed of more modules than any other known Listeria phage genomes (5, 16, 99

21). Major structural component-encoding genes could be identified, such as the terminase 100

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subunits, portal, major capsid protein, major tail protein, head-tail connector and a putative tail 101

fiber. Surprisingly, the lysis cassette of P70 is located at a highly unusual position inbetween 102

genes encoding the large and small terminase subunits. A total of six HNH endonucleases seem 103

to be encoded. Interestingly, P70 also specifies several functions normally associated with a 104

temperate lifestyle, such as ORF44 (holiday junction resolvase), ORF49 (DNA replication 105

inhibitor), or ORF 53 (C1-like repressor protein), albeit the phage is strictly virulent in its nature. 106

It produces clear plaques on any Listeria strain, and all attempts to lysogenize host strains of 107

different species and serovars (EGD-e (SV21/2a), WSLC1001 (SV 1/2c), WSLC1042 (SV 4b) 108

and WSLC3009 (SV 5)) failed. P70 appears to represent an archetype of Listeria phage, similar 109

to P35 and P40 (5, 13), but not featuring consistent protein homologies to known phages of 110

Listeria or other Gram-positive organisms. Further evidence for the unique nature of P70 was 111

obtained from an phylogenetic tree of the large terminase subunit of 109 phages (3, 4) (Figure 112

2A). This places P70 on a separate branch, clearly distinct from all other phages. The closest 113

homologues are found in the PBSX defective prophage of Bacillus subtilis (26), which 114

supposedly employs a P22-like headful packaging mechanism. We investigated the physical 115

genome structure of P70 by restriction profiles, Bal31 nuclease digest (15, 22), PFGE, and 116

Sanger runoff sequencing (Figure 2B). Both restriction profiles and Bal31-digests pointed to a 117

genome with fixed, invariable ends (3, 14, 22). Heating of restriction digests prior to 118

electrophoresis did not change the fragment pattern, making the presence of cohesive genome 119

ends unlikely (data not shown) (3, 14, 22). Finally, runoff sequencing of terminal restriction 120

fragments confirmed P70 to feature terminal repeats of 381 bp length. 121

In conclusion, the unique siphovirus P70 represents a novel species of virulent Listeria 122

bacteriophage, featuring a distinct virion morphology, genome size, and structure unrelated to 123

any other Listeria phage reported previously. 124

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Acknowledgments 125

We are grateful to Steven Hagens, ETH Zurich, for help with isolation of phage P70. 126

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Figure Legends 213

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Figure 1. A. Transmission electron micrographs of P70 show the elongated head and long, non-215

contractile tail, characteristic for the B3 morphotype of the Siphoviridae family in the order 216

Caudovirales. Scale bars are indicated. B. Genome map of P70. Orfs are drawn to scale and 217

numbered consecutively. Numbers above graph indicate nucleotide positions in kb. ORFs with 218

an assigned function for the encoded gene product are colored, and the main functional modules 219

are drawn in different colors. 220

221

Figure 2. A. Snapshot of a terminase phylogeny tree of 109 TerL amino acid sequences and 222

P70 TerL computed by using the Neighbor Joining method from a ClustalW alignment. Numbers 223

at nodes indicate bootstrap values. P70 TerL sequence is highlighted, other Listeria phage TerL 224

sequences are indicated by black dots. B. Restriction profile analysis of P70 genome structure. 225

Panel 1: Restriction digest of purified P70 DNA with nine different restriction endonucleases 226

(lanes c-g and j-m: KpnI, ClaI, SwaI, SalI, EcoRI, EcoRV, PacI, NdeI, HindIII) Lanes a, b, h, i, n, 227

o: 1kb and Lambda mix marker (Fermentas). Panel 2. PFGE run of P70 DNA (lane a). Lanes b 228

and c: Midrange PFG marker I and II (New England Biolabs). Numbers indicate marker band 229

sizes. 230

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