1

1

Novel giant siphovirus from Bacillus anthracis features unusual 2

genome characteristics and relatedness to Spounavirinae 3

4

Holly H. Ganz1#, Christina Law1, Martina Schmuki2, Martin J. Loessner2, Richard Calendar3, Wayne M. 5

Getz1,4, Jonas Korlach5, and Jochen Klumpp2* 6

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1 University of California, Berkeley, Department of Environmental Science, Policy & Management, 8

Berkeley, CA, USA 9

2 Institute of Food, Nutrition and Health, ETH Zurich, Switzerland 10

3 University of California, Berkeley, Department of Molecular and Cellular Biology, Berkeley, CA, USA 11

4 School of Mathematical Sciences, University of KwaZulu-Natal, Durban, South Africa 12

5Pacific Biosciences, Menlo Park, CA, USA 13

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Running title: Giant siphovirus specific for Bacillus ACT group 15

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Key words: Siphovirus, bacteriophage, Bacillus anthracis, genome, PacBio, SMRT sequencing 17

18

* Corresponding author: 19

Jochen Klumpp: Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 20

Zurich, Switzerland. Phone: +41-44-6325378; Fax: +41-44-6321266; email: jklumpp@ethz.ch 21

22

# Present address: 23

Holly Ganz: University of California, Davis, School of Veterinary Medicine, Davis, CA 95616 USA 24

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2

Abstract 26

We present vB_BanS-Etosha, a novel temperate phage isolated from Bacillus 27

anthracis. Etosha is a giant siphovirus, featuring a long, flexible and non-contractile 28

tail of 440 nm and an isometric head of 82 nm. We induced Etosha phage from two 29

different isolates of a B. anthracis genotype responsible for many anthrax outbreaks 30

occurring in wildlife in Etosha National Park, Namibia. The phage genome is the 31

largest sequenced Bacillus siphovirus, containing 168,876 bp and 269 ORFs. 32

3

We present a novel virus species isolated from Bacillus anthracis, the agent 33

responsible for anthrax infections in wildlife, livestock and humans [1]. Along with 34

Bacillus cereus and Bacillus thuringiensis, B. anthracis is a member of the Bacillus 35

ACT group [2]. Genomic studies have identified a number of putative prophages in 36

the Bacillus ACT group (e.g. [3, 4]). Lysogeny occurs commonly in B. anthracis [5, 6] 37

and may play an essential role in its life cycle [7]. 38

We obtained isolates of the siphovirus from two zebra carcass sites in Etosha 39

National Park (ENP), Namibia, a 22,915 km2 wildlife reserve where anthrax infections 40

occur regularly (reviewed in [8]). We isolated bacteriophage from a B. anthracis 41

colony obtained from a swab from 2006 (GPS coordinates: -18.99736, 15.81584) and 42

from a soil sample collected in 2010 (GPS coordinates: -19.1731, 15.92603). Isolates 43

of B. anthracis from the two carcass sites belong to genotype 6 in the A3a group, 44

which has dominated outbreaks in ENP for more than 30 years [8]. We induced 45

phage from bacterial isolates by enrichment culture and mitomycin C induction [9, 46

10]. Both phages were morphologically similar and unusually large. It is intriguing that 47

such a large siphovirus is rarely isolated and yet appeared twice in spatially and 48

temporally separate samples. 49

Phage preparations were purified and concentrated using standard techniques 50

[10, 11]. Plaque assays were performed with a spore preparation of an avirulent B. 51

anthracis strain (6602 R1, [12]) by LB agar soft-agar overlay method [13]. Serial 52

dilutions of plaque-extracts were soft-agar plated with strain 6602 R1 [12]. Phage 53

lysates were PEG precipitated [14] and purified by cesium chloride density gradient 54

centrifugation [10]. 55

We used the spot-on-the-lawn method [15] for host-range testing (Table 1). 56

The phage infects the Bacillus ACT group and is particularly adapted to B. anthracis. 57

4

Etosha lysed 25% (6/24) of B. cereus strains, 40% (2/5) of B. thuringiensis strains 58

and 86% (6/7) of B. anthracis strains tested. The phage did not lyse any other 59

bacteria tested (Table 1). Thus, Etosha is a narrow host-range, species-specific virus, 60

with potential suitability for biocontrol approaches of B. anthracis. 61

TEM images have been acquired from a preparation of pure phage particles 62

negatively stained with 2% uranyl acetate on carbon-coated copper grids (Quantifoil, 63

Jena, Germany) and observed using a Philips CM12 microscope at 120 kV 64

acceleration voltage with a Gatan Orius digital camera. Etosha exhibits typical 65

siphovirus morphology (order Caudovirales), and has a long, flexible and non-66

contractile tail of 440 nm (not including baseplate structure) and an isometric head of 67

82 nm in diameter (Figure 1 A, B). Individual tail striations and a baseplate structure 68

with appendages are visible (Figure 1C). The head features visible capsomers 69

(Figure 1D) similar to those observed in other large bacteriophages [16]. To our 70

knowledge, Etosha is the largest sequenced siphovirus infecting Bacillus. Two larger 71

Bacillus siphoviruses are known but not characterized, B. mycoides phage N5 and B. 72

thuringiensis phage II, which are speculated to be identical (H.-W. Ackermann, 73

personal communication). 74

We sequenced DNA from the isolates using a single-molecule approach 75

(Pacific Biosciences RS) with 10 kb and 800 bp insert libraries (C2 chemistry) and 76

used one SMRT Cell for each library. We used the standard error-correction workflow 77

and SMRT portal software 1.3.1 for assembly of 36166 post-filter reads (2582 bp 78

average read length), resulting in one large contig with an average coverage of 550-79

fold (Figure 2). Both phage isolates were identical. A repeat structure of 284 bp at the 80

genome end was identified during assembly and confirmed in restriction profiles 81

(Figure 3). Methylome analysis revealed no base modifications in the genome. The 82

genome sequence is 168,876 bp in length. Open reading frames were predicted by 83

5

RAST [17] and edited manually. Etosha features 272 open reading frames, 17 tRNA 84

and 2 pseudo-tRNA genes (Figure 2). The GC content is 34%, similar to published 85

genome sequences of B. anthracis. The genome sequence was deposited at 86

GenBank under accession number KC481682. 87

Like many siphoviruses, the genome is structured in functional modules. The 88

early gene cluster (genes for DNA replication, modification and repair, host takeover 89

and nucleotide metabolism) spans roughly 70% of the genome, indicating active 90

participation of virus-encoded genes in the metabolic processes associated with 91

replication in the host cell. It is notable that the Etosha genome encodes three 92

tyrosine integrase/recombinase enzymes of the Cre/XERD type (gp94, gp227, 93

gp255; [18, 19]) which exhibit no nucleotide homology to each other. Etosha features 94

a temperate lifestyle and these three enzymes may serve as means to integrate into 95

different attB sites and ensure a large host range for lysogeny. Further work will 96

elucidate the specificity and activity of the three recombinases. We also note the 97

presence of two Ig-domain containing proteins, gp233 and gp213 [19, 20], which may 98

play accessory roles during infection [21]. 99

Interestingly, Etosha features distributed homologies in its structural proteins 100

to SPO1-related phages A511, A9, LP65 and SPO1 [16]. This finding is very unusual 101

because SPO1-related phages belong to an unrelated family of bacteriophages 102

(Myoviridae); and Etosha is a temperate phage, while SPO1-related phages are 103

strictly virulent. Etosha also displays individual capsomers thought to be a hallmark of 104

the SPO1-related phages (Figure 1) [16]. Virion proteins of Etosha were separated 105

on a 10-20% SDS gradient PAGE. Resulting bands were extracted and protein 106

content identified by mass spectrometry [22]. Six bands were allocated to gene 107

products. The tape measure protein is present in two protein bands of 280 and 100 108

kDa in size, presumably because of instability of the large protein. gp206 features an 109

6

estimated mass of 38 kDa and gp199 and 207 were identified in bands of 26.5 and 110

19 kDa, respectively. Etosha features an unusually long tail of 440 nm, which 111

corresponds with the large size of the tape measure protein (3123 aa) [23, 24] but the 112

protein is disproportionately large in comparison to other sequenced bacteriophages. 113

The large unknown gene 221 likely encodes for a tail fiber component, with the C-114

terminus featuring significant homologies to Cellobiosidase, S-layer associated 115

endoglucanase or glycoside hydrolase domains. 116

In conclusion, we present vB_BanS-Etosha, a novel temperate phage 117

obtained from B. anthracis that is specific to the Bacillus ACT group. To our 118

knowledge Etosha is the largest sequenced siphovirus infecting Bacillus organisms. 119

Its giant head holds a genome of 168 kb. In addition to potential effects on activity 120

and environmental survival of its host, Etosha may have utility in detection and 121

control of B. anthracis. 122

123

7

Acknowledgments 124

We thank Margaret Smith for help with recombinase identification, Matthew Boitano 125

and Tyson A. Clark for genome assembly and initial analysis, and Martina Kusters, 126

Wendy C. Turner and Wilferd Versfeld for field support. Field sampling was 127

authorized by the Namibian Ministry of Environment and Tourism (MET) under permit 128

number 1448/2009 to HHG. We are grateful to the scientific staff at the Etosha 129

Ecological Institute (EEI) for facility resources and logistical support. The isolates 130

studied here were obtained from EEI carcass numbers EB060318-01WV and 131

EB100228-01MK. This research was funded by NIH Grant GM083863 to WMG. CL 132

was supported by the University of California, Berkeley, College of Natural 133

Resources-Biology Scholar’s Program, a Rosberg-Geist Fellowship from the Center 134

for African Studies and an Undergraduate Merit Scholarship from the Institute of 135

International Studies. 136

137

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Figure legends 207

Figure 1: Electron microscopy of Etosha phage. A. Preparation overview. B. Close-208

up of single phage particle. C. Details of the phage tail distal end. D. Details of the 209

phage head structure. Individual capsomers are visible, an observation previously 210

made for SPO1-related phages ([16]). Scale bars represent 100 nm. 211

212

Figure 2: Genome map of phage Etosha. Open reading frames are drawn to scale 213

and transcription direction is indicated by arrows. Selected proteins with putative 214

function are labeled. Genetic modules (i.e. structural genes, early genes) are 215

indicated by coloring. 216

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Figure 3: Restriction profile of Etosha phage. Clear band separation up to 24 kb in 218

size could be achieved and the restriction profiles match with the sequenced genome 219

size. The terminal redundancy location and size was determined from the fragment 220

sizes as previously described [10, 11]. Enzymes used: 1: Alw44I (NEB); 2: Eco91I 221

(Fermentas); 3: NheI (NEB); 4: PacI (NEB); 5: SwaI (NEB); 6: Van91I (Fermentas); 7: 222

XcmI (NEB). M1: Lambda 19 Mix Size standard (Fermentas); M2: 1kb size standard 223

(Fermentas). Numbers to the left indicate band size in kb. 224

Table 1: Host range of Etosha on different Bacillus and non-Bacillus strains. The presence of plaques (successful infection of this strain) is indicated by + and an absence is indicated by -.

Strain name Organism Notes Source Infection6602 R1 Bacillus anthracis pXO1-pXO2 negative {Green, 1985 #228} +Sterne Bacillus anthracis pXO2 negative Institut Pasteur #7702 +Weybridge UM44 Bacillus anthracis pXO2 negative {Green, 1985 #228} +Ames-non reverting

Bacillus anthracis pXO2 negative U.S. Dept. of Agriculture,

Ames, Iowa +

Ames Bacillus anthracis U.S. Dept. of Agriculture,

Ames, Iowa +

Vollum 1b Bacillus anthracis Laboratory Strain +

PAK-1 Bacillus anthracis Pakistan isolate, M. Hugh-

Jones collection -

LA 925 Bacillus cereus CHUV -ATCC 14579 Bacillus cereus ATCC -ATCC 11778 Bacillus cereus ATCC +ATCC 10702 Bacillus cereus ATCC -ATCC 10876 Bacillus cereus ATCC +DSM 2302 Bacillus cereus DSM -BO 366 Bacillus cereus This study -BO 372 Bacillus cereus This study -BO 493 Bacillus cereus This study -DSM 4218 Bacillus cereus DSM -ATCC 33019 Bacillus cereus ATCC +ATCC 14737 Bacillus cereus ATCC +DSM1274 Bacillus cereus DSM -ATCC 27522 Bacillus cereus ATCC -NCTC 11143 Bacillus cereus NCTC -NCIMB 8705 Bacillus cereus NCIMB +ATCC 6464 Bacillus cereus ATCC -B346 Bacillus cereus Mouse isolate -DSM360 Bacillus cereus DSM -HER1399 Bacillus cereus HER +WSBC 10530 Bacillus cereus WSBC -WSBC 10556 Bacillus cereus WSBC -WSBC 10566 Bacillus cereus WSBC -WSBC 10583 Bacillus cereus WSBC -DSM4421 Bacillus thuringiensis DSM -WSBC 10204 Bacillus thuringiensis WSBC -HER1211 Bacillus thuringiensis HER +Kurstaki Bacillus thuringiensis R. Calendar, UC Berkeley -ATCC 10792 Bacillus thuringiensis ATCC +DSM168 Bacillus subtilis DSM -DSM675 Bacillus subtilis DSM -ATCC 23059 Bacillus subtilis ATCC -DSM395 Bacillus sphaericus DSM -DSM90 Bacillus megaterium DSM -WSBC 10550 Bacillus weihenstephanensis WSBC -WSLC 3009 Listeria ivanovii WSLC -ATCC BAA-679 Listeria monocytogenes ATCC -PSK Staphylococcus aureus Laboratory Stock -Twort Staphylococcus aureus Laboratory Stock -414 Staphylococcus epidermidis Laboratory Stock -100655 Staphylococcus epidermidis Laboratory Stock -602 Staphylococcus epidermidis Laboratory Stock -DT7155 Salmonella typhimurium Laboratory Stock -CGSC 4401 Escherichia coli CGSC -DSM 20560 Streptococcus salivarius DSM -NZ9000 Lactococcus lactis Laboratory Stock -ATCC 19433 Enterococcus faecalis ATCC -

CHUV: Strain collection of the Centre Hospitalier universitaire Vaudois, Switzerland; HER: Félix d'Hérelle Reference Center for bacterial viruses, Laval, Canada; ATCC: American Type Culture Collection; NCTC: National Collection of Type Cultures; DSM: Deutsche Sammlung von Mikroorganismen; NCIMB: National Collection of Industrial Bacteria; WSBC: Weihenstephan Bacillus Collection; WSLC= Weihenstephan Listeria Collection. CGSC: Coli Genetic Stock Center, Yale, USA