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Novel giant siphovirus from Bacillus anthracis features unusual
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genome characteristics and relatedness to Spounavirinae
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Holly H. Ganz1#, Christina Law1, Martina Schmuki2, Martin J. Loessner2, Richard Calendar3, Wayne M.
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Getz1,4, Jonas Korlach5, and Jochen Klumpp2*
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University of California, Berkeley, Department of Environmental Science, Policy & Management, Berkeley, CA, USA
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Institute of Food, Nutrition and Health, ETH Zurich, Switzerland
University of California, Berkeley, Department of Molecular and Cellular Biology, Berkeley, CA, USA 4
School of Mathematical Sciences, University of KwaZulu-Natal, Durban, South Africa 5
Pacific Biosciences, Menlo Park, CA, USA
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Running title: Giant siphovirus specific for Bacillus ACT group
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Key words: Siphovirus, bacteriophage, Bacillus anthracis, genome, PacBio, SMRT sequencing
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* Corresponding author:
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Jochen Klumpp: Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092
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Zurich, Switzerland. Phone: +41-44-6325378; Fax: +41-44-6321266; email:
[email protected]
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# Present address:
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Holly Ganz: University of California, Davis, School of Veterinary Medicine, Davis, CA 95616 USA
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Abstract
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We present vB_BanS-Etosha, a novel temperate phage isolated from Bacillus
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anthracis. Etosha is a giant siphovirus, featuring a long, flexible and non-contractile
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tail of 440 nm and an isometric head of 82 nm. We induced Etosha phage from two
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different isolates of a B. anthracis genotype responsible for many anthrax outbreaks
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occurring in wildlife in Etosha National Park, Namibia. The phage genome is the
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largest sequenced Bacillus siphovirus, containing 168,876 bp and 269 ORFs.
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We present a novel virus species isolated from Bacillus anthracis, the agent
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responsible for anthrax infections in wildlife, livestock and humans [1]. Along with
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Bacillus cereus and Bacillus thuringiensis, B. anthracis is a member of the Bacillus
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ACT group [2]. Genomic studies have identified a number of putative prophages in
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the Bacillus ACT group (e.g. [3, 4]). Lysogeny occurs commonly in B. anthracis [5, 6]
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and may play an essential role in its life cycle [7].
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We obtained isolates of the siphovirus from two zebra carcass sites in Etosha
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National Park (ENP), Namibia, a 22,915 km2 wildlife reserve where anthrax infections
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occur regularly (reviewed in [8]). We isolated bacteriophage from a B. anthracis
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colony obtained from a swab from 2006 (GPS coordinates: -18.99736, 15.81584) and
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from a soil sample collected in 2010 (GPS coordinates: -19.1731, 15.92603). Isolates
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of B. anthracis from the two carcass sites belong to genotype 6 in the A3a group,
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which has dominated outbreaks in ENP for more than 30 years [8]. We induced
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phage from bacterial isolates by enrichment culture and mitomycin C induction [9,
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10]. Both phages were morphologically similar and unusually large. It is intriguing that
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such a large siphovirus is rarely isolated and yet appeared twice in spatially and
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temporally separate samples.
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Phage preparations were purified and concentrated using standard techniques
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[10, 11]. Plaque assays were performed with a spore preparation of an avirulent B.
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anthracis strain (6602 R1, [12]) by LB agar soft-agar overlay method [13]. Serial
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dilutions of plaque-extracts were soft-agar plated with strain 6602 R1 [12]. Phage
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lysates were PEG precipitated [14] and purified by cesium chloride density gradient
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centrifugation [10].
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We used the spot-on-the-lawn method [15] for host-range testing (Table 1).
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The phage infects the Bacillus ACT group and is particularly adapted to B. anthracis. 3
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Etosha lysed 25% (6/24) of B. cereus strains, 40% (2/5) of B. thuringiensis strains
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and 86% (6/7) of B. anthracis strains tested. The phage did not lyse any other
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bacteria tested (Table 1). Thus, Etosha is a narrow host-range, species-specific virus,
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with potential suitability for biocontrol approaches of B. anthracis.
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TEM images have been acquired from a preparation of pure phage particles
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negatively stained with 2% uranyl acetate on carbon-coated copper grids (Quantifoil,
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Jena, Germany) and observed using a Philips CM12 microscope at 120 kV
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acceleration voltage with a Gatan Orius digital camera. Etosha exhibits typical
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siphovirus morphology (order Caudovirales), and has a long, flexible and non-
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contractile tail of 440 nm (not including baseplate structure) and an isometric head of
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82 nm in diameter (Figure 1 A, B). Individual tail striations and a baseplate structure
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with appendages are visible (Figure 1C). The head features visible capsomers
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(Figure 1D) similar to those observed in other large bacteriophages [16]. To our
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knowledge, Etosha is the largest sequenced siphovirus infecting Bacillus. Two larger
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Bacillus siphoviruses are known but not characterized, B. mycoides phage N5 and B.
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thuringiensis phage II, which are speculated to be identical (H.-W. Ackermann,
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personal communication).
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We sequenced DNA from the isolates using a single-molecule approach
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(Pacific Biosciences RS) with 10 kb and 800 bp insert libraries (C2 chemistry) and
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used one SMRT Cell for each library. We used the standard error-correction workflow
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and SMRT portal software 1.3.1 for assembly of 36166 post-filter reads (2582 bp
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average read length), resulting in one large contig with an average coverage of 550-
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fold (Figure 2). Both phage isolates were identical. A repeat structure of 284 bp at the
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genome end was identified during assembly and confirmed in restriction profiles
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(Figure 3). Methylome analysis revealed no base modifications in the genome. The
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genome sequence is 168,876 bp in length. Open reading frames were predicted by 4
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RAST [17] and edited manually. Etosha features 272 open reading frames, 17 tRNA
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and 2 pseudo-tRNA genes (Figure 2). The GC content is 34%, similar to published
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genome sequences of B. anthracis. The genome sequence was deposited at
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GenBank under accession number KC481682.
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Like many siphoviruses, the genome is structured in functional modules. The
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early gene cluster (genes for DNA replication, modification and repair, host takeover
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and nucleotide metabolism) spans roughly 70% of the genome, indicating active
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participation of virus-encoded genes in the metabolic processes associated with
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replication in the host cell. It is notable that the Etosha genome encodes three
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tyrosine integrase/recombinase enzymes of the Cre/XERD type (gp94, gp227,
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gp255; [18, 19]) which exhibit no nucleotide homology to each other. Etosha features
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a temperate lifestyle and these three enzymes may serve as means to integrate into
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different attB sites and ensure a large host range for lysogeny. Further work will
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elucidate the specificity and activity of the three recombinases. We also note the
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presence of two Ig-domain containing proteins, gp233 and gp213 [19, 20], which may
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play accessory roles during infection [21].
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Interestingly, Etosha features distributed homologies in its structural proteins
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to SPO1-related phages A511, A9, LP65 and SPO1 [16]. This finding is very unusual
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because SPO1-related phages belong to an unrelated family of bacteriophages
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(Myoviridae); and Etosha is a temperate phage, while SPO1-related phages are
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strictly virulent. Etosha also displays individual capsomers thought to be a hallmark of
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the SPO1-related phages (Figure 1) [16]. Virion proteins of Etosha were separated
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on a 10-20% SDS gradient PAGE. Resulting bands were extracted and protein
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content identified by mass spectrometry [22]. Six bands were allocated to gene
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products. The tape measure protein is present in two protein bands of 280 and 100
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kDa in size, presumably because of instability of the large protein. gp206 features an 5
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estimated mass of 38 kDa and gp199 and 207 were identified in bands of 26.5 and
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19 kDa, respectively. Etosha features an unusually long tail of 440 nm, which
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corresponds with the large size of the tape measure protein (3123 aa) [23, 24] but the
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protein is disproportionately large in comparison to other sequenced bacteriophages.
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The large unknown gene 221 likely encodes for a tail fiber component, with the C-
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terminus featuring significant homologies to Cellobiosidase, S-layer associated
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endoglucanase or glycoside hydrolase domains.
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In conclusion, we present vB_BanS-Etosha, a novel temperate phage
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obtained from B. anthracis that is specific to the Bacillus ACT group. To our
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knowledge Etosha is the largest sequenced siphovirus infecting Bacillus organisms.
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Its giant head holds a genome of 168 kb. In addition to potential effects on activity
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and environmental survival of its host, Etosha may have utility in detection and
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control of B. anthracis.
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Acknowledgments
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We thank Margaret Smith for help with recombinase identification, Matthew Boitano
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and Tyson A. Clark for genome assembly and initial analysis, and Martina Kusters,
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Wendy C. Turner and Wilferd Versfeld for field support. Field sampling was
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authorized by the Namibian Ministry of Environment and Tourism (MET) under permit
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number 1448/2009 to HHG. We are grateful to the scientific staff at the Etosha
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Ecological Institute (EEI) for facility resources and logistical support. The isolates
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studied here were obtained from EEI carcass numbers EB060318-01WV and
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EB100228-01MK. This research was funded by NIH Grant GM083863 to WMG. CL
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was supported by the University of California, Berkeley, College of Natural
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Resources-Biology Scholar’s Program, a Rosberg-Geist Fellowship from the Center
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for African Studies and an Undergraduate Merit Scholarship from the Institute of
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International Studies.
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207
Figure legends
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Figure 1: Electron microscopy of Etosha phage. A. Preparation overview. B. Close-
209
up of single phage particle. C. Details of the phage tail distal end. D. Details of the
210
phage head structure. Individual capsomers are visible, an observation previously
211
made for SPO1-related phages ([16]). Scale bars represent 100 nm.
212 213
Figure 2: Genome map of phage Etosha. Open reading frames are drawn to scale
214
and transcription direction is indicated by arrows. Selected proteins with putative
215
function are labeled. Genetic modules (i.e. structural genes, early genes) are
216
indicated by coloring.
217 218
Figure 3: Restriction profile of Etosha phage. Clear band separation up to 24 kb in
219
size could be achieved and the restriction profiles match with the sequenced genome
220
size. The terminal redundancy location and size was determined from the fragment
221
sizes as previously described [10, 11]. Enzymes used: 1: Alw44I (NEB); 2: Eco91I
222
(Fermentas); 3: NheI (NEB); 4: PacI (NEB); 5: SwaI (NEB); 6: Van91I (Fermentas); 7:
223
XcmI (NEB). M1: Lambda 19 Mix Size standard (Fermentas); M2: 1kb size standard
224
(Fermentas). Numbers to the left indicate band size in kb.
10
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 6602 R1 Sterne Weybridge UM44 Ames-non reverting
Organism Bacillus anthracis Bacillus anthracis Bacillus anthracis
Ames
Bacillus anthracis
Vollum 1b
Bacillus anthracis
PAK-1
Bacillus anthracis
LA 925 ATCC 14579 ATCC 11778 ATCC 10702 ATCC 10876 DSM 2302 BO 366 BO 372 BO 493 DSM 4218 ATCC 33019 ATCC 14737 DSM1274 ATCC 27522 NCTC 11143 NCIMB 8705 ATCC 6464 B346 DSM360 HER1399 WSBC 10530 WSBC 10556 WSBC 10566 WSBC 10583 DSM4421 WSBC 10204 HER1211 Kurstaki ATCC 10792 DSM168 DSM675 ATCC 23059 DSM395 DSM90 WSBC 10550 WSLC 3009 ATCC BAA-679 PSK Twort 414 100655 602 DT7155 CGSC 4401 DSM 20560 NZ9000 ATCC 19433
Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus Bacillus thuringiensis Bacillus thuringiensis Bacillus thuringiensis Bacillus thuringiensis Bacillus thuringiensis Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus sphaericus Bacillus megaterium Bacillus weihenstephanensis Listeria ivanovii Listeria monocytogenes Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Salmonella typhimurium Escherichia coli Streptococcus salivarius Lactococcus lactis Enterococcus faecalis
Bacillus anthracis
Notes pXO1-pXO2 negative pXO2 negative pXO2 negative pXO2 negative
Source {Green, 1985 #228} Institut Pasteur #7702 {Green, 1985 #228} U.S. Dept. of Agriculture, Ames, Iowa U.S. Dept. of Agriculture, Ames, Iowa Laboratory Strain Pakistan isolate, M. HughJones collection CHUV ATCC ATCC ATCC ATCC DSM This study This study This study DSM ATCC ATCC DSM ATCC NCTC NCIMB ATCC Mouse isolate DSM HER WSBC WSBC WSBC WSBC DSM WSBC HER R. Calendar, UC Berkeley ATCC DSM DSM ATCC DSM DSM WSBC WSLC ATCC Laboratory Stock Laboratory Stock Laboratory Stock Laboratory Stock Laboratory Stock Laboratory Stock CGSC DSM Laboratory Stock ATCC
Infection + + + + + + + + + + + + + + -
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
