Genomic characterization of six novel Bacillus pumilus bacteriophages

Virology 444 (2013) 374–383

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Genomic characterization of six novel Bacillus pumilus bacteriophages Laura Lorenz a, Bridget Lins b, Jonathan Barrett a, Andrew Montgomery a, Stephanie Trapani a, Anne Schindler a, Gail E. Christie c, Steven G. Cresawn d, Louise Temple a,n a

Department of Integrated Science and Technology, James Madison University, Harrisonburg, VA 22807, USA Department of Biochemistry and Molecular biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA c Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298, USA d Department of Biology, James Madison University, Harrisonburg, VA 22807, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 1 March 2013 Returned to author for revisions 7 April 2013 Accepted 4 July 2013 Available online 30 July 2013

Twenty-eight bacteriophages infecting the local host Bacillus pumilus BL-8 were isolated, purified, and characterized. Nine genomes were sequenced, of which six were annotated and are the first of this host submitted to the public record. The 28 phages were divided into two groups by sequence and morphological similarity, yielding 27 cluster BpA phages and 1 cluster BpB phage, which is a BL-8 prophage. Most of the BpA phages have a host range restricted to distantly related strains, B. pumilus and B. simplex, reflecting the complexities of Bacillus taxonomy. Despite isolation over wide geographic and temporal space, the six cluster BpA phages share most of their 23 functionally annotated protein features and show a high degree of sequence similarity, which is unique among phages of the Bacillus genera. This is the first report of B. pumilus phages since 1981. & 2013 Elsevier Inc. All rights reserved.

Keywords: Bacillus pumilus Bacteriophage Genomics Host range Bacillus taxonomy

Introduction The genus Bacillus contains a diverse group of 165 bacterial species that are generally Gram-positive, aerobic sporeformers (Logan and Halket, 2011). The wide range of phenotypic diversity and ecological origin of species in this genus has complicated Bacillus taxonomy; for example, the Bacillus cereus group, comprised of the six species B. cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis, do not resolve by 16S rDNA sequence analysis but show high phenotypic variability (Maughan and Van, 2011). Despite difficulties in classifying taxonomy, a number of specific Bacillus species are used in industry, i.e. in molecule production and food fermentation. In addition, two Bacillus species infect humans; B. cereus and B. anthracis are the causative agents of foodborne illness and anthrax, respectively. A number of other Bacillus species have intermittently been implicated in food-associated illness, including Bacillus licheniformis, Bacillus subtilis, and Bacillus pumilus (From et al., 2007; Logan, 2012; Salkinoja-Salonen et al.,

n

Correspondence to: MSC 4415, 701 Carrier Drive, Harrisonburg, VA 22807, USA. E-mail addresses: [email protected] (L. Lorenz), [email protected] (B. Lins), [email protected] (J. Barrett), [email protected] (A. Montgomery), [email protected] (S. Trapani), [email protected] (A. Schindler), [email protected] (G.E. Christie), [email protected] (S.G. Cresawn), [email protected] (L. Temple). 0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.07.004

1999). B. pumilus spores have been shown to survive in extreme environments, even in spacecraft and space-related facilities after strict sterilization techniques (La Duc et al., 2004). The only fully sequenced and assembled B. pumilus genome, strain SAFR-032, was isolated from a spacecraft in an assembly facility and showed resistance up to three times the concentration of hydrogen peroxide and 7.5 times the concentration of UV exposure than related Bacillus species (Gioia et al., 2007). A number of bacteriophages (phages) in the genus Bacillus have been described. Bacteriophages are arguably the most abundant organisms on Earth, with an estimated global population of 1031 phage particles, and the potential genetic diversity is nearly limitless (Hatfull et al., 2010). Phages are utilized in applications as diverse as therapy for human bacterial infections, biocontrol of pathogens on produce or animal products, gene or vaccine delivery systems, protein display systems, and bacterial typing (Haq et al., 2012). Bacillus species phages show great diversity in morphology, sequence length, sequence content, and host range, reflecting the high variability among and within species in this genus. Sequenced phage records exist in the GenBank database for B. anthracis, B. subtilis, B. thuringiensis, Bacillus clarkii, and B. cereus, including the extensively studied superfamily reference phages SPO1 and φ29 (Klumpp et al., 2010; Meijer et al., 2001) 2010 and B. anthracis typing phage Gamma (Abshire et al., 2005). Prior to this study, at least 33 other B. pumilus phages were reported (Bramucci et al., 1977; Reanney and Teh, 1976; van Elsas and

L. Lorenz et al. / Virology 444 (2013) 374–383

Penido, 1981), but none have been sequenced and the latest study was published in 1981 (van Elsas and Penido, 1981). In this study, 28 B. pumilus phages were isolated, purified, and characterized. Nine genomes were sequenced, and the annotation of six of them is described here. This report is the first genomic analysis of B. pumilus phages and nearly doubles the number of B. pumilus phage isolates shown in the literature.

Results Isolation and characterization of novel B. pumilus phages Twenty-eight phages (Table 1) were isolated from various soil samples over a period of four years using the locally isolated host B. pumilus BL-8. Viral morphology of 21 of the phages was visualized by negative stain transmission electron microscopy. The siphoviridae morphotype was observed for 20 of the imaged phages and the podoviridae for one phage. Representative TEM images are depicted in Fig. 1. Nine of the isolated phages were sequenced. Six of these sequenced phages (Andromeda, Taylor, Eoghan, Gemini, Finn, Curly) have been annotated and accessioned in the GenBank database. The final three sequenced phages (Pleiades, Polaris, and BC) are in varying stages of sequence finishing and annotation.

375

Finn ranged between 49,259 and 50,161 bp in length and between 75 and 79 number of genes, and contain a terminal redundancy between 794 and 852 bp. These and other genometrics of the Cluster BpA phages are described in Table 2. The accessioned Cluster BpA phages, Andromeda, Taylor, Eoghan, Gemini, Curly, and Finn, were annotated and their gene organization analyzed. Putative functions were described for 23 genes in each genome and they showed similar organizational structure to each other, schematically depicted in Fig. 2. The Cluster BpB phage, Pleiades, was uncovered as a separate contig in the Polaris (Cluster BpA phage) sequence assembly and visualized as a podovirus by electron microscopy of lysates resulting from infection with Cluster BpA phages (Fig. 1). Pleiades was discovered to exist as a prophage when Pleiades-specific primers produced amplicons in samples of B. pumilus BL-8 genomic DNA (Fig. 3). Pleiades appears to be uninducible by either UV or mitomicin-C; however, no sensitive strain for plaque tests exists for Pleiades, hampering efforts at further analysis. Preliminary experiments with additional B. pumilus strains revealed a dependence of Cluster BpA phage infection on the presence of a similar prophage in partially sequenced strain ATCC 7061 (accession number ABRX00000000.1) (data not shown). Pleiades-specific sequences have been found in varying ratios in all of the Cluster BpA phage sequence assemblies, suggesting there is some relationship between Cluster BpA phage infection and expression of the BL-8 prophage; these studies are ongoing.

Analysis of the sequenced phages Genome comparisons of the nine sequenced genomes yielded two groups of genomically distinct phages: the Cluster BpA phages (Andromeda, Taylor, Eoghan, Gemini, Curly, Finn, BC, and Polaris) and the Cluster BpB phage, Pleiades. The sequenced Cluster BpA phages shared an average of 82% genome similarity to each other. The genomes of Andromeda, Taylor, Eoghan, Gemini, Curly, and

Genome organization and annotation of the finished cluster BpA phages The six accessioned Cluster BpA phages showed a uniform structural organization consisting of three putative gene cassettes for structural genes, lysis genes, and replication genes, based on the 23 functionally annotated features. All genomes began with a

Table 1 Isolation information of the 28 phages presented in this study ordered by isolation date. Shaded rows are phages that have been sequenced. All phages except for phages Taylor and Eoghan came from distinct soil samples. Name

Isolation date

Isolation source

GPS coordinates

Polarisa Pleiades BC a JG a Leonitasa Andromeda a Gemini a Taylor a Eoghan a Aladdinb Habu a Dextera Maximillian a Strevson a DRNb Valhallaa SB1a SB2a TDC a LCDa Cheesea Davea Samboa Tweety a Kylea Sigmaa Curly Finn

2008 2008 2008 2008 2008 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2010 2010

Greenhouse soil, JMU campus Greenhouse soil, JMU campus Greenhouse soil, JMU campus Greenhouse soil, JMU campus Greenhouse soil, JMU campus Grassy area, Harrisonburg VA Near a reservoir, JMU campus Compost pile, NJ Compost pile, NJ Fairly dry residential compost, NJ Unknown Small hill surrounded by shrubs Edge of lake, JMU campus Mulch near statue, JMU campus Mulch under tree, JMU campus Mulch under tree, JMU campus Zion National Park, Utah Zion National Park, Utah Unknown Grassy area, Harrisonburg VA Near horse barn, 4 miles NW of Harrisonburg, VA Soil near pond Unknown Compost pile, NJ Base of pine tree, JMU campus Compost pile, NJ Compost pile, Harrisonburg VA Under a tree, Harrisonburg VA

38.439329,
78.872169 38.439329,
78.872169 38.439329,
78.872169 38.439329,
78.872169 38.439329,
78.872169 38.4234667,
78.8846667 38.4344258,
78.874342 40.7455556,
74.4808333 40.7455556,
74.4808333 40.7455556,
74.4975000 Unknown Unknown 38.423327,
78.881133 38.4340500,
78.8636890 38.433364,
78.861773 38.433364,
78.861773 Unknown Unknown Unknown 38.4234667,
78.8846667 Unknown 38.4340500,
78.8636890 Unknown 40.7455556,
74.4808333 38.4340100,
78.8679740 40.7455556,
74.4808333 38.4817750,
78.8756778 38.4444722,
078.9176806

a b

Infects B. pumilus BL-8 (original host) and B. simplex RWR1, does not infect B. cereus 113, B. megaterium 113, B. weihenstaphanensis LazoA1, or B. pseudomycoides 113. Only infects B. pumilus BL-8 (original host), does not infect B. simplex RWR1, B. cereus 113, B. megaterium 113, B. weihenstaphanensis LazoA1, or B. pseudomycoides 113.

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L. Lorenz et al. / Virology 444 (2013) 374–383

Fig. 1. Representative TEMs of observed phage particle morphology. Phages pictured are: A, Curly; B, Gemini; C, Eoghan; D, Taylor; E, Pleiades. Pleiades image was derived from an Andromeda infection that yielded a mixed burst of phage.

Table 2 Genometrics and morphological data of the nine sequenced phages. Shaded phages have completed sequencing and annotation and are available in Genbank. Unshaded phages are incomplete and the metrics shown are based on incomplete sequence assembly and auto-annotation. Phage particle dimensions are averages in cases where the phage was imaged more than once with the following sample sizes and standard deviations of head length, head width, and tail length measurements: Taylor, n¼ 2, SD 7 1 nm, 7 4 nm, 7 7 nm; Eoghan, n ¼4, SD 7 5 nm, 7 3 nm, 7 4 nm; Curly, n¼6, SD 73 nm, 72 nm, 79nm; Pleiades, n¼ 3, SD 7 2 nm, 7 1 nm. Name

Length (bp)

Number of genes

GC content (%)

Percentage coding

Terminal repeat length Particle (bp) morphology

Head length (nm)

Head width (nm)

Tail length (nm)

Andromeda Gemini Taylor Eoghan Curly Finn Polaris Pleiades BC

49259 49362 49492 49558 49425 50161 49096 65676 48729

79 79 75 76 77 77 83 114 88

41.91 41.9 42.29 42.22 41.82 41.69 42.13 47.57 41.96

91.54 91.22 91.82 91.74 91.78 90.19

852 852 831 794 852 849

55 47 58 56 57 45

56 48 60 54 54 41

115 149 114 125 110 153

59

37

highly conserved terminally redundant region, followed by seven highly variable small genes of unknown function and no significant similarity in the BLASTp non-redundant database. These were followed by a terminase gene, beginning the putative structural cassette that also included the portal gene, phage head

Siphoviridae Siphoviridae Siphoviridae Siphoviridae Siphoviridae Siphoviridae Siphoviridae Podoviridae Siphoviridae

morphogenesis gene, scaffold protein gene, phage virion morphogenesis gene, tape measure protein gene, and tailspike protein gene. After a gap of three unknown genes, the lysis cassette followed, including an autolysin and a holin gene. The remaining annotated genes encode proteins either directly or indirectly

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377

Fig. 2. Genome schematic of B. pumilus phage Curly, overlaid with annotated gene functions. Terminally redundant regions are shaded purple, structural cassette annotated genes in green, lysis cassette genes in orange, and replication cassette genes in blue. The six BpA cluster phage genomes share similar organization and features.

Table 4 PCR results of the seven unsequenced Bacillus phages that failed to amplify one or more of the seven conserved regions found in five of the sequenced BpA phages. Presence or absence of the PCR product are denoted by the (+) and (
) signs, respectively.

Maximillian Aladdin Dave Cheese Valhalla DRN Leonitas Fig. 3. Gel of PCR results of B. pumilus BL-8 genomic DNA amplified using primers designed from the Pleiades (A–C) and Polari (D–F) sequences. Lane A: Pleiades a/b primers; Lane B: Pleiades c/d primers; Labe C: Pleiades e/f primers; Lane D: Polaris a/b primers; Lane E: Polaris c/d primers; Lane F: Polaris e/f primers; Lane G: 1 kb ladder; Lane H: no template with Pleiades a/b primers.

related to replication, including a helicase, primase, polymerase subunits and factors, and dNTP regulating enzymes.

P1

P2

P3

P4

P5

P6

P7

+
+ + + +


+ + + + + + +

+




+

+ + + + + +


+
+ + +





+ + + + +

+ + + + +



Host range The host range of 25 of the phages was compared between the known host, B. pumilus BL-8, and four different Bacillus species isolated locally: B. simplex RWR1, B. cereus-113, B. thuringiensis-113, and Bacillus megaterium-113. All but two phage isolates, Aladdin and DRN, infected strain RWR1, and none infected any other Bacillus strains tested (Table 1).

Clustering of the unsequenced phages Comparison with other sequenced Bacillus phages Sequence similarity of the unsequenced phages was investigated using two approaches: PCR of regions conserved in the sequenced phages and restriction digests of genomic DNA. PCR primers were designed for seven conserved regions in five of the sequenced BpA phages (Andromeda, Taylor, Eoghan, Gemini, Polaris) and the presence or absence of amplicons in 19 unsequenced phages was observed. Twelve of the unsequenced phages, along with the five sequenced phages from which the primers were designed, amplified in all seven conserved regions. The remaining seven phages did not amplify in at least one of the regions. Table 3 depicts the location of the primer sets and Table 4 lists a summary of the PCR results of the seven phages that failed to amplify at least one of the conserved regions. All primer sets correctly amplified the anticipated sequence in the 5 sequenced BpA phages and in the following unsequenced phages: Tweety, LCD, LEG, AP, SB1, SB2, TDC, SH, Laurel, JG, KC, Dexter. In addition, a restriction digest of the genomes of 24 phages using restriction enzyme EcoRI was performed. Gel analysis yielded 11 distinct banding patterns that are depicted in Fig. 4.

Andromeda, Taylor, Eoghan, Gemini, Curly, and Finn were compared in the program Phamerator with 18 other previously sequenced Bacillus phages: five B. anthracis phages (AP50, Fah, Cherry, WBeta, Gamma), four B. thuringiensis phages (Bam35c, GIL16c, IEBH, 0305phi8–36), six B. subtilis phages (SPBc2, SPO1, SPP1, Φ29, B103, phi105), one B. clarkii phage (BCJA1c), one B. cereus phage (TP21-L) and one Lactobacillus plantarum phage (LP65). Only small regions of sequence similarity (E o10–4) existed between our phages and phages of other host genera (Supplementary Fig. 1). In fact, sequence similarity of this degree was rare among all the Bacillus phages, except for those within our B. pumilus group and the four B. anthracis phages Cherry, Gamma, WBeta, and Fah, which have been shown to be derivatives of a single B. anthracis prophage named W (Fouts et al., 2006; Minakhin et al., 2005). Thirteen of the non-Bacillus pumilus phages with a gene annotated or matching a CDD domain indicating a terminase function were used for phylogenetic analysis against the terminase genes of our six annotated phage genomes by protein

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Table 3 Primer set genome coordinates, location, and gene function within the phage Andromeda. Primers were used in PCR analysis of unsequenced phages. Primer set

Coordinates in Andromeda

Location

Function

P1 P2 P3 P4 P5 P6 P7

27210–27399 6355–6508 13698–13889 23193–23405 31290–21527 43714–43905 47989–48236

gp38 gp11 gp24 Before In and In and In and

DNA primase Head morphogenesis protein No significant similarity by BLASTn No significant similarity by BLASTn DNA polymerase Bacillus conserved unknown protein No significant similarity by BLASTn

and in gp33 between gp41 and gp42 between gp66 and gp67 between gp76 and gp77

Fig. 4. Gel electrophoresis of B. pumilus phage genomic DNA digested with EcoRI. Lane A: Andromeda; Lane B: Taylor; Lane C: Gemini; Lane D: SB2; Lane E: Cheese; Lane F: Strevson; Lane G: SB1; Lane H: LCD; Lane I: Tweety; Lane J: Sigma; Lane K: Eoghan; Lane L: 1 kb Ladder.

alignment with Clustal Omega and ClustalW and visualization in Njplot (Fig. 5). Identification of proteins by mass spectrometry Fifteen proteins were identified by 1D LC-MS/MS of Curly lysate and Curly phage particles (Table 5). Of these 15, eight had been assigned functional annotation from bioinformatic analysis: gp 32 (endolysin), gp10 (portal), gp9 (terminase), gp42 (DNA polymerase), gp25 (tape measure), gp55 (kinase), gp22 (major tail protein) and gp39 (primase). Although LC-MS/MS analysis did not identify gp16, I-TASSER bioinformatic analysis showed significant hits to the HK9 fold of capsid proteins (Pietila et al., 2013; Roy et al., 2010).

Discussion This report contains the first description of novel B. pumilus phages since 1981 and the first analysis of sequenced B. pumilus phages published. Of the phages tested, all but one displayed the siphoviridae morphotype and all but two displayed uniform host specificity. Sequenced phages were divided into Cluster BpA or Cluster BpB based on sequence and morphological similarity. Among the sequenced Cluster BpA phages, pairwise alignments produce an average of 82% nucleotide similarity. Twelve unsequenced phages joined the six sequenced Cluster BpA phages due to sequence conservation by PCR in all seven tested regions, while

Fig. 5. Phylogenetic relationship of terminase genes in 19 Bacillus phages. Phages are color coded according to their host bacterium. Pink is Lactobacillus planarum, cyan is B. subtilis, red is B. anthracis, green is B. thuringiensis, orange is B. clarkii, purple is B. cereus, and dark blue is B. pumilus.

the other seven phages tested showed conservation in at least three of the seven regions. By these measures, our population of Cluster BpA phage appears to share significant similarity, though restriction digest analysis shows they are not identical. These Cluster BpA phages also appear to be ubiquitous in the environment, varying greatly in their geographic and temporal origin over four years and locations as far apart as 300 miles. The sequenced Cluster BpA phages displayed a uniform genomic structure, and all contained roughly the same 20 annotated

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379

Table 5 Proteins identified by mass spectrometry. Tryptic digests of gel fragments comprising molecular weights from 4–150 kD, inclusive, were analyzed, and 15 predicted proteins were identified. Gene product #

Predicted function of protein

E-value for best peptide

# of unique peptides

Sequence of best peptide(s)

gp32 gp10 gp29 gp09 gp42 gp52 gp25 gp07 gp13 gp22 gp55 gp37 gp36 gp69 gp39

Endolysin Portal Hypothetical Terminase DNA polymerase Hypothetical Tape measure Hypothetical Hypothetical Major tail protein Kinase Hypothetical Hypothetical Hypothetical Primase

1.50E-04 1.50E-04 1.50E-04 1.50E-04 1.50E-04 1.50E-04 1.50E-04 1.50E-04 1.50E-04 1.90E-14 1.90E-14 3.60E-14 0.14 0.024 0.1

2 3 1 2 2 2 1 1 1 1 1 1 1 1 1

R.NTAMSAILTESLFINNPADAK.L K.IMESLLVPPSLLGLSRGQSGSYALSSNQFDIFMIHLESIQR.D K.IQPWVIFQMPDGGEIEFPLGIFYLSTPTR.N K.KKLLIVHMESAPNMDTDER.I K.AIGLTDHGAMHGIPEFMNLAK.Q R.ELDLGEVSPRRTSSEHICLYGAPILMDYNVNTGELSVWSASK.T MASNSLINIVVSATDMASAQIGKVGGALEGMASK.A VIVLYKQVYMKDVNVVALLNGEESYGHR.K K.VGLPVLLRYAK.N MASQSHGFDNTIVFGK.E R.VVGENVCDIHSK.S K.PERRDSPTWFR.E K.GNFQDIK.Y MLEVTSKQGR.A R.MMRKQDK.K

Table 6 Evidence of proteins encoded in terminally redundant regions for four other Bacillus phages. Accession numbers for proteins follow below. Phage

Terminal Terminally redundant proteins redundancy length

Bacillus clarkii phage BCJA1c (39) Bacillus subtilis phage SPO1 (40) Bacillus subtilis phage SPP1 (41) Bacillus thurengiensis phage 0305phi8-36 (42)

350

Hypothetical protein gp1a

13,185

Host shut off proteins gp38-40, transcription regulation proteins gp44, gp50 and gp51, cell division inhibitor gp56, and 23 hypothetical proteins between gp36.1 and gp60b Terminase small subunit ORF1, terminase large subunit ORF2, hypothetical protein ORF 2.1 and its complement ORF2.2c Rel-Spo-like synthetase/hydrolase gp102, and hypothetical proteins gp101, and gp 103-107d

1320 (circularly permuted) 6479

a

YP_164380.1 gp36.1 YP_002300277.1, gp36.2 YP_002300278.1, gp36.3 YP_002300279.1, gp35.1 YP_002300280.1, gp37 YP_002300281.1, gp38 YP_002300282.1, gp39 YP_002300283.1, gp40 YP_002300284.1, gp41 YP_002300285.1, gp42 YP_002300286.1, gp43 YP_002300287.1, gp44 YP_002300288.1, gp45 YP_002300289.1, gp46 YP_002300290.1, gp47 YP_002300291.1, gp48 YP_002300292.1, gp49 YP_002300293.1, gp50 YP_002300294.1, gp51 YP_002300295.1, gp52 YP_002300296.1, gp52.2 YP_002300297.1, gp52.1 YP_002300298.1, gp53 YP_002300299.1, gp54 YP_002300300.1, gp55 YP_002300301.1, gp56 YP_002300302.1, gp57 YP_002300303.1, gp58 YP_002300304.1, gp59 YP_002300305.1, gp60 YP_002300306.1 c ORF1 NP_690652.1, ORF 2 NP_690654.1, ORF 2.1 NP_690655.1, ORF 2.2 NP_690656.1. d gp 102 YP_001429592.1, gp 101 YP_001429591.1, gp 103 YP_001429593.1, gp 104 YP_001429594.1, gp 105 YP_001429595.1, gp 106 YP_001429596.1, gp 107 YP_001429597.1 b

features. Though broad regions of functional similarity were assigned to this organization, most of the predicted genes did not match significantly to the BLAST non-redundant database or matched only with genes encoding proteins of unknown function. All sequenced Cluster BpA genomes began with a highly conserved terminally redundant region. Though not all terminal redundancies encode proteins, of the 18 Bacillus and Lactobacillus phages utilized for comparative analysis in this paper, four Bacillus phages with terminally redundant ends contain coding potential: B. clarkii phage BCJA1c (Kropinski et al., 2005), B. subtilis phage SPO1 (Stewart et al., 2009), B. subtilis phage SPP1 (Alonso et al., 1997) and B. thuringiensis phage 0305phi8-36 (Thomas et al., 2007). Gene calls in these regions range from hypothetical proteins to critical enzymes such as terminase subunits (Table 6), but the gene observed in the terminal redundancy in the Cluster BpA phages did not show homology by BLASTp to any of these proteins, nor did HHPred analysis confidently identify any structure similarities to the June 1, 2013 PDB70 database. Using phage Curly for reference, the putative structural cassette began with the terminase at gp9 and also included the portal protein gene (gp10), phage head morphogenesis gene (gp11), scaffold protein gene (gp14), major head protein (gp16), phage virion morphogenesis gene (gp19), major tail protein (gp22), tape measure protein gene (gp25), and tailspike protein gene (gp28). Of particular interest in this cassette are the phage head

morphogenesis gene gp11, a putative translational frameshift between gp23 and 24, the tape measure gene gp25, and the tailspike protein gene gp 28. CDD-BATCH and BLASTp results linked gp11 to the domain family pfam04233 that includes the well-studied GP7 gene product from B. subtilis phage SPP1, a protein that has been implicated in head assembly (Becker et al., 1997), though lack of GP7 mutants inhibited further analysis. Gp24 may be expressed as a C-terminal extension of gp23 by a translational frameshift, implied by the lack of a good candidate RBS upstream of gp24 and a “slippery” run of adenosine bases overlapping the end of gp23 and the beginning of gp24, much like the translational frameshift observed in tail maturation proteins in E. coli phages λ and P2 (Christie et al., 2002; Levin et al., 1993; Xu et al., 2004). The tape measure gene, the length of which has been shown to control tail length in tailed phages (Katsura and Hendrix, 1984), is nearly identical in this set of phages except for that of Finn, whose tape measure gene is 175 bases longer. This roughly corresponds to Finn's longer observed tail length, though the sample size for tail length measurements in this family of phages are prohibitively small for statistical analysis. Finally, HHPred analysis of the tailspike protein gp28 yielded many high probability domains, including many fungal and bacterial sugar degrading enzymes implicated in cell wall degradation and a 100% probability match (E ¼2  1026, p ¼6.6  1031) between gp28 and the self-cleaving trimeric B. subtilis phage Φ29's tail

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appendage protein (Φ29 gp12). Xiang et al. identified that the sequence of the tail appendage proteins from Φ29 shows similarity to various polysaccharide binding enzymes due to its activity in digesting the bacterial cell wall during irreversible attachment (Xiang et al., 2009). The strong similarity between the BpA tailspike protein gp28, various polygalactoronases, and Φ29's tail appendage protein, may suggest that gp28 shares functional and mechanistic similarity to the well-described Φ29 gp12 in vivo. Mass spectrometry analysis provided identification of 15 proteins from a crude lysate of Curly. Six of the identified proteins fall into the predicted structural gene region. Many of the functionally annotated genes described here are present in other phages, but their sequence and order are unique among phages of the same host genus. The scaffold protein of the structural cassette and holin protein of the lysis cassette belong to gene families that exhibit little sequence conservation, so BLASTp or CDD searches yielded no significant hits. As a result, functions of the scaffold and holin genes were assigned instead by analyzing candidate genes′ protein secondary structure elements using the program PSIPRED and looking for structural features as described by Morais (Morais et al., 2003) and Young (Young and Bläsi, 1995). The sequence and order of the holin and its accompanying endolysin gene have an unusual genome placement conserved among the six sequenced B. pumilus phages. These genes are usually found directly adjacent in other phage genomes, as in B. subtilis phages Φ29, PZA, and SPO1 (Stewart et al., 2009; Young, 1992), but all phages described by this study have a small variable region containing one to two genes of unknown function situated between the two canonical lysis genes. Despite the high similarities in sequence and genomic structure between them, the sequenced phage genomes varied in their length, %GC content, and number of ORFs, particularly in regions with smaller genes. Previous research in Mycobacteriophages demonstrates that smaller genes are generally in greater genetic flux, likely due to illegitimate recombination events that favor smaller independent movable units (Hatfull et al., 2010). Our data suggest this may be a more widespread phenomenon in phage of host genera other than Mycobacteria. To examine gene transfer among phages infecting the Bacillus genera, the terminase genes of 19 sequenced Bacillus phages, including the novel six accessioned B. pumilus phage genomes, were aligned and a phylogenetic tree produced (Fig. 5). Terminases have previously been used to assess phylogenetic relationships between phages (Hatfull et al., 2010) as they are one of the most highly conserved gene families among phages (Casjens, 2005). Interestingly, categorization by initial host is not always preserved in this analysis; only the B. pumilus and B. anthracis phages remained grouped by this method, while the latter likely share high sequence similarity due to a shared prophage ancestor (Fouts et al., 2006; Minakhin et al., 2005). The arrangement does not seem to be grouped by similar secondary hosts, either, as the only known multi-species infecting phages in this group, Cherry, Gamma, and IEBH, are not aligned close to B. cereus phage TP21, which shares a secondary host species with them. These data suggest that horizontal gene transfer is not prevalent in the previously published sample of Bacillus phages, even when infection of a shared species is possible. This implies that the B. pumilus phages have either recently diverged (despite the wide geographic and temporal distance of the isolates) or have a uniquely high rate of horizontal gene transfer compared to other Bacillus phages. Most published Bacillus phages are limited to a single species and in some cases to only a few strains, though B. subtilis phage Φ29 has shown the ability to infect up to four different Bacillus species (Meijer et al., 2001). Phages with narrow host range can be utilized for bacterial typing purposes, whereas broad host range phage can be utilized for sterilization or for their potential ability to infect and destroy animal pathogens. All of these are critical issues in the Bacillus

genus, in which species are difficult to resolve even with modern molecular methods. Hardy spores persist in the most sterile of environments, and two species are potentially lethal human pathogens. Host range studies performed with 25 novel B. pumilus phages showed all but two (Aladdin, DRN) cross-infected in B. simplex but not one infected B. thuringiensis, B. cereus, B. megaterium, B. weihenstephanensis, or B. pseudomycoides (Table 1). The host range results have implications for Bacillus taxonomy. Modern taxonomy separates the seven species used for host range testing into three distinct clades; B. pumilus in one, B. cereus sensu lato (including B. cereus, B. thuringiensis, B. weihenstephanensis, and B. pseudomyoides) in another, and B. simplex and B. megaterium in the last (Maughan and Van, 2011). Interestingly, all but two tested phages cross-infected B. simplex but not B. megaterium, two closely related bacterial species by 16S rDNA analysis. However, Maughen et al. showed that rearranging the phylogeny to minimize 11 phenotypic changes along the timeline places these two species further apart, with B. pumilus, our phage host, evolutionarily between them (Maughan and Van, 2011). This suggests that some specific phenotype shared between B. pumilus and B. simplex, two genetically distant species, is related to infection by these phages, possibly one of the phenotypes that Maughen identified in her analysis (Maughan and Van, 2011). Further studies with the two restricted host range phages, Aladdin and DRN, may elucidate the connection between these three Bacillus species. Inability of these 25 phages to infect strains of B. cereus sensu lato may indicate that these phages will not infect other B. cereus group species including B. anthracis, but host range studies with this group could yield a potential bacterial typing system if these phages preferentially infect one species in the group. Interestingly, we have recently isolated a new myovirus on B. pumilus BL-8 that can crossinfect B. thuringiensis. This is the first B. pumilus phage we have isolated that is a myovirus and that can cross-infect B. thuringiensis (data not shown). Sequencing of this phage genome may reveal genomic differences that define phage host range among these Bacillus species. In summary, the 28 novel Bacillus phages presented here provide insight into three main questions. First, the terminase phylogenetic tree implies a slow rate of genetic transfer among Bacillus phages except among B. pumilus and B. anthracis phages. Studies are ongoing to characterize the BpB cluster B. pumilus BL-8 prophage, Pleiades, in order to explain the prophage–phage interaction between it and the BpA phages we have observed. Their interaction may also explain the observed high genetic transfer rate among the BpA phages and how it relates to the B. anthracis phages and their ancestor prophage W. Second, the restricted host range phages Aladdin and DRN and the unique B. pumilus/B. thuringiensis cross-infecting phage Glitter may help identify useful typing variations in difficult to resolve Bacillus species such as B. simplex, B. megaterium, and the uniquely complicated B. cereus sensu lato group. Finally, the contribution of sequenced B. pumilus phages to the literature provides insight into the wide range of Bacillus phage diversity and provides methods of clustering and categorization for future investigators.

Materials and methods Isolation and identification of strains Brain heart infusion (BHI) broth and agar plates were used at 37 1C for the routine cultivation of bacteria. Cultures were aerated in BHI overnight on a rotary shaker at 220 rpm. Plate lawns or isolation streaks were grown on 1.5% agar BHI plates. Bacterial strains were maintained on plates at 4 1C for routine use and in 50% glycerol stocks at
70 1C for long-term storage.

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Isolation and identification of strains Several Bacillus strains were isolated from soil on the campus of James Madison University (Harrisonburg, VA) and used in this study: B. pumilus BL-8, B. simplex RWR1, B. cereus-113, B. thuringiensis-113, B. megaterium-113, B. weihenstephanensis LazoA1 and B. pseudomycoides-113. To isolate strains, approximately 1 ml of soil was suspended in 10 ml 1  phosphate buffered saline (PBS) and mixed. After the soil settled by gravity, serial dilutions of the supernatant were performed using 1  PBS, which were then plated onto BHI plates and incubated at 37 1C overnight. Individual colonies were restreaked to ensure isolation. To identify strains, amplification of 16S rDNA genes from the isolates were performed as described by Fierer et al. (Fierer and Jackson, 2006) and sequencing was performed by Elim Biopharmaceuticals (Hayward, CA). 16S rDNA sequence comparison and putative species identification was accomplished using BLAST analysis and the Ribosomal Database Project web tool (Cole et al., 2009). Phage isolation To obtain a population of B. pumilus specific particles, approximately 1 ml of soil was enriched with B. pumilus BL-8 in 10 ml BHI was incubated overnight with shaking at 37 1C. The resulting mixture was centrifuged and the supernatant sterilized by either the addition of chloroform or filtered through a 0.22 μm filter (Thermo Fischer Scientific, Waltham, MA). Phage were isolated and purified by plaque purification. Briefly, liquid suspensions of B. pumilus BL-8 were infected with the filtered soil solution and agar was added. Mixtures were plated and allowed to harden before incubation overnight at 37 1C. To purify individual phage, plaques were picked and up to 8 sequential rounds of plaque purification were performed until a single plaque morphology was assured. High-titer lysates (4109 pfu/ml) were obtained by flooding infected plates displaying ∼2  103–5  103 plaques with phage buffer (PB: 10 mM Tris, pH 7.5, 10 mM MgSO4, 68 mM NaCl, 1 mM CaCl2), incubating six hours to overnight at 4 1C, then collecting and filtering the liquid. Glycerol stocks of phage isolates were stored in 40% glycerol at
70 1C. Phage characterization To test host range or titer calculation, spot tests were performed by dropping high-titer lysate or dilutions of high-titer lysate onto plates containing a top agar consisting of 0.7% BHI-agar mixed with 400 μl of the target bacterial species at OD500∼1.0. To extract phage DNA, a modification of the DNA clean-up protocol from Promega (2010) was used. Briefly, a high titer phage lysate was incubated with a nuclease mixture to remove contaminating bacterial DNA (0.25 mg/ml each DNaseI and RNaseA, New England Biolabs, Ipswich, MA), then precipitation solution (30% polyethylene glycol, PEG8000, and NaCl to a final concentration of 3.3 M) was added before incubation at 4 1C overnight. Phage particles were pelleted by centrifugation and resuspended in sterile dH20. Two ml of DNA clean-up resin (ProMega, Madison, WI) was added and mixed gently. This slurry was added to DNA purification columns (Zymo, Irvine, CA) and spun at 13,000  g. The resin was washed with 80% isopropanol and the DNA was eluted in dH2O. DNA was quantified using a Nanodrop spectrophotometer (Thermo Fischer Scientific, Waltham, MA). To perform restriction digests, phage DNA was incubated with EcoRI restriction endonuclease (New England Biolabs, Ipswich, MA) overnight at 37 1C, and products were visualized on a 1% agarose gel. PCR for clustering of unsequenced phages was performed in a Bio-Rad Thermal Cycler (Bio-Rad, Hercules, CA) under the following cycle: 95 1C for 30 s, 55 1C for 30 s, and 72 1C for 30 s for 30 cycles, and a final 72 1C for

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10 min. For forward and reverse primers see Supplementary Table 1. Prophage identification The B. pumilus BL-8 prophage Pleiades was identified by PCR of BL-8 genomic DNA with Pleiades-specific primers (Supplementary Table 2). PCR was performed in a Bio-Rad Thermal Cycler (Bio-Rad, Hercules, CA) under the following cycle: 3 min at 95 1C, then 95 1C for 30 s, 54 1C for 30 s, and 72 1C for 60 s for 30 cycles, and a final 72 1C for 5 min. For forward and reverse primers see Supplementary Table 2. Sequencing and genome finishing Phages were sequenced at Virginia Commonwealth University's Sequencing Center (Richmond, VA). Assembly was done in the program Newbler (454 Life Sciences, Branford, CT) and analyzed in Consed version 16.0 (Gordon et al., 1998). Regions of weak or missing reads for incomplete phage genomes were amplified using PCR, sequenced at Elim Biopharmaceuticals (Hayward, CA) and reassembled in Sequencher version 5.0 (Gene Codes Corporation, Ann Arbor, MI). Terminally redundant regions identified by double read coverage and defined read ends in Consed and were manually fitted into the sequenced genome after read assembly. Electron microscopy High titer phage lysate was adhered to EM grids (Ted Pella, Redding, CA), washed with sterile H20, and stained with 3.0% uranyl acetate. Images were taken at Virginia Commonwealth University (Richmond, VA). Image analysis was done using ImageJ (Schneider et al., 2012). Genome annotation Phage genomes were auto-annotated primarily using the statistical gene and RBS prediction program Genemark.hmm-P version 2.8 (Lukashin and Borodovsky, 1998) using the B. pumilus model species, but also with Glimmer version 3.02 (Delcher et al., 1999), and tRNA-scan-SE version 1.21 (Schattner et al., 2005). Annotations were refined manually using Apollo version 1.11.6 (Lewis et al., 2002), Artemis version 14 (Rutherford et al., 2000), and DNA Master (Lawrence Lab, University of Pittsburgh, http:// cobamide2.bio.pitt.edu/). Final gene calls were optimized by comparing program predictions, as well as BLASTp results (Altschul et al., 1990), and preferentially choosing or manually refining gene calls for matches with existing genes, longer ORFs (minimum 100 bp in length), higher Shine-Dalgarno scores, minimizing intergene overlap or gap, and including all coding potential. Putative gene functions were assigned by corroborating BLASTp, CD-BATCH Search (Marchler-Bauer et al., 2011), PSIPRED (McGuffin et al., 2000), HHPred (Söding et al., 2005), and I-TASSER (Roy et al., 2010; Zhang, 2008) results. GenBank accession numbers are as follows: Curly, KC330679; Eoghan, KC330680; Gemini, KC330681; Taylor, KC330682; Finn, KC330683; Andromeda, KC330684. Comparative genomic analysis Phage genomes were analyzed using the program Phamerator (Cresawn et al., 2011). Comparisons were made between the six B. pumilus phages and 18 other sequenced Bacillus phages (GenBank accession numbers NC_011523, NC_007814, NC_007457, NC_007734, NC_007458, NC_005258, NC_006945, NC_011167, NC_009760, NC_011048, NC_011421, NC_001884, NC_004166, NC_004165, NC_004167, NC_006557, NC_011645, NC_006565).

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Multiple alignments were performed with Clustal Omega (Sievers et al., 2011), phylogenetic trees were constructed using ClustalW2 (Larkin et al., 2007) and phylogenetic trees were visualized using Njplot (Perrière and Gouy, 1996). Genome maps were generated using SnapGene Viewer (GSL Biotech, Chicago IL). Protein identification by mass spectrometry Proteins were identified using 1D LC-MS/MS on tryptic digested protein gels of precipitated phage particles and high-titer lysate of phage Curly. Phage particles from a high-titer lysate were precipitated overnight at 4 1C with precipitate solution (30% polyethylene glycol, PEG8000, and NaCl to a final concentration of 3.3 M), centrifuged, and the pellet resuspended in dH2O. Both the phage precipitate and crude Curly lysate were concentrated with 10 kD Microcon microconcentrators (Millipore, Billeria, MA) and run on a 4–16% Bis-Tris protein gel in MES running buffer (Novex by Life Technologies, Grand Island, NY). Peptides were destained, digested, and extracted following the procedure by Shevchenko, et al. (Shevchenko et al., 2006). Briefly, four gel fragments representing all the proteins in a single gel lane were excised and destained using ammonium bicarbonate and acetonitrile. Gel pieces were saturated with trypsin buffer (13 ng/ul trypsin in 10 mM ammonium bicarbonate containing 10% acetonitrile by volume) and incubated at 55 1C for 30 min. Peptides were extracted using C18 ZipTips (Millipore, Billeria, MA) and the sequence derived from the mass spectra were compared to a database of Curly proteins by sequence query search in Mascot (Matrix Science, Boston, MA) (Perkins et al., 1999).

Acknowledgments The authors wish to acknowledge Dr. Ronald Raab and Dr. Stephanie Stockwell of James Madison University for their involvement in the Viral Discovery program, Dr. Mark Radosevich of University of Tennessee, Knoxville, for funding sequencing efforts, Dan Russell and Graham Hatfull of the University of Pittsburgh for sequencing, Virginia Commonwealth University for sequencing and electron microscopy, Dr. Dave Anderson of SRI International for mass spectrometry, Howard Hughes Medical Institute Science Education Alliance for initial support of the Viral Discovery program and to James Madison University′s Department of Integrated Science and Technology and Department of Biology for continuing support of the Viral Discovery program. The authors also wish to acknowledge phage hunters Brendan Cowcer, Mary Kathryn Dickinson, Drew Loso, Robert Procida, Blake Richardson, Madison Shinaberry, Nick Wright, Ali Blackman, Shannon Chambers, Lesley Chambers, Karen Corbett, Terri Corbett, Leo Duke, Lauren Gerson, Jessica Gyourko, Samantha Howell, Catherine Lopez, Matthew Mattson, Kent McQueen, Dave Motsek, Dorothy Nugent, Laurel Owens, and Ashley Parra, all of whom contributed to the isolation and characterization of the phages detailed in this work.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2013.07.004.

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