Characterization of Paenibacillus larvae bacteriophages and their genomic relationships to firmicute bacteriophages
© Merrill et al.; licensee BioMed Central Ltd. 2014
Received: 13 February 2014
Accepted: 26 August 2014
Published: 30 August 2014
Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.
P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively.
This paper represents the first comparison of phage genomes in the Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae.
KeywordsPaenibacillus larvae Bacteriophage Jimmer1 Jimmer2 Emery Abouo Davies phiIBB_Pl23 Phamerator Phage genomics
Paenibacillus larvae (P. larvae) is a sporulating Firmicute. It is the causative agent of American Foulbrood (AFB), a disease that infects and destroys the larvae of honeybees (Apis mellifera). The first eight P. larvae phages were reported between 1955 and 1999 and included BLA , L3 , BL2 , PBL1 , PBL0.5 , PBL2 , PBL3 , and PPL1c . These phages have not been sequenced. Most of these phages were isolated from lysogens and were used to characterize different strains of P. larvae [8–10]. Phages that infect P. larvae were originally identified as Bacillus larvae phages; however, the names of these phages were changed to Paenibacillus larvae phages following the reclassification of the bacteria [11, 12].
Recent advances in DNA sequencing technology have made it possible to sequence many bacteriophage genomes. When these sequences are analyzed, putative protein functions can be determined. Other studies have used comparative genomics to organize phages into related clusters , correlate phage packaging mechanisms with large terminase protein sequences , and study gene transfer, phylogenetic relationships, and impacts on host virulence [15, 16].
Comparative genomics can be accomplished using software specialized for phage genomes such as the computer program, Phamerator . Phamerator incorporates available data for each genome entered into its database, such as bacterial host, annotations of genes from GenBank, and conserved domains . Phamerator compares each gene product in the database to each other using BLASTP  and ClustalO , the scores of which are used to create phamilies (phams) of related gene products. Phamerator provides visual tools such as full genome comparison maps and can display the relationships between proteins within a pham using a circular diagram (pham circle). Proteins within each pham must meet or exceed user-defined cutoffs for E-values and percent identity for at least one other gene product in the pham. Strict cutoffs result in phams that indicate a shared similar function and predict phylogenetic relationships.
In 2013, six P. larvae phages were isolated. These phages were fully sequenced and their genomes published [21, 22]. P. larvae siphovirus phiIBB_Pl23 was isolated in Portugal  and P. larvae myoviruses Abouo, Davies, Emery, Jimmer1, and Jimmer2 were isolated in Utah . In this report we compare the genomes of the six fully-sequenced P. larvae phages, categorize all published P. larvae phages into three groups based on structural morphology, use Phamerator to analyze previously unexplored Paenibacillus phages, and explore genetic relationships of the P. larvae phages with 107 other phages that infect Firmicute hosts. We identify gene products with conserved domains including a putative bacteriocin, serine recombinase, and antirepressor, and investigate their conservation among P. larvae phages. Results from Phamerator and CoreGenes indicate a relationship between four P. larvae phages and four small genome myoviruses that infect Lactobacillus, Clostridium and Streptococcus. These results show that comparisons can be drawn between phages that infect a phylum and provide a basis for analyzing and comparing newly isolated phages that infect P. larvae.
Bacterial identification, phage isolation, and phage sequencing
Bacterial isolates were collected from spores found in honey samples. All characteristic tests for P. larvae were positive: the isolates grew on PLA plates and were catalase negative and Gram-positive. PCR products from 16S rRNA primers were sequenced using BigDye sequencing. BLAST results from nine of the ten 16S rRNA sequences showed more than 99% similarity with Paenibacillus larvae subsp. pulvifaciens strains DSM 8442 and DSM 8443 as well as the related bacteria Brevibacillus laterosporus. Of nine isolates, PL2 and PL6 were used for phage isolation. Phages Abouo, Davies, and Emery were isolated using PL6, while phages Jimmer1 and Jimmer2 were isolated using PL2.
Characteristics of sequenced Paenibacillus larvae bacteriophages
Host strain of P. larvae
Number of genes
Phage cross-infectivity, lysogeny, and lysogen superinfection
Plaques from the sample containing Emery and Abouo were clear while plaques from the other three phages were hazy. Phages Jimmer1 and Jimmer2 were isolated independently using PL2 and neither of these phages could infect PL6. Phages Davies and Emery/Abouo were isolated using PL6 and these phages could not infect PL2. None of these phages were able to produce plaques on lawns of B. cereus, B. subtilis, L. acidophilus, S. aureus, or S. epidermidis. Of the four phage samples, three formed stable lysogens: Jimmer1 in PL2, Jimmer2 in PL2, and Davies in PL6. Stable lysogens were identified when phages were not able to superinfect bacteria lysogenic for the same phage. The lysate containing Emery and Abouo induced lysis in the PL6 Davies lysogen. No other superinfection or induced lysis was observed in any other lysogen-phage combinations. The sample containing Emery and Abouo did not form a stable lysogen and no superinfection data were obtained.
Electron microscopy reveals myovirus structure for five P. larvaephages
Sequence similarities between phages
Distinguishing between phages Emery and Abouo
Comparison of Paenibacillus larvae phage structures from electron microscopy data
Capsid height (nm)
Capsid width (nm)
Tail length (nm)
Tail width (nm)
Contracted sheath length (nm)
Contracted sheath width (nm)
68.3 ± 5.6
65.1 ± 3.6
113.6 ± 2.6
12.7 ± 3.8
45.7 ± 5.0
24.0 ± 2.3
68.1 ± 4.5
61.7 ± 3.2
125.4 ± 0.2
9.8 ± 0.7
83.0 ± 0
19.2 ± 0
73.5 ± 3.2
66.0 ± 3.3
162.2 ± 5.2
11.0 ± 1.4
82.9 ± 0
25.5 ± 0
68.2 ± 3.6
61.3 ± 3.9
98.9 ± 14.7
11.9 ± 1.4
43.1 ± 1.5
23.3 ± 2.5
62.9 ± 2.5
66.3 ± 2.2
75.6 ± 8.7
7.7 ± 0.3
42.9 ± 2.4
20.6 ± 0.4
PBL0.5c* (and PBL2 *)
66.3 ± 3
66.3 ± 3
142.0 ± 1
9.2 ± 0.9
92.0 ± 1
22.5 ± 1.2
64.1 ± 1.1**
60.1 ± 3.9**
62.2 ± 2.2**
70 ± 2.1
140 ± 2.8
10.1 ± 0.9
PBL1* (and PPL1c *)
95.9 ± 2.6
52.5 ± 1.9
131.8 ± 1.5
9.4 ± 0.6
Measurements from electron micrographs
P. larvae phages were separated into three distinct groups based on structural morphology. The first group of phages were myoviruses (icosahedral capsids and contractile tails) and included phages Abouo, Davies, Emery, Jimmer1, Jimmer2, PBL0.5 , and PBL2 . There were two distinct groups of phages that were siphoviruses. The first siphovirus group contains phages BLA , PBL1 , and PPL1c . These phages had long, non-contractile tails and elongated capsids. The second siphovirus group contained only PBL3 which had a round capsid . Phage phiIBB_Pl23 was also a siphovirus , but could not be categorized into one of the two siphovirus groups because images or measurements were not yet available. There was no apparent correlation between the type of phage and where the phage was isolated. Measurements taken from all published electron micrographs of P. larvae phages are shown in Table 2. Measurements for phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 were taken from at least three different intact phages. Phages grouped into categories by morphology type were found to have similar structural measurements. The structures of the P. larvae myoviruses were similar in size with an average capsid height of 67.2 ± 3.2 nm and an average width of 64.1 ± 2.6 nm. The average tail length was 122.0 ± 27.3 nm and was the most variable of the measured phage features.
Frameshift in P. larvaephage Emery
Phage Emery exhibited a putative ribosomal slippage site in gp4 that encoded for a head morphogenesis protein in the SPP1 gp7 family. This frameshift was identified by the online frameshift finding tool FrameD . The two products in Emery are predicted to be 82.5 kDa following ribosomal slippage and 58.9 kDa if there is no slippage. The presence of both head morphogenesis proteins in the Emery virion has not yet been verified. We were unable to detect a putative frameshift via FrameD in Bacillus phage SPP1 or any protein sequence homology using BLASTP between the morphogenesis proteins in Emery and SPP1.
P. larvaephage genomic comparison using Phamerator software
A database of phage genomes related to the P. larvae phages was assembled for analysis using the phage genome comparison program Phamerator . The finished Phamerator database contained a diverse set of phages that infected Firmicute bacteria including the 6 P. larvae phage genomes, 71 Bacillus phages, 1 Clostridium phage, 3 Enterococcus phages, 2 Geobacillus phages, 7 Lactobacillus phages, 6 Listeria phages, one Paenibacillus glucanolyticus phage, 15 Staphylococcus phages, and 1 Streptococcus phage (Additional file 1). Phages included in the database were selected based on BLAST hits to gene products identified during annotation of the P. larvae phage genomes. Phamerator grouped the 13,697 putative proteins annotated in the 113 phage genomes into 6,181 phamilies (phams). Only 2,233 phams (36.1%) contained two or more members. These 2,233 phams contained 9,749 (71.1%) of the 13,697 putative proteins in the database. The remaining 3,948 (28.8%) putative proteins could not be grouped with other proteins and were designated as "orphams" .
Comparison of P. larvae phage genes
Genes in pham with non-PL phage
Genes only in PL phages
# of genes
# of genes with BLAST* hits (%)
# of genes with BLAST* functions (%)
# of genes with CD (%)
All P. larvae phages
Pham groupings reflected the genetic relationships of P. larvae phages. The genomic sequence comparison of Jimmer1 and Jimmer2 using ClustalW identified differences in 80 bp of the 54,312 bp genomes (99.85% similar) . All corresponding genes between Jimmer1 and Jimmer2 shared the same phams. Of the 80 P. larvae phage genes in Jimmer1 and Jimmer2, 31 were unique to these two phages and were not found in any other phages in the database. These 31 genes would be orphams if Jimmer2 were not isolated (Table 3).
Phamerator identified conserved domains in at least one gene in 1,501 phams (24.3%) of the total 6,181 phams in the database. Although many P. larvae phage genes encoded proteins with significant BLAST hits, less than half of the proteins had a known function. Of all P. larvae phage putative proteins, 86% had a BLAST hit with an E-value less than 1 × 10-4 (see Table 3), yet only 48% of the proteins returned BLAST hits listing a function. Conserved domains were identified in only 43% of the P. larvae phage putative proteins (Table 3). Phages Emery and phiIBB_Pl23 contained the most orphams, the fewest BLAST hits, and the most putative proteins with no identifiable conserved domains.
P. larvaephages share structural and regulatory genes with similar functions
P. larvae phage assembly proteins and structural proteins; gene product # and (pham #, specific to this analysis)
Jimmer1 and 2
Terminase small subunit
Terminase large subunit
Phage portal protein
SPP1 Gp7 family head morphogenesis protein
gp4 and 5 (5611)
Phage virion morphogenesis family protein
Phage minor structural GP20 family protein
Prohead core scaffolding/protease
Phage major capsid protein E
DNA packaging protein
Head-tail joining protein
gp7 (5912), gp8 (2096)
Phage tail sheath protein
Tail length tape measure protein
Baseplate J family protein
gp30 (30), gp31 (31)
gp30 (30), gp31 (31)
gp12 (5618), gp18 (5624)
gp10 (5914), gp11 (5915), gp15 (5918), gp17 (5920)
Regulatory proteins and other non-structural proteins; gene product # and (pham #, specific to this analysis)
Jimmer1 and Jimmer2
LysM domain-containing protein (peptidoglycan binding)
Cell wall hydrolase/late control D
Subtilisin-like serine protease
Phage-like element PBSX protein
gp26 (26), gp28 (28)
gp26 (26), gp28 (28)
gp28 (26), gp30 (28)
gp35 (35), gp44 (44)
gp35 (35), gp44 and gp45 (44)
gp34 (5637), gp53 and gp54 (44)
gp41 (41), gp68 (71)
gp41 (41), gp72 (71)
gp19 (951), gp79 (71)
Tyrosine Recombinase XerC
gp1 (5608), gp47 (43)
Accessory gene regulator B family protein
AbrB family transcriptional regulator
RuvC Holliday junction resolvase
ArpU family transcriptional regulator
ABC transporter-like protein
Arc-like DNA binding domain
gp62 and gp63 (53)
Single-stranded DNA-binding protein
gp62 (65), gp63 (66)
gp66 (65), gp67 (66)
gp73 (65), gp74 (66)
SOS-response repressor and protease LexA
Recombinational DNA repair protein RecT
Phage replication protein O
DNA replication protein
Site-specific DNA methylase
RNA polymerase sigma-70 factor
DNA-dependent DNA polymerase family A
Virulence-associated E family protein
Stage V sporulation protein K
VRR-NUC domain-containing protein
Transcriptional regulator (HTH, XRE)
gp49 (50), gp50 (51), gp51 (52), gp54 (57), gp56 (59)
gp51 (50), gp52 (51), gp53 (52), gp56 (55), gp57 (56), gp58 (57), gp60 (59)
gp52 and gp56 (50), gp57 (51), gp58 (52), gp67 (59), gp18 (3031), gp17 (3030), gp65 (3057), gp60 (3054), gp61 (3055)
gp60 (5656), gp89 (5678), gp40 (2605), gp65 (2691), gp64 (2692), gp48 (5649), gp59 (5655), gp49 (5650)
gp32 (5935), gp55 (5957)
Most of the functions listed in Tables 4 and 5 describe proteins found in more than one phage. For example, all five of the P. larvae myoviruses contained seven proteins that belonged to the same family or superfamily but not always to the same pham. The function of these proteins includes head morphogenesis, tape measure, baseplate (see Table 4), LysM peptidoglycan binding, peptidoglycan hydrolase, PBSX, and bacteriocin (see Table 5).
Few proteins with known functions were identified as putative virulence factors. BLAST results indicate that gp26 in P. larvae phage phiIBB_Pl23 is a protein that is toxic to insect larvae. No toxin genes were identified in the P. larvae myoviruses. Other host-related proteins include an ABC transporter-like protein found in P. larvae phages Abouo, Davies, Jimmer1, Jimmer2, and phiIBB_Pl23 as well as an XRE-family transcriptional regulator found in all P. larvae phages. The five myoviruses contained between five and ten of these regulators per genome compared to only two in the siphovirus phiIBB_Pl23. Abouo gp51, Jimmer1 gp58, Jimmer2 gp58 and Emery gp40, gp64, and gp65 (Table 5) are the only transcriptional regulators that share a pham with a non-P. larvae phage. All others are only found in P. larvae phages. It is not known what effects these transcriptional regulators have on the host, but they do contain a canonical helix-turn-helix (HTH) domain. Very few of the regulatory genes in these phages have known functions.
Phage genome organization and pham groupings indicate relatedness of four P. larvaephages
Phamily relationships of large terminase proteins indicate putative DNA packaging strategies
P. larvaephages exhibit genetic and structural similarity with other small genome myoviruses.
Genetic comparison of small genome myovirus phams and gene products within each pham
Phages Abouo, Davies
Phages Jimmer1, Jimmer2
Clostridium phage PhiC2
Lactobacillus phage KC5a
Streptococcus phage EJ-1
Terminase (small subunit)
Head morphogenesis protein
Minor structural protein
Tail sheath protein
Baseplate J family protein
XRE family transcriptional regulator
Arc-like DNA-binding protein
The electron micrographs of P. larvae phages Abouo, Davies, Jimmer1, and Jimmer2 reveal a structure similar to those reported for Clostridium phages phiC2 , phiCD27 , and phiCD119 , Lactobacillus phages KC5a  which is reported as similar to Lactobacillus phages KC21T  and phiAQ113 , and Streptococcus phage EJ-1 . The capsid of Clostridium phage phiC2 is 65 nm in diameter and the tail is 148 nm long. The capsid of Streptococcus phage EJ-1 is 57 nm in diameter and the tail is 130 nm long. The capsid of Lactobacillus phage KC5a was not measured, however it was reported as similar to KC21T, which had a capsid diameter of 45 nm and a tail length of 160 nm. KC5a was also reported to be genetically similar to phiAQ113, which has a capsid diameter of 55 nm, a tail length of 147 nm, and a tail sheath width of 22 nm. Phamerator grouped all KC5a gene products listed in Table 6 into the same phams as phiAQ113 genes except the small terminase (absent in phiAQ113) and the head morphogenesis protein.
Although P. larvae phage Emery contained gene products with the same functions as those listed in Table 6, the proteins were all orphams. However, the first five gene products encoded in P. larvae phage phiIBB_Pl23 (small terminase, large terminase, portal protein, protease, and major capsid proteins) all shared a pham with similar proteins from five siphovirus Staphylococcus phages (3A, 47, phi12, phiSLT, and tp310-2).
CoreGenes analysis indicates relationships among small genome myoviruses
Reference genome (total # of gene products)
phiCD119 (53325 bp)
phiC2 (56,538 bp)
EJ-1 (42935 bp)
KC5a (38239 bp)
AQ113 (36566 bp)
Abouo (45552 bp)
Davies (58572 bp)
Jimmer1/2 (54312 bp)
phiCD119 (79 gp)
phiC2 (82 gp)
phiCD27 (75 gp)
EJ-1 (73 gp)
KC5a (61 gp)
AQ113 (56 gp)
Abouo (94 gp)
Davies (94 gp)
Jimmer1/2 (102 gp)
Emery (102 gp)
Only one pham includes all five myoviruses, and very few phams are shared between unrelated P. larvaephages
Phages Emery and phiIBB_Pl23 are significantly different from each other and from the four similar P. larvae myoviruses, as is evident from the genome maps in Figure 3. However, Tables 4 and 5 demonstrate that some proteins encoded by these phages grouped into similar phams.
Pham 34 is the only pham in the database that included proteins from all five of the new myovirus P. larvae phages. These gene products are Abouo gp34, Davies gp34, Emery gp29, Jimmer1 gp36, and Jimmer2 gp36 and encode a bhlA/bacteriocin protein (Figure 3). No other gene products in the Phamerator database were grouped into this pham. The conserved domain in these proteins was DUF2762, a putative holin-like protein. When comparing amino acid sequences, these five proteins shared > 87% identity and an E-value less than 5 × 10-43.
The siphovirus Paenibacillus glucanolyticus phage PG1 also contained three gene products that shared a pham with P. larvae phages Abouo, Davies, Jimmer1, and Jimmer2. Pham 90 encoded an RNA polymerase sigma-70 factor and had 5 members: PG1 gp62, Abouo gp91, Davies gp91, Jimmer1 gp99, Jimmer2 gp99. Pham 75 encoded a recombination protein U and had 9 members, including PG1 gp42, Abouo gp73, Davies gp76, Jimmer1 gp83, and Jimmer2 gp83. Pham 78 encoded a dUTPase and had 13 members, including PG1 gp43, Abouo gp76, Davies gp79, Jimmer1 gp86, and Jimmer2 gp86.
Four P. larvaephages contain duplicated genes
Phages Abouo and phiIBB_Pl23 did not contain any proteins that belonged to the same pham. However, gp52 and gp56 in phages Jimmer1 and Jimmer2 shared 52.1% identity (E-value is 1.39 × 10-36), belonged to pham 50, and encode an XRE family transcriptional regulator that contains a helix-turn-helix DNA binding domain. Additionally, gp62 and gp63 in phages Jimmer1 and Jimmer2 shared 40.3% amino acid identity (E-value is 8.32 × 10-11), belonged to pham 53, and contain an arc-like DNA-binding domain.
Prior to this report, nine P. larvae phages were described but were never analyzed collectively or grouped based on similar characteristics. Structural and morphological characteristics are the only published information for grouping the reported P. larvae phages to date. Therefore, for general comparison, P. larvae phages were identified as myovirus, elongated-capsid siphovirus, round-capsid siphovirus, or unknown siphovirus. The five P. larvae myoviruses characterized in this paper are structurally similar to previously isolated P. larvae myoviruses and may also be genetically similar. Since few phages infecting P. larvae have been sequenced, it is useful to compare structural similarity observed in electron micrographs. Now that sequencing data has been published for six P. larvae phage genomes and sequencing of others is sure to follow, genomic grouping will prevail and clusters will likely emerge as occurred with the mycobacteriophages .
The five myoviruses were isolated from three soil samples each from a separate location: Jimmer1 and Jimmer2 were isolated independently from the same sample , Emery and Abouo were isolated together, and Davies was isolated separately. P. larvae phage PBL2 was isolated from a different sample than BL2, yet all tests indicated no obvious structural or genetic differences between these phages . Similar host properties and selective pressures can result in isolation of similar phages from different locations . More P. larvae siphoviruses need to be sequenced before further correlations between genome and structural morphology can be drawn. As demonstrated in this work and by others, the sequence of the tape measure protein gene may be used to identify individual phages being studied if co-isolation occurs again in the future .
Bacteriophages are often unable to superinfect an existing lysogen if the entering and lysogenic phages are genetically similar . The portion of the genome responsible for superinfection immunity has been determined for some phages . Repressor genes involved in superinfection immunity have been characterized and are known to defend the prophage from premature lysis by silencing genes related to lysis . This system does not work against phages that are always lytic or temperate phages that are not sensitive to the prophage repressor genes. Lysogens of Jimmer1, Jimmer2, and Davies displayed superinfection immunity when incubated with the same phage. Jimmer1 and Jimmer2 exhibited nearly identical sequences and were also immune to superinfections of each other.
The host specificity and correlating genome similarity between Jimmer1 and Jimmer2 (infect PL2) and Davies and Abouo (infect PL6) reflect common evolutionary ancestry. The high degree of similarity (over 80% average nucleotide identity) between the four phages may indicate that these phages infect a common host that has not yet been isolated or tested or that two phages recently switched hosts as is common in phages . Jimmer1, Jimmer2, Davies, and Abouo likely coevolved.
Many bacteriophages contain genes that affect the virulence of the bacterial hosts. One toxin gene has been identified in phiIBB_Pl23 (gp26), and no toxin genes have been identified in the five P. larvae myoviruses. P. larvae phages Abouo, Davies, Jimmer1, Jimmer2, and phiIBB_Pl23 encode an ABC transporter-like protein. This was characterized as an extracellular protein produced by P. larvae , but it is not known how this protein is involved in host virulence. Future experiments involving the many putative XRE transcriptional regulators encoded by these phages may show a correlation with the virulence of P. larvae. Most of the transcriptional regulators found in the six P. larvae phages do not share phams with phages that infect any other bacterial host, indicating that these regulators are both phage- and host-specific. Two of these transcriptional regulators were duplicated in Jimmer1 and Jimmer2. The differences between these genes indicate that they are ohnologous and arose by gene duplication and subsequent divergence . The duplicated genes in Emery and Davies are putative membrane proteins and likely evolved in a similar fashion. Abouo contains only one copy of this gene (Figure 6). BLAST hits for all six of the sequenced P. larvae phages show similarity to many proteins encoded by Paenibacillus and Brevibacillus bacteria. BLAST hits to these bacteria are not surprising because the genera Paenibacillus and Brevibacillus both belong to the family Paenibacillaceae and are closely related .
Analysis of large terminase protein phamilies revealed that Abouo, Davies, Jimmer1, and Jimmer2 likely use the headful packaging mechanism, while phiIBB_Pl23 likely has 3′ cohesive ends. Because of the stringent cutoff values required for inclusion in a pham, these results identify one way experimentally determined properties of a protein can be inferred on others sharing the same phamily.
Several gene products in P. larvae phages have similar functions but do not share phamilies. These include head morphogenesis, tape measure, baseplate, LysM peptidoglycan binding, peptidoglycan hydrolase, PBSX, and bacteriocin proteins. The conserved genes either diverged a long time ago or were acquired via convergent evolution. Additionally, the antirepressor protein in P. larvae phages phiIBB_Pl23, Jimmer1, Jimmer2 shares a pham with antirepressors from 13 other myoviruses and siphoviruses that infect host bacteria in the genera Bacillus, Enterococcus, Geobacillus, Lactobacillus, Listeria, Paenibacillus, and Staphylococcus (Figure 5). The presence of a similar antirepressor among phages of diverse Firmicute hosts may indicate the usefulness of the gene products and their associated conserved domains to regulate production of phage proteins within a diverse set of host bacteria. These data indicate that P. larvae phages have been subjected to multiple evolutionary pressures.
The head morphogenesis protein in phage Emery belongs to the SPP1 gp7 family and contains a ribosomal slippage site that is not found in Bacillus phage SPP1. Although two gene products are produced by the head morphogenesis gene in SPP1 that are 34 kDa and 28 kDa (compared to predicted proteins of 82.5 kDa and 58.9 kDa in Emery), the two SPP1 proteins are thought to be due to an alternative start site, not a frameshift caused by ribosomal slippage . The lack of homology between protein sequences indicates these proteins further illustrates that Emery is not closely related to any other known bacteriophages.
Most of the putative encoded proteins in the P. larvae phages are not grouped into phams containing proteins from other phage types. These data indicate that most P. larvae phage genes are novel among currently identified genes of phages or bacteria. More than half of the P. larvae phage proteins have no identified conserved domains or putative functions, illustrating the diversity of bacteriophages and the vast number of unknown genes yet to be explored.
CoreGenes was previously used to verify current taxonomic relationships between phages in the Podoviridae  and Myoviridae families . It was also used analyze other "dwarf" myoviruses and group them based on the similarity of core genes . Analysis of core genes and shared phams indicates that P. larvae phages Abouo, Davies, Jimmer1, and Jimmer2 are distantly related to phages in the phiCD119likevirus family as well as phages EJ-1, KC5a, and AQ113. Because proteins grouped into similar phams are phylogenetically related, these proteins likely share a common ancestry. The structural similarities between phages Abouo, Davies, Jimmer1, Jimmer2, phiC2, KC5a, AQ113, and EJ-1 may correlate with their genetic similarities because the conserved core genes include the structural module of each genome. However, the current accepted threshold of 40% for a sufficiently strong CoreGenes percentage prevents any of these phages from being grouped taxonomically (except perhaps KC5a and AQ113, which is not within the scope of this paper). The differences in genome lengths may also prevent the formation of a taxonomic family of these phages as CoreGenes reflects the percentage based on the number of genes, which means that genome length differences and subsequent differences in total gene numbers within a genome can influence the score.
The results of the CoreGenes analysis indicate that P. larvae phages Abouo, Davies, Jimmer1, and Jimmer2 are related phylogenetically. They are also distantly related to phiC2, KC5a, AQ113, and EJ-1 which infect other bacterial hosts. This relationship indicates that these four phages are the closest known phylogenetic relatives to these four P. larvae phages. The conservation of primarily structural genes among the eight small genome myoviruses may indicate that the phages adapted to maintain infectivity as their bacterial hosts diverged, but retained ancestral structural genes that were under less selective pressure.
Conserved genes between different phages may indicate important genes. The bacterial hosts PL2 and PL6 are similar (according to the 16S rRNA sequences and physical properties), and similar BhlA/bacteriocin genes such as found in the shared pham of Jimmer1, Jimmer2, Emery, Abouo, and Davies (pham 34) can likely be used to lyse the bacterial host. It is interesting to note that the only two genes shared between phiIBB_Pl23 and any other P. larvae phage encode a serine recombinase and an antirepressor, shared with the PL2 phages Jimmer1 and Jimmer2. This correlation may indicate similar host interactions, as these genes help regulate the lytic and lysogenic cycles. The PL6 phages do not contain any antirepressor gene products belonging to this pham. Although P. larvae phages Emery and phiIBB_Pl23 do not show significant genetic relatedness to any other sequenced phages, similar genes and phages will likely be discovered in the future. The six newly sequenced genomes of the P. larvae phages compared in this report are an initial foundation for future studies.
This first comparison of P. larvae phage genomes provides insight into the genus Paenibacillus and the important honeybee bacterial pathogen, P. larvae. Although six P. larvae phages show some relatedness to phages that infect other Firmicute bacteria, most P. larvae phage genes do not share phams with non-P. larvae phages and many gene products still have unknown functions. Efforts to characterize these gene products and to isolate, sequence, and analyze new P. larvae phages will help us better understand the genetics of these phages and their bacterial host.
Identification of field isolates
Paenibacillus larvae spores were extracted from local honey samples using the process described by Hornitzky . Pelleted spores were streaked on PLA  plates that contained nalidixic and pipemidic acid and plates were incubated for 48–72 hours at 37°C. Colonies were streaked to purity on PLA plates. Isolates were tested with hydrogen peroxide for the presence of the catalase enzyme  and were tested by gram stain [45, 51].
A single colony from each bacterial field isolate was boiled at 98°C for five minutes, and 3 μL of the lysate was used as a PCR template. The 16S rRNA gene region was amplified using universal primers 27 F and 907R , and the standard protocol for Taq DNA polymerase (New England Biolabs). Following PCR, amplicon size was checked by agarose gel electrophoresis. Samples producing a ~1 kb band were submitted for BigDye (Applied Biosystems, Life Technologies) sequencing to the BYU DNA Sequencing Center. Resulting 16S sequences were analyzed using BLAST .
Superinfection of P. larvaelysogens with phage
Phages described by Sheflo et al.  were used in lysogenic superinfection studies using a protocol adapted from . Some agar was removed from the center of an isolated plaque, streaked out on an LB plate, and incubated at 37°C for 24 hours to allow any lysogens to grow. One colony was removed, incubated at 37°C in 1 ml of LB broth for two hours, and then plated using the method described above. When the top agar was solid, 5 μL of each phage lysate was placed on the plate. The plate was incubated agar side down at 37°C for 24 hours. Clearing under the spots indicated superinfection had occurred, while no clearing indicated that the lysogenic bacteria were immune to superinfection.
Electron microscopy of P. larvaephages
Electron microscopy was performed at Brigham Young University in the Life Sciences Microscopy Lab using an FEI Tecnai 12 Spirit transmission electron microscope. To prepare the samples for imaging, 20 μl of high-titer phage lysate was placed on a 200-mesh copper carbon type-B electron microscope grid for one minute. The lysate was wicked away and the grids were stained for two minutes using 2% phosphotungstic acid (pH = 7). Residual liquid was wicked away and the grid was allowed to dry before being imaged. Phage structures in electron micrographs were measured using ImageJ . The average and standard deviation for each measurement was calculated from a minimum of three separate measurements.
Genomic comparison of sequenced phages
The DNA sequences for the six sequenced P. larvae phages were downloaded from GenBank using reported accession numbers [21, 22]. Dotplots of nucleic acid and protein sequences were generated using Gepard  and then compared. ClustalW  was used to calculate Average Nucleotide Identity (ANI) percentages comparing each of the P. larvae phage genomes. The online tool FrameD  was used to search for frameshift mutations. Core genes were identified using the program CoreGenes 3.5 [57, 58] with the default BLASTP threshold of 75.
Phages genes were analyzed using Phamerator , an open-source program (GNU general public license) designed to compare phage genes and genomes. For this study, Phamerator was adapted and stored in a GitHub repository (http://github.com/byuphamerator/phamerator-dev) separate from the original version. Phamerator uses BLASTP  and ClustalO  to compare each protein encoded by the genes in the database. E-values and percent identity scores are used to sort proteins into groups referred to as phamilies (phams) based on user-defined cutoffs for each score. Conserved domains in each protein are then identified. The Phamerator database used in this study was populated with 71 Bacillus phages, one Clostridium phage, 3 Enterococcus phages, 2 Geobacillus phages, 7 Lactobacillus phages, 6 Listeria phages, 6 P. larvae phages, one Paenibacillus glucanolyticus phage, 15 Staphylococcus phages, and one Streptococcus phage. The non-Bacillus phages were included in the database because proteins from these phages appeared in low E-value (<0.0001) BLAST hits for P. larvae phage proteins. In this Phamerator database, genes with E-values smaller than 1 × 10-50 or greater than 32.5% identity with at least one other protein were grouped into phams. These parameters were identical to those used by Cresawn et al. . Conserved domains in proteins were identified using RPS-BLAST  to search the Conserved Domain Database (CDD) released by NCBI on 21 March 2013 . The 100 published phages in the Phamerator database are listed below with the respective bacterial hosts and accession numbers. The Bacillus_Draft database can be accessed through Phamerator (see http://phagesdb.org/Phamerator/faq/). The Phamerator database for this study (bphage5) is available at http://phagehunters.byu.edu/BeeProject.aspx.
Bacillus phages vB_BceM_Bc431v3 (B. cereus) [NC_020873], 0305phi8-36 (B. thuringiensis) [NC_009760], 250 (B. cereus) [GU229986], Andromeda (B. pumilus) [KC330684], AP50 (B. anthracis) [NC_011523], B103 (B. subtilis) [NC_004165], B4 (B. cereus) [JN790865], B5S (B. cereus) [JN797796], Bam35c (B. thuringiensis) [NC_005258], Basilisk (B. cereus) [KC595511], Bastille (B. cereus) [NC_018856], BCD7 (B. cereus) [NC_019515], BceA1 (B. cereus) [HE614282], BCJA1c (B. clarkii) [NC_006557], BCP78 (B. cereus) [NC_018860], BCU4 (B. cereus) [JN797798], BMBtp2 (B. thuringiensis) [JX887877], BPS13 (B. cereus) [NC_018857], BtCS33 (B. thuringiensis) [NC_018085], CAM003 (B. thuringiensis) [NC_024216.1], Cherry (B. anthracis) [NC_007457], Curly (B. pumilus) [KC330679], Eoghan (B. pumilus) [KC330680], Evoli, (B. thuringiensis) [NC_024207.1], Fah (B. anthracis) [NC_007814], Finn (B. pumilus) [KC330683], G (B. megaterium) [JN638751], GA-1 (Bacillus sp.) [NC_002649], Gir1 (Bacillus sp.) [Bacillus_Draft], Gamma 51 (B. cereus) [DQ222853], Gamma 53 (B. anthracis) [DQ222855], Gamma isolate d’Herelle (B. cereus) [DQ289556], Gemini (B. pumilus) [KC330681], GIL16c (B. thuringiensis) [NC_006945], Hakuna (B. thuringiensis) [NC_024213.1], Hoody T (B. thuringiensis) [NC_024205], IEBH (B. thuringiensis) [NC_011167], JL (B. cereus) [KC595512], JPB9 (B. thuringiensis) [Bacillus_Draft], Megatron (B thuringiensis) [NC_024211.1], MG-B1 (B. weihenstephanensis) [NC_021336], Nf (B. subtilis) [EU622808], Pappano (B. pumilus) [Bacillus_Draft], PBC1 (B. cereus) [NC_017976], Pegasus (Bacillus sp.) [bacillus.phagesdb.org], pGIL01 (B. thuringiensis) [AJ536073], phi105 (B. subtilis) [NC_004167], phi29 (B. Subtilis) [NC_011048], phiAGATE (B. pumilus) [JX238501], phiNIT1 (B. subtilis) [NC_021856], phIS3501 (B. thuringiensis) [NC_019502], Pleiades (B. pumilus) [Bacillus_Draft], PM1 (B. subtilis) [NC_020883], Polaris (B. pumilus) [Bacillus_Draft], PZA (B. subtilis) [M11813], Riley (B. thuringiensis) [KJ489402], Shanette (B. cereus) [KC595513], SP10 (B. subtilis) [NC_019487], SPbeta (SPBc2) (B. subtilis) [NC_001884], SPO1 (B. subtilis) [NC_011421], SPP1 (B. Subtilis) [NC_004166], Stitch (Bacillus sp.) [Bacillus_Draft], Taylor (B. pumilus) [KC330682], TP21-L (B. cereus) [NC_011645], Troll (B. thuringiensis) [KF208639], W.Ph. (B. cereus) [NC_016563], WBeta (B. cereus) [NC_007734], Wip1 (B. anthracis) [KF188458], phBC6A51 (B. cereus) [NC_004820], phBC6A52 (B. cereus) [NC_004821], Bacillus virus 1 (Bacillus. sp. 6 k512) [NC_009737]; Clostridium phage phiC2 (C. difficile) [NC_009231]; Enterococcus phages phiEf11 (E. faecalis) [NC_013696], phiEF24C (E. faecalis) [NC_009904], phiFL3A (E. faecalis) [NC_013648]; Geobacillus phages GBSV1 (Geobacillus sp. 6 k51) [NC_008376], E2 (Geobacillus) [NC_009552]; Lactobacillus phages A2 (L. casei) [NC_004112], KC5a (L. gasseri) [NC_007924], Lb338-1 (L. paracasei) [NC_012530], Lc-Nu (L. rhamnosus) [NC_007501], LP65 (L. plantarum) [NC_006565], phiAT3 (L. casei) [NC_005893], phig1e (Lactobacillus) [NC_004305]; Listeria phages P100 (L. monocytogenes) [DQ004855], A118 (L. monocytogenes) [NC_003216], A500 (L. monocytogenes) [NC_009810], A511 (L. monocytogenes) [NC_009811], B054 (L. monocytogenes) [NC_009813], P40 (L. monocytogenes) [NC_011308]; Paenibacillus phages Abouo (P. larvae) [KC595517], Davies (P. larvae) [KC595518], Emery (P. larvae) [KC595516], Jimmer1 (P. larvae) [KC595515], Jimmer2 (P. larvae) [KC595514], PG1(P. glucanolyticus) [HQ332138], phiIBB_Pl23 (P. larvae) [KF010834]; Staphylococcus phages phi 12 (S. aureus) [AF424782], 37 (S. aureus) [NC_007055], 3A (S. aureus) [NC_007053], 47 (S. aureus) [NC_007054], 55 (S. aureus) [NC_007060], 88 (S. aureus) [NC_007063], G1 (S. aureus) [NC_007066], K (S. aureus) [NC_005880], PH15 (S. epidermidis) [NC_008723], phiSLT (S. aureus) [NC_002661], SA11-v (S. aureus) [NC_019511], SpaA1 (S. pasteuri) [NC_018277], Twort (S. aureus) [NC_007021], X2 (S. aureus) [NC_007065], tp310-2 (S. aureus) [NC_009762]; Streptococcus prophage EJ-1 (S. pneumoniae) [NC_005294].The neighbor-joining phylogenetic tree (Figure 4) was generated using ClustalO. The phage large terminase proteins included in this tree are listed below with their respective accession numbers.
Bacillus phages 0305phi8-36 gp117 [YP_001429607.1], 250 gp5 [ADB28373.1], Andromeda gp9 [YP_007517474.1], B4 gp4 [YP_006908233.1], B5S gp4 [AEW47238.1], Basilisk gp3 [AGR46580.1], Bastille gp267 [AEQ34197.1], BCD7 gp104 [YP_007005955.1], BceA1 gp4 [CCE73839.1], BCJA1c gp34 [YP_164412.1], BCP78 gp12 [YP_006907847.1], BCU4 gp5 [AEW47511.1], BMBtp2 gp40 [YP_007236398.1], BPS13 gp6 [YP_006907565.1], BtCS33 gp2 [YP_006488672.1], Cherry gp2 [YP_338134.1], Curly gp9 [YP_007517553.1], Eoghan gp9 [YP_007517399.1], Fah gp2 [YP_512312.1], Finn gp9 [YP_007517630.1], G gp1 [YP_009015312.1], Gamma51 gp2 [ABA46445.1], Gamma isolate d’Herelle gp2 [ABC40454.1], Gemini gp9 [AGE60848.1], IEBH gp49 [YP_002154374.1], JL gp4 [AGR46722.1], PBC1 gp2 [YP_006383455.1], phBC6A52 gp2583 [NP_852588.1], phiAGATE gp196 [YP_007349220.1], phiNIT1 gp79 [YP_008318309.1], phIS3501 gp23 [YP_007004362.1], Shanette gp4 [AGR46934.1], SP10 gp119 [YP_007003376.1], SPO1 gp2.11 [YP_002300330.1], Taylor gp9 [AGE60927.1], Troll gp61 [YP_008430845.1], vB_BceM_Bc431v3 gp10 [YP_007676908.1], W.Ph. gp3 [YP_004957018.1], WBeta gp2 [YP_459966.1], and e virus 1 gp18 [YP_001425601.1]; Clostridium phage phiC2 gp2 [YP_001110720.1], Enterococcus phages phiEf11 gp2 [YP_003358792.1], phiEF24C gp5 [YP_001504114.1], and phiFL3A gp43 [YP_003347605.1]; Geobacillus phages GBSV1 gp17 [YP_764473.1] and E2 gp2 [YP_001285808.1]; Lactobacillus phages A2 gp2 [NP_680484.1], KC5a gp35 [YP_529870.1], Lb338-1 gp89 [YP_002790768.1], Lc-Nu gp2 [YP_358760.1], LP65 gp113 [YP_164748.1], phiAT3 gp2 [YP_025027.1], and phig1e gp37 [NP_695170.1]; Listeria phages A118 gp2 [NP_463463.1], A500 gp2 [YP_001468388.1], A511 gp5 [YP_001468454.1], B054 gp2 [YP_001468706.1], P100 gp5 [AAY53308.1], and P40 gp2 [YP_002261418.1]; Paenibacillus phages Abouo gp2 [AGR47449.1], Davies gp2 [YP_008858637.1], Emery gp3 [AGR47349.1], Jimmer1 gp2 [AGR47249.1], Jimmer2 gp2 [AGR47149.1], and phiIBB_Pl23 gp2 [YP_008320338.1]; Staphylococcus phages PH15 gp2 [YP_950664.1], phi 12 gp30 [NP_803336.1], phiSLT gp39 [BAB21732.1], SpaA1 gp4 [YP_006560692.1], and tp310-2 gp45 [ABS87507.1]; Streptococcus phage EJ-1 gp39 [NP_945278.1]; and Mycobacterium phage Nigel gp4 [YP_002003843.1].
Availability of supporting data
The data set supporting the results of this article is included within the article (and its Additional files). The same files are also available at http://phagehunters.byu.edu/BeeProject.aspx.
The authors thank Dr. Steven Cresawn for his assistance in setting up the Phamerator database and the support of Byron Doyle at Brigham Young University for adaptation of the computer code to run Phamerator on our local computers. We appreciate the knowledgeable advice of Dr. Graham Hatfull in preparing this manuscript. We appreciate the assistance of Michael Standing from the BYU Microscopy Lab and Dr. Edward Wilcox from the BYU DNA Sequencing Center. We are grateful for the help of student researchers Parker A. Ayer, Alexander P. Beckstead, Christopher P. Fajardo, Nicholas C. Ferguson, Joshua N. B. Fisher, Adam V. Gardner, Kiel A. Graves, Kaitlin A. Hartmann, Johnathan E. Liu, Bryce L. Lunt, Ashley K. Kennedy, Curtis A. Merrill, Elizabeth Newey, Robert C. Russell, Jonathan E. Schoenhals, Michael A. Sheflo, Braden N. Wake, Kurt R. Williams, and Lauren J. Zimmerman.
This project was funded by the Department of Microbiology and Molecular Biology at Brigham Young University (BYU), a student grant from the BYU Office of Research and Creative Activities, and a BYU Technology Transfer grant.
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