BACKGROUND
paenibacillus larvae  is a sporulating firmicute.
it is the causative agent of american foulbrood , a disease that infects and destroys the larvae of honeybees .
the first eight p. larvae phages were reported between  <dig> and  <dig> and included bla
<cit> , l3
<cit> , bl2
<cit> , pbl1
<cit> , pbl <dig>
<cit> , pbl2
<cit> , pbl3
<cit> , and ppl1c
<cit> .
these phages have not been sequenced.
most of these phages were isolated from lysogens and were used to characterize different strains of p. larvae
.
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
<cit> .
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
<cit> , correlate phage packaging mechanisms with large terminase protein sequences
<cit> , and study gene transfer, phylogenetic relationships, and impacts on host virulence
<cit> .
comparative genomics can be accomplished using software specialized for phage genomes such as the computer program, phamerator
<cit> .
phamerator incorporates available data for each genome entered into its database, such as bacterial host, annotations of genes from genbank, and conserved domains
<cit> .
phamerator compares each gene product in the database to each other using blastp
<cit>  and clustalo
<cit> , the scores of which are used to create phamilies  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 .
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  <dig>  six p. larvae phages were isolated.
these phages were fully sequenced and their genomes published
<cit> .
p. larvae siphovirus phiibb_pl <dig> was isolated in portugal
<cit>  and p. larvae myoviruses abouo, davies, emery, jimmer <dig>  and jimmer <dig> were isolated in utah
<cit> .
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  <dig> 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.
RESULTS
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  <dig> and dsm  <dig> as well as the related bacteria brevibacillus laterosporus.
of nine isolates, pl <dig> and pl <dig> were used for phage isolation.
phages abouo, davies, and emery were isolated using pl <dig>  while phages jimmer <dig> and jimmer <dig> were isolated using pl <dig>
each p. larvae phage sample was plaque-purified at least three times, sequenced, and published
<cit> .
prior to genome sequencing and electron microscopy we were unaware that one phage sample still contained two different phages.
plaque purification did not successfully separate these two phages.
however, assembly of the genomes revealed two clearly independent genomes that separated with ease with over 100-fold coverage of the genomes.
these co-isolated phages were named emery and abouo.
results from sequencing and annotation of the six p. larvae phages are found in table
<dig>  the genomes varied in length from ~40 kb to ~58 kb.
most of the genes in each genome were located on the forward strand .
the average g + c content for these phages is  <dig> % ±  <dig> %.
blast hits for proteins within these phages included both paenibacillus and brevibacillus bacterial strains as well as bacteriophages that infected other firmicutes.table  <dig>
characteristics of sequenced
paenibacillus larvae
bacteriophages
abouo
davies
emery
jimmer1
jimmer2
phiibb_pl <dig> *
*paenibacillus phage phiibb_pl <dig> data from
<cit> .
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 jimmer <dig> and jimmer <dig> were isolated independently using pl <dig> and neither of these phages could infect pl <dig>  phages davies and emery/abouo were isolated using pl <dig> and these phages could not infect pl <dig>  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: jimmer <dig> in pl <dig>  jimmer <dig> in pl <dig>  and davies in pl <dig>  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 pl <dig> 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
electron microscopy revealed that these five p. larvae phages were myoviruses, marked by the presence of a contractile tail sheath .
figure
1a shows tail structures separated from the phage capsids.
these tail structures were more abundant than intact phages in all samples submitted for electron microscopy.
figure
1b,
1c,
1e, and
1f show intact phages with contracted tail sheaths, while figure
1d shows an extended tail sheath.
because phages emery and abouo were not separated, micrographs for these phages were taken from the same copper grid.figure  <dig>
electron micrographs of
p. larvae
phages.
a) tails and tail sheaths of p. larvae phages separated from the capsids .
these structures were more abundant than intact phages in electron micrographs.
scale bar represents  <dig>  μm.
b) phage abouo.
c) phage davies.
d) phage emery.
e) phage jimmer <dig>  f) phage jimmer <dig>  scale bars represent 50 nm for panels b-f.
sequence similarities between phages
gepard dotplots of the full genome sequences for all six p. larvae phages are shown in figure
<dig>  diagonal lines within the dotplots indicated that phages abouo, davies, jimmer <dig>  and jimmer <dig> were very similar to each other.
host specificity was also reflected in similarities between these four phages.
for instance, the pl <dig> phages jimmer <dig> and jimmer <dig> shared  <dig> % average nucleotide identity and the pl <dig> phages abouo and davies shared  <dig> % identity.
however, the average nucleotide identity between pl <dig> phages jimmer <dig> and jimmer <dig> and pl <dig> phages abouo and davies was only  <dig> %.
the lack of diagonal lines in the dotplot indicated that phages emery and phiibb_pl <dig> were very different from the other p. larvae phages examined.
while the emery and abouo phages were found in the same sample, sequences assembled independently without conflict with over 100-fold coverage for each genome
<cit> .
no similarities between the sequences of emery and abouo were apparent in the dotplot .
however, a black line indicates a small section of homology between emery and davies that davies does not share with abouo.
because emery and abouo were co-isolated and sequenced together, individual reads in these sections of emery, davies, and abouo were scrutinized using consed
<cit>  to ensure the assemblies were correct.
the fold coverage before, throughout, and after these sections of emery, davies, and abouo was at least  <dig> figure  <dig>
dotplots of six
p. larvae
phage genomes.
a) nucleic acid comparison of full genomes.
b) amino acid comparison of coding regions in each genome.
distinguishing between phages emery and abouo
in addition to the separation of unique dna sequences for emery and abouo in the same sample, two markedly different phages were present in electron micrographs based on measurements of capsid height and tail length.
putative tape measure protein  genes were identified in each phage genome.
the tmp gene found in emery  was  <dig>  bp long, while the tmp gene in abouo  was  <dig>  bp long, showing that the tmp gene in emery was  <dig>  times longer than the tmp gene in abouo.
the positive correlation between tmp gene length and tail length
<cit>  was used to suggest a correlation between phages emery and abouo in electron micrographs and their respective genome sequences.
the average tail length for emery was  <dig>  nm long while the average tail length for abouo was  <dig>  nm, making the tail of phage emery  <dig>  times longer than the tail of phage abouo .
in comparison, the tmp gene of emery was  <dig>  times longer than the tmp gene in abouo.
based on this data, we matched the long tmp gene and tail length with emery, and the short tmp gene and tail length with abouo .table  <dig>
comparison of
paenibacillus larvae
phage structures from electron microscopy data
myoviridae
siphoviridae
round capsid
elongated capsid
*data for paenibacillus larvae phages bla, bl <dig>  pbl <dig>  pbl <dig>  pbl <dig> c, pbl <dig>  ppl1c, and phiibb_pl <dig> were taken from referenced publications
.
independent electron microscopy data for phages pbl <dig> and ppl1c were not published; instead, they were reported to be indistinguishable from pbl <dig> c and pbl <dig>  respectively.
**measurements were taken from published electron micrographs instead of reported data which were either not presented or were inaccurate.
— phage measurements were not taken.
measurements from electron micrographs
p. larvae phages were separated into three distinct groups based on structural morphology.
the first group of phages were myoviruses  and included phages abouo, davies, emery, jimmer <dig>  jimmer <dig>  pbl <dig>
<cit> , and pbl2
<cit> .
there were two distinct groups of phages that were siphoviruses.
the first siphovirus group contains phages bla
<cit> , pbl1
<cit> , and ppl1c
<cit> .
these phages had long, non-contractile tails and elongated capsids.
the second siphovirus group contained only pbl <dig> which had a round capsid
<cit> .
phage phiibb_pl <dig> was also a siphovirus
<cit> , 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
<dig>  measurements for phages abouo, davies, emery, jimmer <dig>  and jimmer <dig> 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  <dig>  ±  <dig>  nm and an average width of  <dig>  ±  <dig>  nm.
the average tail length was  <dig>  ±  <dig>  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 gp <dig> that encoded for a head morphogenesis protein in the spp <dig> gp <dig> family.
this frameshift was identified by the online frameshift finding tool framed
<cit> .
the two products in emery are predicted to be  <dig>  kda following ribosomal slippage and  <dig>  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 spp <dig> or any protein sequence homology using blastp between the morphogenesis proteins in emery and spp <dig>
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
<cit> .
the finished phamerator database contained a diverse set of phages that infected firmicute bacteria including the  <dig> p. larvae phage genomes,  <dig> bacillus phages,  <dig> clostridium phage,  <dig> enterococcus phages,  <dig> geobacillus phages,  <dig> lactobacillus phages,  <dig> listeria phages, one paenibacillus glucanolyticus phage,  <dig> staphylococcus phages, and  <dig> streptococcus phage .
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  <dig>  putative proteins annotated in the  <dig> phage genomes into  <dig>  phamilies .
only  <dig>  phams  contained two or more members.
these  <dig>  phams contained  <dig>   of the  <dig>  putative proteins in the database.
the remaining  <dig>   putative proteins could not be grouped with other proteins and were designated as "orphams"
<cit> .
the phamerator database allowed comparison of p. larvae phage genes to each other and to phages infecting other bacteria.
a spreadsheet was exported from the phamerator database to report all phage gene products in the database, the phams to which the gene products are assigned, and the conserved domains found in gene products in those phams .
table
<dig> indicates how many putative proteins in each p. larvae phage are orphams, are shared only with p. larvae phages, or are shared with phages infecting other bacterial hosts.
putative proteins from jimmer <dig> and jimmer <dig> shared phams with  <dig> non-p. larvae phages, while abouo shared phams with  <dig>  davies with  <dig>  emery with  <dig>  and philbb_p <dig> with  <dig> other non-p. larvae phages.
between the six p. larvae phages, there are phams shared with  <dig> non-p. larvae phages of the  <dig>  included in the database.
of  <dig> genes in the six p. larvae phages, only  <dig>  encoded proteins grouped into phams with proteins from other types of phages in the database.
of the remaining genes,  <dig>  encoded proteins that were grouped into phams containing only p. larvae phage proteins and  <dig>  were orphams.table  <dig>
comparison of
p. larvae
phage genes
*blast hits had an e-value of less than  <dig> × 10- <dig>
**of the  <dig> p. larvae phage genes in jimmer <dig> and jimmer <dig>   <dig> are not found in any other phages in the database.
pham groupings reflected the genetic relationships of p. larvae phages.
the genomic sequence comparison of jimmer <dig> and jimmer <dig> using clustalw identified differences in 80 bp of the  <dig>  bp genomes
<cit> .
all corresponding genes between jimmer <dig> and jimmer <dig> shared the same phams.
of the  <dig> p. larvae phage genes in jimmer <dig> and jimmer <dig>   <dig> were unique to these two phages and were not found in any other phages in the database.
these  <dig> genes would be orphams if jimmer <dig> were not isolated .
phamerator identified conserved domains in at least one gene in  <dig>  phams  of the total  <dig>  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  <dig> × 10- <dig> , 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 .
phages emery and phiibb_pl <dig> 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
conserved domains and blast hits matching phage or bacterial proteins were used to assign functions to  <dig> gene products in the six p. larvae phages, indicating that these genes were not novel and were characteristically found in other phages.
the assembly and structural proteins were grouped according to function in table
<dig>  the regulatory and non-structural proteins are listed in table
<dig>  the pham assignment for each gene is shown in parentheses.
pham numbers are specific to the phamerator database used for this analysis.table  <dig>
p. larvae
phage assembly proteins and structural proteins; gene product # and
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig> , gp <dig>
gp <dig> , gp <dig>
gp <dig> , gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig> and gp <dig>
gp <dig> , gp <dig>
gp <dig> , gp <dig>
gp <dig> , gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig>
gp <dig> , gp <dig> , gp <dig> , gp <dig> , gp <dig>
gp <dig> , gp <dig> , gp <dig> , gp <dig> , gp <dig> , gp <dig> , gp <dig>
most of the functions listed in tables
<dig> and
<dig> 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 , lysm peptidoglycan binding, peptidoglycan hydrolase, pbsx, and bacteriocin .
few proteins with known functions were identified as putative virulence factors.
blast results indicate that gp <dig> in p. larvae phage phiibb_pl <dig> 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, jimmer <dig>  jimmer <dig>  and phiibb_pl <dig> 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_pl <dig>  abouo gp <dig>  jimmer <dig> gp <dig>  jimmer <dig> gp <dig> and emery gp <dig>  gp <dig>  and gp <dig>  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  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
a linear genome map of the six p. larvae phages shows that the genes in phages jimmer <dig>  jimmer <dig>  abouo, and davies are organized similarly .
identically colored genes encode products that share a pham, while white genes encode orpham gene products.
there are  <dig> phams that each contained gene products from phages abouo, davies, jimmer <dig>  and jimmer <dig>  proteins in  <dig> of these phams had identifiable functions based on blast hits and are italicized in tables
<dig> and
<dig>  of the  <dig> conserved phams,  <dig> did not contain homologs from any other phage in the database.
of the remaining  <dig> phams that have homologs from other phage types, three of the most populated phams are those containing small terminase , large terminase , and portal protein .
of the  <dig> other phages that shared one of these phams with the four similar p. larvae myoviruses, only three phages shared all three phams: staphylococcus phages  <dig>   <dig>  and ph <dig> figure  <dig>
linear genome map of the six
p. larvae
phages.
connecting lines between jimmer <dig>  abouo, davies, and emery represent nucleotide similarity .
genes are color-coded according to phams of the encoded protein.
gene numbers are indicated inside gene boxes.
functions matching gene products described in tables
<dig> and
<dig> are also displayed.
phamily relationships of large terminase proteins indicate putative dna packaging strategies
phage gene products must meet stringent parameters in order be grouped into a pham with other genes that encode similar proteins.
because gene products in a pham are highly similar, phylogenetic analysis indicates that these proteins will be more closely related than others with the same function.
a neighbor-joining phylogenetic tree grouped large terminase proteins in the phamerator database by phamily .
amino acid sequences of large terminase proteins can indicate the dna packaging strategy
<cit> .
phages abouo, davies, jimmer <dig>  and jimmer <dig> likely use headful packaging and have circularly permuted terminal repeats based on close association with the large terminases of well-characterized headful packaging phages p40
<cit> , and spp1
<cit>  which share a pham.
phage phiibb_pl <dig> likely has 3′ cohesive ends based on close association with phage phislt
<cit> .
further analysis of experimental data indicated that no phams generated by phamerator contained terminases belonging to phages with different packaging strategies .
the packaging strategy for phage emery is still undetermined because its large terminase protein is an orpham.figure  <dig>
neighbor-joining phylogenetic tree of the large terminase gene products from the phamerator database indicate proposed packaging strategies.
colored boxes indicate proteins belonging to the same pham.
proteins that are not highlighted are orphams.
large terminase proteins grouped into similar phams are closely related on the tree and share a packaging strategy.
*experimentally determined headful packaging, circularly permuted terminal repeats
<cit> , **experimentally determined 3′ cohesive ends
<cit> .
***experimentally determined, long direct terminal repeats
<cit> .
p. larvaephages exhibit genetic and structural similarity with other small genome myoviruses.
the putative proteins encoded in p. larvae phages abouo, davies, jimmer <dig>  and jimmer <dig> were often grouped into phams with proteins in clostridium phage phic <dig>  lactobacillus phage kc5a, and streptococcus phage ej- <dig>  the similar proteins were mostly structural  and included the terminase , portal, head morphogenesis, minor structural, tail sheath, and baseplate proteins.
all of the gene products listed in the table were grouped into the same pham except for three proteins that narrowly missed the pham cutoff values and are marked by asterisks.
the tape measure proteins in abouo, davies, jimmer <dig>  and jimmer <dig> were somewhat similar to those found in streptococcus phage ej- <dig>  and clostridium phage phic <dig>  but were not near the pham cutoff values of  <dig> × 10- <dig> table  <dig>
genetic comparison of small genome myovirus phams and gene products within each pham
no genes in emery were grouped into any of the phams listed in this table.
genes followed by an asterisk were not grouped into the same phams as the p. larvae phages using phamerator.
the average e-value and percent identity when compared to abouo, davies, jimmer <dig>  and jimmer <dig> is reported below:
*kc5a gp37 =  <dig> × 10- <dig>  31% identity.
**ej- <dig> gp41 =  <dig> × 10- <dig>  31% identity.
***ej- <dig> gp46 =  <dig> × 10- <dig>  32% identity.
the electron micrographs of p. larvae phages abouo, davies, jimmer <dig>  and jimmer <dig> reveal a structure similar to those reported for clostridium phages phic2
<cit> , phicd27
<cit> , and phicd119
<cit> , lactobacillus phages kc5a
<cit>  which is reported as similar to lactobacillus phages kc21t
<cit>  and phiaq113
<cit> , and streptococcus phage ej-1
<cit> .
the capsid of clostridium phage phic <dig> is 65 nm in diameter and the tail is 148 nm long.
the capsid of streptococcus phage ej- <dig> 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 phiaq <dig>  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
<dig> into the same phams as phiaq <dig> genes except the small terminase  and the head morphogenesis protein.
although p. larvae phage emery contained gene products with the same functions as those listed in table
<dig>  the proteins were all orphams.
however, the first five gene products encoded in p. larvae phage phiibb_pl <dig>  all shared a pham with similar proteins from five siphovirus staphylococcus phages .
the program coregenes  <dig>  was used to further compare the genes in the p. larvae phages with small-genome myoviruses.
using the default blastp threshold of  <dig>  core proteins were identified in the five p. larvae myoviruses with respect to clostridium phage phicd <dig>  streptococcus phage ej- <dig>  lactobacillus phage kc5a, and lactobacillus phage aq <dig>  the number of core proteins shared between comparison and reference genomes are listed in table
<dig>  the percent of core proteins with respect to the reference genome are also reported.
clostridium phage phicd <dig> was the only one of these phages that belonged to a genus ; the other three are currently unclassified.
previous analyses of the podoviridae and myoviridae families grouped phages together when phages share 40% of core proteins with a reference phage genome
<cit> .
based on this cutoff value, abouo, davies, jimmer <dig> and jimmer <dig> formed a new group of small genome myoviruses.table  <dig>
coregenes analysis indicates relationships among small genome myoviruses
*the number of genes in common is followed by a percentage in parenthesis that represents the% genes in common with the reference genome.
families are typically grouped based on a coregenes score of 40% or higher.
only one pham includes all five myoviruses, and very few phams are shared between unrelated p. larvaephages
phages emery and phiibb_pl <dig> are significantly different from each other and from the four similar p. larvae myoviruses, as is evident from the genome maps in figure
<dig>  however, tables
<dig> and
<dig> demonstrate that some proteins encoded by these phages grouped into similar phams.
pham  <dig> 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 gp <dig>  davies gp <dig>  emery gp <dig>  jimmer <dig> gp <dig>  and jimmer <dig> gp <dig> and encode a bhla/bacteriocin protein .
no other gene products in the phamerator database were grouped into this pham.
the conserved domain in these proteins was duf <dig>  a putative holin-like protein.
when comparing amino acid sequences, these five proteins shared > 87% identity and an e-value less than  <dig> × 10- <dig>
p. larvae siphovirus phiibb_pl <dig> contained only two proteins that shared a pham with any new myovirus p. larvae phages.
the conserved domains in one gene product suggest it encodes a serine recombinase protein  .
the conserved domains in the other gene product suggest it encodes a phage antirepressor protein  .
antirepressors from  <dig> other phages are also assigned to this pham , including an antirepressor from paenibacillus glucanolyticus phage pg <dig> gp <dig>  a phamily circle links the  <dig> phages in the database containing a gene product in pham  <dig> .figure  <dig>
phamily circle connects  <dig> phages containing a related antirepressor gene.
the phamily circle of pham  <dig> connects all phages with genes encoding proteins in this pham.
the phages containing gene products in pham  <dig> infect bacillus, enterococcus, lactobacillus, listeria, clostridium, paenibacillus, and staphylococcus bacteria.
the antirepressor gene is found in  <dig> different phages, including staphylococcus phages  phislt ,  ph <dig> , and   <dig> ; paenibacillus phages  phiibb_pl <dig> ,  jimmer <dig> ,  jimmer <dig> , and  pg <dig> ; clostridium phage  phic <dig> ; listeria phages  b <dig> ,  a <dig> , and  a <dig> ; lactobacillus phages  phiat <dig> , and  a <dig> ; enterococcus phage  phief <dig> ; and bacillus phages  spbc <dig>  and  pm <dig> .
the siphovirus paenibacillus glucanolyticus phage pg <dig> also contained three gene products that shared a pham with p. larvae phages abouo, davies, jimmer <dig>  and jimmer <dig>  pham  <dig> encoded an rna polymerase sigma- <dig> factor and had  <dig> members: pg <dig> gp <dig>  abouo gp <dig>  davies gp <dig>  jimmer <dig> gp <dig>  jimmer <dig> gp <dig>  pham  <dig> encoded a recombination protein u and had  <dig> members, including pg <dig> gp <dig>  abouo gp <dig>  davies gp <dig>  jimmer <dig> gp <dig>  and jimmer <dig> gp <dig>  pham  <dig> encoded a dutpase and had  <dig> members, including pg <dig> gp <dig>  abouo gp <dig>  davies gp <dig>  jimmer <dig> gp <dig>  and jimmer <dig> gp <dig>
four p. larvaephages contain duplicated genes
phages abouo and phiibb_pl <dig> did not contain any proteins that belonged to the same pham.
however, gp <dig> and gp <dig> in phages jimmer <dig> and jimmer <dig> shared  <dig> % identity , belonged to pham  <dig>  and encode an xre family transcriptional regulator that contains a helix-turn-helix dna binding domain.
additionally, gp <dig> and gp <dig> in phages jimmer <dig> and jimmer <dig> shared  <dig> % amino acid identity , belonged to pham  <dig>  and contain an arc-like dna-binding domain.
davies gp <dig> and gp <dig> and emery gp <dig> and gp <dig> belonged to pham  <dig> and encoded a putative membrane protein .
comparisons indicated that homologous proteins encoded on the two genomes were more similar than duplicated proteins encoded within one of the genomes.
davies gp <dig> and emery gp <dig> shared  <dig> % identity , and davies gp <dig> and emery gp <dig> shared  <dig> % identity .
however, davies gp <dig> and gp <dig> shared  <dig> %  and emery gp <dig> and gp <dig> shared  <dig> % identity .
abouo gp <dig> also belongs to pham  <dig> but the nucleotide sequence for this gene is different from the genes encoding the four gene products in emery and davies.
abouo gp44- <dig>  davies gp44- <dig>  and emery gp53- <dig> are identified in figure
2a and
2b by the dark line indicating homology between emery and davies and the white gap between abouo and davies at the same location.figure  <dig>
davies and emery share a duplicated gene while abouo has only one copy.
this genome map shows two gene products in both davies and emery that belong to pham  <dig> and encode a putative membrane protein.
davies gp44- <dig> and emery gp53- <dig> are more similar to each other than they are to abouo gp44- <dig>  gene product numbers are located inside the colored boxes.
the numbers above each gene product indicate the pham number specific to this analysis and the .
gene products with the same color share a pham.
discussion
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
<cit> .
the five myoviruses were isolated from three soil samples each from a separate location: jimmer <dig> and jimmer <dig> were isolated independently from the same sample
<cit> , emery and abouo were isolated together, and davies was isolated separately.
p. larvae phage pbl <dig> was isolated from a different sample than bl <dig>  yet all tests indicated no obvious structural or genetic differences between these phages
<cit> .
similar host properties and selective pressures can result in isolation of similar phages from different locations
<cit> .
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
<cit> .
bacteriophages are often unable to superinfect an existing lysogen if the entering and lysogenic phages are genetically similar
<cit> .
the portion of the genome responsible for superinfection immunity has been determined for some phages
<cit> .
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
<cit> .
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 jimmer <dig>  jimmer <dig>  and davies displayed superinfection immunity when incubated with the same phage.
jimmer <dig> and jimmer <dig> exhibited nearly identical sequences and were also immune to superinfections of each other.
the host specificity and correlating genome similarity between jimmer <dig> and jimmer <dig>  and davies and abouo  reflect common evolutionary ancestry.
the high degree of similarity  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
<cit> .
jimmer <dig>  jimmer <dig>  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_pl <dig> , and no toxin genes have been identified in the five p. larvae myoviruses.
p. larvae phages abouo, davies, jimmer <dig>  jimmer <dig>  and phiibb_pl <dig> encode an abc transporter-like protein.
this was characterized as an extracellular protein produced by p. larvae
<cit> , 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 jimmer <dig> and jimmer <dig>  the differences between these genes indicate that they are ohnologous and arose by gene duplication and subsequent divergence
<cit> .
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 .
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
<cit> .
analysis of large terminase protein phamilies revealed that abouo, davies, jimmer <dig>  and jimmer <dig> likely use the headful packaging mechanism, while phiibb_pl <dig> 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_pl <dig>  jimmer <dig>  jimmer <dig> shares a pham with antirepressors from  <dig> other myoviruses and siphoviruses that infect host bacteria in the genera bacillus, enterococcus, geobacillus, lactobacillus, listeria, paenibacillus, and staphylococcus .
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 spp <dig> gp <dig> family and contains a ribosomal slippage site that is not found in bacillus phage spp <dig>  although two gene products are produced by the head morphogenesis gene in spp <dig> that are 34 kda and 28 kda , the two spp <dig> proteins are thought to be due to an alternative start site, not a frameshift caused by ribosomal slippage
<cit> .
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
<cit>  and myoviridae families
<cit> .
it was also used analyze other "dwarf" myoviruses and group them based on the similarity of core genes
<cit> .
analysis of core genes and shared phams indicates that p. larvae phages abouo, davies, jimmer <dig>  and jimmer <dig> are distantly related to phages in the phicd119likevirus family as well as phages ej- <dig>  kc5a, and aq <dig>  because proteins grouped into similar phams are phylogenetically related, these proteins likely share a common ancestry.
the structural similarities between phages abouo, davies, jimmer <dig>  jimmer <dig>  phic <dig>  kc5a, aq <dig>  and ej- <dig> 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 .
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, jimmer <dig>  and jimmer <dig> are related phylogenetically.
they are also distantly related to phic <dig>  kc5a, aq <dig>  and ej- <dig> 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 pl <dig> and pl <dig> are similar , and similar bhla/bacteriocin genes such as found in the shared pham of jimmer <dig>  jimmer <dig>  emery, abouo, and davies  can likely be used to lyse the bacterial host.
it is interesting to note that the only two genes shared between phiibb_pl <dig> and any other p. larvae phage encode a serine recombinase and an antirepressor, shared with the pl <dig> phages jimmer <dig> and jimmer <dig>  this correlation may indicate similar host interactions, as these genes help regulate the lytic and lysogenic cycles.
the pl <dig> phages do not contain any antirepressor gene products belonging to this pham.
although p. larvae phages emery and phiibb_pl <dig> 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.
CONCLUSIONS
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.

 paenibacillus larvae is a sporulating firmicute. it is the causative agent of american foulbrood, a disease that infects and destroys the larvae of honeybees. the first eight p. larvae phages were reported between and and included bla, l3, bl2, pbl1, pbl, 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. 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. 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. 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 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. 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 six p. larvae phages were isolated. these phages were fully sequenced and their genomes published. p. larvae siphovirus phiibb_pl was isolated in portugal and p. larvae myoviruses abouo, davies, emery, jimmer and jimmer 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 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 and dsm as well as the related bacteria brevibacillus laterosporus. of nine isolates, pl and pl were used for phage isolation. phages abouo, davies, and emery were isolated using pl while phages jimmer and jimmer were isolated using pl each p. larvae phage sample was plaque-purified at least three times, sequenced, and published. prior to genome sequencing and electron microscopy we were unaware that one phage sample still contained two different phages. plaque purification did not successfully separate these two phages. however, assembly of the genomes revealed two clearly independent genomes that separated with ease with over 100-fold coverage of the genomes. these co-isolated phages were named emery and abouo. results from sequencing and annotation of the six p. larvae phages are found in table  the genomes varied in length from ~40 kb to ~58 kb. most of the genes in each genome were located on the forward strand. the average g + c content for these phages is % ±  %. blast hits for proteins within these phages included both paenibacillus and brevibacillus bacterial strains as well as bacteriophages that infected other firmicutes.table characteristics of sequenced paenibacillus larvae bacteriophages abouo davies emery jimmer1 jimmer2 phiibb_pl * *paenibacillus phage phiibb_pl data from. 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 jimmer and jimmer were isolated independently using pl and neither of these phages could infect pl phages davies and emery/abouo were isolated using pl and these phages could not infect pl 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: jimmer in pl jimmer in pl and davies in pl 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 pl 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 electron microscopy revealed that these five p. larvae phages were myoviruses, marked by the presence of a contractile tail sheath. figure  1a shows tail structures separated from the phage capsids. these tail structures were more abundant than intact phages in all samples submitted for electron microscopy. figure  1b, 1c, 1e, and 1f show intact phages with contracted tail sheaths, while figure  1d shows an extended tail sheath. because phages emery and abouo were not separated, micrographs for these phages were taken from the same copper grid.figure electron micrographs of p. larvae phages. a) tails and tail sheaths of p. larvae phages separated from the capsids. these structures were more abundant than intact phages in electron micrographs. scale bar represents  μm. b) phage abouo. c) phage davies. d) phage emery. e) phage jimmer f) phage jimmer scale bars represent 50 nm for panels b-f. sequence similarities between phages gepard dotplots of the full genome sequences for all six p. larvae phages are shown in figure  diagonal lines within the dotplots indicated that phages abouo, davies, jimmer and jimmer were very similar to each other. host specificity was also reflected in similarities between these four phages. for instance, the pl phages jimmer and jimmer shared % average nucleotide identity and the pl phages abouo and davies shared % identity. however, the average nucleotide identity between pl phages jimmer and jimmer and pl phages abouo and davies was only %. the lack of diagonal lines in the dotplot indicated that phages emery and phiibb_pl were very different from the other p. larvae phages examined. while the emery and abouo phages were found in the same sample, sequences assembled independently without conflict with over 100-fold coverage for each genome. no similarities between the sequences of emery and abouo were apparent in the dotplot. however, a black line indicates a small section of homology between emery and davies that davies does not share with abouo. because emery and abouo were co-isolated and sequenced together, individual reads in these sections of emery, davies, and abouo were scrutinized using consed to ensure the assemblies were correct. the fold coverage before, throughout, and after these sections of emery, davies, and abouo was at least figure dotplots of six p. larvae phage genomes. a) nucleic acid comparison of full genomes. b) amino acid comparison of coding regions in each genome. distinguishing between phages emery and abouo in addition to the separation of unique dna sequences for emery and abouo in the same sample, two markedly different phages were present in electron micrographs based on measurements of capsid height and tail length. putative tape measure protein genes were identified in each phage genome. the tmp gene found in emery was  bp long, while the tmp gene in abouo was  bp long, showing that the tmp gene in emery was times longer than the tmp gene in abouo. the positive correlation between tmp gene length and tail length was used to suggest a correlation between phages emery and abouo in electron micrographs and their respective genome sequences. the average tail length for emery was  nm long while the average tail length for abouo was  nm, making the tail of phage emery times longer than the tail of phage abouo. in comparison, the tmp gene of emery was times longer than the tmp gene in abouo. based on this data, we matched the long tmp gene and tail length with emery, and the short tmp gene and tail length with abouo.table comparison of paenibacillus larvae phage structures from electron microscopy data myoviridae siphoviridae round capsid elongated capsid *data for paenibacillus larvae phages bla, bl pbl pbl pbl c, pbl ppl1c, and phiibb_pl were taken from referenced publications. independent electron microscopy data for phages pbl and ppl1c were not published; instead, they were reported to be indistinguishable from pbl c and pbl respectively. **measurements were taken from published electron micrographs instead of reported data which were either not presented or were inaccurate. — phage measurements were not taken. measurements from electron micrographs p. larvae phages were separated into three distinct groups based on structural morphology. the first group of phages were myoviruses and included phages abouo, davies, emery, jimmer jimmer pbl, 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 pbl which had a round capsid. phage phiibb_pl 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  measurements for phages abouo, davies, emery, jimmer and jimmer 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  ±   nm and an average width of  ±   nm. the average tail length was  ±   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 gp that encoded for a head morphogenesis protein in the spp gp family. this frameshift was identified by the online frameshift finding tool framed. the two products in emery are predicted to be  kda following ribosomal slippage and  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 spp or any protein sequence homology using blastp between the morphogenesis proteins in emery and spp 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 p. larvae phage genomes, bacillus phages, clostridium phage, enterococcus phages, geobacillus phages, lactobacillus phages, listeria phages, one paenibacillus glucanolyticus phage, staphylococcus phages, and streptococcus phage. 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 putative proteins annotated in the phage genomes into phamilies. only phams contained two or more members. these phams contained of the putative proteins in the database. the remaining putative proteins could not be grouped with other proteins and were designated as "orphams". the phamerator database allowed comparison of p. larvae phage genes to each other and to phages infecting other bacteria. a spreadsheet was exported from the phamerator database to report all phage gene products in the database, the phams to which the gene products are assigned, and the conserved domains found in gene products in those phams. table  indicates how many putative proteins in each p. larvae phage are orphams, are shared only with p. larvae phages, or are shared with phages infecting other bacterial hosts. putative proteins from jimmer and jimmer shared phams with non-p. larvae phages, while abouo shared phams with davies with emery with and philbb_p with other non-p. larvae phages. between the six p. larvae phages, there are phams shared with non-p. larvae phages of the included in the database. of genes in the six p. larvae phages, only encoded proteins grouped into phams with proteins from other types of phages in the database. of the remaining genes, encoded proteins that were grouped into phams containing only p. larvae phage proteins and were orphams.table comparison of p. larvae phage genes *blast hits had an e-value of less than × 10- **of the p. larvae phage genes in jimmer and jimmer are not found in any other phages in the database. pham groupings reflected the genetic relationships of p. larvae phages. the genomic sequence comparison of jimmer and jimmer using clustalw identified differences in 80 bp of the  bp genomes. all corresponding genes between jimmer and jimmer shared the same phams. of the p. larvae phage genes in jimmer and jimmer were unique to these two phages and were not found in any other phages in the database. these genes would be orphams if jimmer were not isolated. phamerator identified conserved domains in at least one gene in phams of the total 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 × 10-, 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. phages emery and phiibb_pl 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 conserved domains and blast hits matching phage or bacterial proteins were used to assign functions to gene products in the six p. larvae phages, indicating that these genes were not novel and were characteristically found in other phages. the assembly and structural proteins were grouped according to function in table  the regulatory and non-structural proteins are listed in table  the pham assignment for each gene is shown in parentheses. pham numbers are specific to the phamerator database used for this analysis.table p. larvae phage assembly proteins and structural proteins; gene product # and gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp, gp gp, gp gp, gp gp gp gp gp gp gp gp gp gp and gp gp, gp gp, gp gp, gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp gp, gp, gp, gp, gp gp, gp, gp, gp, gp, gp, gp most of the functions listed in tables  and 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, lysm peptidoglycan binding, peptidoglycan hydrolase, pbsx, and bacteriocin. few proteins with known functions were identified as putative virulence factors. blast results indicate that gp in p. larvae phage phiibb_pl 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, jimmer jimmer and phiibb_pl 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_pl abouo gp jimmer gp jimmer gp and emery gp gp and gp 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 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 a linear genome map of the six p. larvae phages shows that the genes in phages jimmer jimmer abouo, and davies are organized similarly. identically colored genes encode products that share a pham, while white genes encode orpham gene products. there are phams that each contained gene products from phages abouo, davies, jimmer and jimmer proteins in of these phams had identifiable functions based on blast hits and are italicized in tables  and of the conserved phams, did not contain homologs from any other phage in the database. of the remaining phams that have homologs from other phage types, three of the most populated phams are those containing small terminase, large terminase, and portal protein. of the other phages that shared one of these phams with the four similar p. larvae myoviruses, only three phages shared all three phams: staphylococcus phages and ph figure linear genome map of the six p. larvae phages. connecting lines between jimmer abouo, davies, and emery represent nucleotide similarity. genes are color-coded according to phams of the encoded protein. gene numbers are indicated inside gene boxes. functions matching gene products described in tables  and are also displayed. phamily relationships of large terminase proteins indicate putative dna packaging strategies phage gene products must meet stringent parameters in order be grouped into a pham with other genes that encode similar proteins. because gene products in a pham are highly similar, phylogenetic analysis indicates that these proteins will be more closely related than others with the same function. a neighbor-joining phylogenetic tree grouped large terminase proteins in the phamerator database by phamily. amino acid sequences of large terminase proteins can indicate the dna packaging strategy. phages abouo, davies, jimmer and jimmer likely use headful packaging and have circularly permuted terminal repeats based on close association with the large terminases of well-characterized headful packaging phages p40, and spp1 which share a pham. phage phiibb_pl likely has 3′ cohesive ends based on close association with phage phislt. further analysis of experimental data indicated that no phams generated by phamerator contained terminases belonging to phages with different packaging strategies. the packaging strategy for phage emery is still undetermined because its large terminase protein is an orpham.figure neighbor-joining phylogenetic tree of the large terminase gene products from the phamerator database indicate proposed packaging strategies. colored boxes indicate proteins belonging to the same pham. proteins that are not highlighted are orphams. large terminase proteins grouped into similar phams are closely related on the tree and share a packaging strategy. *experimentally determined headful packaging, circularly permuted terminal repeats, **experimentally determined 3′ cohesive ends. ***experimentally determined, long direct terminal repeats. p. larvaephages exhibit genetic and structural similarity with other small genome myoviruses. the putative proteins encoded in p. larvae phages abouo, davies, jimmer and jimmer were often grouped into phams with proteins in clostridium phage phic lactobacillus phage kc5a, and streptococcus phage ej- the similar proteins were mostly structural and included the terminase, portal, head morphogenesis, minor structural, tail sheath, and baseplate proteins. all of the gene products listed in the table were grouped into the same pham except for three proteins that narrowly missed the pham cutoff values and are marked by asterisks. the tape measure proteins in abouo, davies, jimmer and jimmer were somewhat similar to those found in streptococcus phage ej- and clostridium phage phic but were not near the pham cutoff values of × 10- table genetic comparison of small genome myovirus phams and gene products within each pham no genes in emery were grouped into any of the phams listed in this table. genes followed by an asterisk were not grouped into the same phams as the p. larvae phages using phamerator. the average e-value and percent identity when compared to abouo, davies, jimmer and jimmer is reported below: *kc5a gp37 =  × 10- 31% identity. **ej- gp41 =  × 10- 31% identity. ***ej- gp46 =  × 10- 32% identity. the electron micrographs of p. larvae phages abouo, davies, jimmer and jimmer 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 phic is 65 nm in diameter and the tail is 148 nm long. the capsid of streptococcus phage ej- 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 phiaq 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  into the same phams as phiaq genes except the small terminase and the head morphogenesis protein. although p. larvae phage emery contained gene products with the same functions as those listed in table  the proteins were all orphams. however, the first five gene products encoded in p. larvae phage phiibb_pl all shared a pham with similar proteins from five siphovirus staphylococcus phages. the program coregenes was used to further compare the genes in the p. larvae phages with small-genome myoviruses. using the default blastp threshold of core proteins were identified in the five p. larvae myoviruses with respect to clostridium phage phicd streptococcus phage ej- lactobacillus phage kc5a, and lactobacillus phage aq the number of core proteins shared between comparison and reference genomes are listed in table  the percent of core proteins with respect to the reference genome are also reported. clostridium phage phicd was the only one of these phages that belonged to a genus; the other three are currently unclassified. previous analyses of the podoviridae and myoviridae families grouped phages together when phages share 40% of core proteins with a reference phage genome. based on this cutoff value, abouo, davies, jimmer and jimmer formed a new group of small genome myoviruses.table coregenes analysis indicates relationships among small genome myoviruses *the number of genes in common is followed by a percentage in parenthesis that represents the% genes in common with the reference genome. families are typically grouped based on a coregenes score of 40% or higher. only one pham includes all five myoviruses, and very few phams are shared between unrelated p. larvaephages phages emery and phiibb_pl are significantly different from each other and from the four similar p. larvae myoviruses, as is evident from the genome maps in figure  however, tables  and demonstrate that some proteins encoded by these phages grouped into similar phams. pham 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 gp davies gp emery gp jimmer gp and jimmer gp and encode a bhla/bacteriocin protein. no other gene products in the phamerator database were grouped into this pham. the conserved domain in these proteins was duf a putative holin-like protein. when comparing amino acid sequences, these five proteins shared > 87% identity and an e-value less than × 10- p. larvae siphovirus phiibb_pl contained only two proteins that shared a pham with any new myovirus p. larvae phages. the conserved domains in one gene product suggest it encodes a serine recombinase protein. the conserved domains in the other gene product suggest it encodes a phage antirepressor protein. antirepressors from other phages are also assigned to this pham, including an antirepressor from paenibacillus glucanolyticus phage pg gp a phamily circle links the phages in the database containing a gene product in pham.figure phamily circle connects phages containing a related antirepressor gene. the phamily circle of pham connects all phages with genes encoding proteins in this pham. the phages containing gene products in pham infect bacillus, enterococcus, lactobacillus, listeria, clostridium, paenibacillus, and staphylococcus bacteria. the antirepressor gene is found in different phages, including staphylococcus phages phislt, ph, and; paenibacillus phages phiibb_pl, jimmer, jimmer, and pg; clostridium phage phic; listeria phages b, a, and a; lactobacillus phages phiat, and a; enterococcus phage phief; and bacillus phages spbc and pm. the siphovirus paenibacillus glucanolyticus phage pg also contained three gene products that shared a pham with p. larvae phages abouo, davies, jimmer and jimmer pham encoded an rna polymerase sigma- factor and had members: pg gp abouo gp davies gp jimmer gp jimmer gp pham encoded a recombination protein u and had members, including pg gp abouo gp davies gp jimmer gp and jimmer gp pham encoded a dutpase and had members, including pg gp abouo gp davies gp jimmer gp and jimmer gp four p. larvaephages contain duplicated genes phages abouo and phiibb_pl did not contain any proteins that belonged to the same pham. however, gp and gp in phages jimmer and jimmer shared % identity, belonged to pham and encode an xre family transcriptional regulator that contains a helix-turn-helix dna binding domain. additionally, gp and gp in phages jimmer and jimmer shared % amino acid identity, belonged to pham and contain an arc-like dna-binding domain. davies gp and gp and emery gp and gp belonged to pham and encoded a putative membrane protein. comparisons indicated that homologous proteins encoded on the two genomes were more similar than duplicated proteins encoded within one of the genomes. davies gp and emery gp shared % identity, and davies gp and emery gp shared % identity. however, davies gp and gp shared % and emery gp and gp shared % identity. abouo gp also belongs to pham but the nucleotide sequence for this gene is different from the genes encoding the four gene products in emery and davies. abouo gp44- davies gp44- and emery gp53- are identified in figure  2a and 2b by the dark line indicating homology between emery and davies and the white gap between abouo and davies at the same location.figure davies and emery share a duplicated gene while abouo has only one copy. this genome map shows two gene products in both davies and emery that belong to pham and encode a putative membrane protein. davies gp44- and emery gp53- are more similar to each other than they are to abouo gp44- gene product numbers are located inside the colored boxes. the numbers above each gene product indicate the pham number specific to this analysis and the. gene products with the same color share a pham. discussion 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: jimmer and jimmer were isolated independently from the same sample, emery and abouo were isolated together, and davies was isolated separately. p. larvae phage pbl was isolated from a different sample than bl 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 jimmer jimmer and davies displayed superinfection immunity when incubated with the same phage. jimmer and jimmer exhibited nearly identical sequences and were also immune to superinfections of each other. the host specificity and correlating genome similarity between jimmer and jimmer and davies and abouo reflect common evolutionary ancestry. the high degree of similarity 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. jimmer jimmer 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_pl, and no toxin genes have been identified in the five p. larvae myoviruses. p. larvae phages abouo, davies, jimmer jimmer and phiibb_pl 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 jimmer and jimmer 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. 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, jimmer and jimmer likely use the headful packaging mechanism, while phiibb_pl 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_pl jimmer jimmer shares a pham with antirepressors from other myoviruses and siphoviruses that infect host bacteria in the genera bacillus, enterococcus, geobacillus, lactobacillus, listeria, paenibacillus, and staphylococcus. 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 spp gp family and contains a ribosomal slippage site that is not found in bacillus phage spp although two gene products are produced by the head morphogenesis gene in spp that are 34 kda and 28 kda, the two spp 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, jimmer and jimmer are distantly related to phages in the phicd119likevirus family as well as phages ej- kc5a, and aq because proteins grouped into similar phams are phylogenetically related, these proteins likely share a common ancestry. the structural similarities between phages abouo, davies, jimmer jimmer phic kc5a, aq and ej- 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. 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, jimmer and jimmer are related phylogenetically. they are also distantly related to phic kc5a, aq and ej- 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 pl and pl are similar, and similar bhla/bacteriocin genes such as found in the shared pham of jimmer jimmer emery, abouo, and davies can likely be used to lyse the bacterial host. it is interesting to note that the only two genes shared between phiibb_pl and any other p. larvae phage encode a serine recombinase and an antirepressor, shared with the pl phages jimmer and jimmer this correlation may indicate similar host interactions, as these genes help regulate the lytic and lysogenic cycles. the pl phages do not contain any antirepressor gene products belonging to this pham. although p. larvae phages emery and phiibb_pl 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. 
the correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages emery and abouo in electron micrographs. paenibacillus larvae is a firmicute bacterium that causes american foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. paenibacillus larvaebacteriophagejimmer1jimmer2emeryabouodaviesphiibb_pl23phameratorphage genomicsissue-copyright-statement© the author 2014 an annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. although p. larvae phages were isolated prior to 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, jimmer jimmer and siphovirus phiibb_pl to each other and to other known phages. p. larvae phages abouo, davies, emery, jimmer and jimmer are myoviruses with ~ kbp genomes. the six p. larvae phages form three distinct groups by dotplot analysis. sixty phage assembly or structural protein genes and regulatory or other non-structural protein genes were identifiable among the six p. larvae phages. a phamerator database was assembled with the p. larvae phage genomes and genomes of firmicute-infecting phages, including bacillus phages. phamerator identified conserved domains in of phamilies encoded by genes in the database and revealed that p. larvae phage genomes shared at least one phamily with of the other phages. 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. jimmer jimmer and davies formed stable lysogens resistant to superinfection by genetically similar phages. analyses from coregenes, phamerator, and electron micrograph measurements indicated jimmer jimmer abouo and davies were related to phages phic ej- kc5a, and aq which are small-genome myoviruses that infect streptococcus, lactobacillus, and clostridium, respectively. the phamily relationship of large terminase proteins was used to indicate putative dna packaging strategies.
 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 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, jimmer jimmer and siphovirus phiibb_pl to each other and to other known phages.  p. larvae phages abouo, davies, emery, jimmer and jimmer are myoviruses with ~ 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 regulatory or other non-structural protein genes were identifiable among the six p. larvae phages. jimmer jimmer 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 genomes of firmicute-infecting phages, including bacillus phages. phamerator identified conserved domains in of phamilies encoded by genes in the database and revealed that p. larvae phage genomes shared at least one phamily with of the 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 jimmer jimmer abouo and davies were related to phages phic ej- kc5a, and aq 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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords paenibacillus larvaebacteriophagejimmer1jimmer2emeryabouodaviesphiibb_pl23phameratorphage genomicsissue-copyright-statement© the author 2014 