BACKGROUND
campylobacter is one of the most important foodborne pathogens worldwide.
the genus comprises many species of which the thermophilic c. jejuni and its close relative c. coli are reported as the most common causes of acute bacterial enteritis  <cit> .
the bacteria are commensals of the gastrointestinal tract of various mammals and birds and are frequently found in chicken flocks where they may spread rapidly  <cit> .
human infections are caused mainly by the consumption of undercooked meats, especially poultry  <cit> .
to reduce the number of campylobacter along the food chain, various biosecurity measures and post-slaughter decontamination procedures have been tested.
as these measures are expensive and not always efficient and since suitable vaccines are not available, phages might be an appropriate means to reduce campylobacter counts in chicken and on chicken products  <cit> .
indeed phage administration in the laboratory reduced c. jejuni colonization of the broiler gut and the contamination on chicken skin by several orders of magnitude .
a phage cocktail applied via drinking water also efficiently reduced campylobacter counts in chicken on a commercial broiler farm  <cit> .
phages intended for therapeutic applications or for the control of pathogens in food production have to fulfil a number of requirements to avoid any undesired side effect.
amongst others, the phage genomes have to be stable and free from critical genes   <cit> .
therefore, a basic understanding of the biology and genetics of the phages to be used is of great importance  <cit> .
according to their genome size, campylobacter phages described so far are classified into three groups.
while group i phages  have been rarely isolated and have not been used for applications as yet, members of group ii  and group iii  are very common  <cit> .
group ii phages might be best suited for the reduction of campylobacter since they can infect both c. jejuni and c. coli strains.
though, the successive application of a group iii and a group ii phage reduced the numbers of c. jejuni in chickens most efficiently  <cit> .
up to now three  group ii phages have been completely sequenced while vb_ccom-ibb_ <dig>  is represented, in genbank, in the form of five contigs  <cit> .
the phages revealed strong dna homologies to each other whereas only little dna sequence similarities have been detected to group iii phages  <cit> .
group ii campylobacter phages contain e.g., genes for membrane proteins, transposases, and metabolic enzymes including up to twelve proteins with s-adenosylmethionine  domains that are absent in group iii  <cit> .
conversely, group iii phages show an expansion of homing endonucleases, represented by up to nine orfs, that are much less frequently occurring in group ii.
however, at the protein level both groups showed some relationship to t4-type phages  <cit> .
a striking feature of cp <dig>  cpt <dig> and cp <dig> is that their genomes are composed of large modules separated by long dna repeat regions, which could lead to rearrangements  <cit> .
while the genomes of cp <dig> and cpt <dig> are collinear, the genome of cp <dig> exhibits considerable rearrangement of homologous modules  <cit> .
functional modules are common in temperate and virulent phages  <cit> .
they are shuffled by recombination which may result in new combinations of modules and the emergence of novel phages  <cit> .
the frequency of recombination events largely depends on the phylogenetic distance between the respective hosts  <cit> .
illegitimate recombination events are rather rare and may often lead to defective particles but can take place at nearly random points across the genome.
on the other hand, homologous recombinations of modules at specific linker sequences occur more frequently, particularly in closely related phages like those belonging to the lambdoid phage family  <cit> .
in this work, the genome organization of cp <dig> was analysed in detail with special focus on the repeat regions which impede the elucidation of the genome structure of group ii phages.
on the basis of the available group ii sequences, a pcr typing system was established to study the modular genome arrangement of other yet not characterized group ii phages.
the system was also used to unravel the genome organization of phage ibb_ <dig>  we show that group ii phages can be allocated to at least two subgroups which revealed distinct host ranges.
methods
bacterial strains and culture conditions
most strains originated from the campynet  strain collection hosted by the leibniz institute dsmz-german collection of microorganisms and cell cultures, braunschweig, germany, .
the remaining strains were taken from the campylobacter strain collection of the national reference laboratory for campylobacter at the federal institute for risk assessment ).
if not otherwise indicated, the c. coli strain nctc <dig>  was used for phage propagation and plaque assays  <cit> .
the cultivation of bacteria was performed as previously described  <cit> .
isolation, propagation and purification of group ii campylobacter phages
the phages cp <dig>  cp <dig>  and cp <dig> were isolated from poultry produced on organic farms in berlin, germany.
to recover phages, samples were suspended in sm-buffer and incubated overnight at 45 °c on a stirrer.
thereafter, the samples were centrifuged at  <dig>  × g followed by filtration of the supernatants through  <dig>  μm nitrocellulose membrane filters .
phage activity was determined by spot tests on lawns of the reference strains c. jejuni nctc <dig> and c. coli nctc <dig>  <cit> .
three previously described  <cit>  group ii phages , obtained from nctc were included in the study.
all phages were purified by three consecutive single plaque-assays.
high-titre lysates of the phages were obtained by infecting 1 l cultures of the indicator strain  with phages at a moi of ~ <dig>  for 24 h. to remove remaining cells and debris, lysates were centrifuged for 30 min at  <dig>  × g and then filtrated .
phages were concentrated by ultracentrifugation and purified by cscl step gradients  <cit> .
plaque assays, single plaque isolations, and propagation of the phages were performed as previously described  <cit> .
host range determination
the host range of the phages was determined by activity assays.
400 μl of the respective indicator strain were mixed with 6 ml prewarmed nzcym soft agar  and poured onto a lb agar plate.
ten μl of each lysate  were spotted onto the overlay agar.
plates were incubated overnight at 42 °c under microaerobic conditions  <cit> .
transmission electron microscopy
cscl-purified phages were investigated by tem using the negative staining procedure with ammonium molybdate.
briefly, drops of phage preparations were applied to pioloform-carbon-coated, 400-mesh copper grids , incubated for 10 min and fixed with  <dig>  % aqueous glutaraldehyde  for 1 min.
thereafter, phages were stained with a 3 % aqueous ammonium molybdate  solution  for 3 min.
specimens were examined by tem using a jem- <dig>  at 80 kv accelerated voltage.
mass spectrometrical analysis of structural proteins
for the determination of cp <dig> structural proteins, cscl-purified phages were disintegrated by boiling in sds buffer for 10 min.
proteins were subjected to sds-page and separated at 20 ma on a one-dimensional 15 %  gel at 15 °c  <cit> .
bands of interest were excised and prepared for tryptic in gel-digests as previously described.
the prepared samples were subjected to tandem matrix-assisted laser desorption ionization-time-of-flight mass spectrometry  analysis.
mass spectra were analyzed and interpreted using the mascot software  followed by ncbi nr database  searches.
isolation of phage dna, restriction analysis and pcr
isolation of phage dna was conducted by incubating purified phage particles  in 10 mm tris–hcl , 1 mm edta,  <dig>  % sds supplemented with 2 μl of proteinase k  at 56 °c for 2 h followed by ethanol precipitation.
to analyse restriction patterns, phage dnas were digested with restriction endonucleases  according to the manufacturer’s recommendations.
restriction fragments were separated by agarose gel electrophoresis.
in general, pcr analyses were performed in an eppendorf mastercycler ep gradient  according to standard protocols as previously described  <cit> .
single reactions were carried out in a final volume of 50 μl by the use of the dreamtaq dna polymerase amplification components .
the mastermix of a reaction comprised  <dig>  μl rnase-free water,  <dig>  μl 10 × dreamtaq buffer, 5 μl dntp solution ,  <dig>  μl of dreamtaq enzyme, 5 μl of each primer and  <dig>  μl of template dna .
due to the low g + c content of campylobacter phage genomes, conditions  of the thermal/time profile of the pcr protocol had to be adapted as stated below.
for the determination of the modular structure of the campylobacter group ii phages, the same pcr protocol was used: initial pcr activation and template denaturation at 94 °c for 120 s followed by 35 cycles including denaturation phase at 94 °c for 15 s, annealing at 47 °c for 15 s and elongation for 210 s at 72 °c.
in addition, the protocol contained a final elongation step at 72 °c for 1 min before the pcr reactions were stored at 4 °c until further processing.
sequence determination and bioinformatic analysis of the cp <dig> genome
whole-genome sequencing was performed by lgc genomics  as previously described  <cit> .
library generation for  <dig> flx sequencing was carried out according to the manufacturer’s standard protocols .
phage dna was sheared by nebulization into fragments ranging in size from  <dig> to 1000 bp.
the fragments were end-polished and the  <dig> a and b adaptors required for the emulsion pcr and sequencing were added to the ends of the fragments by ligation.
the concentration of the resulting fragment library was measured by fluorometry  and sequencing was performed on 1/ <dig> picotiterplate  on the gs flx using roche/ <dig> titanium chemistry.
a total of  <dig>  sequence reads were assembled using the roche/ <dig> newbler software at default settings ).
assembly resulted in three independent contigs  with an average sequence coverage of more than  <dig> per consensus base.
gaps were closed by pcr and sanger sequencing.
to determine the sequences of repeat regions , pcr primers were deduced from their flanking sequences.
the repeat regions were amplified by pcr and used as targets for in vitro transposon mutagenesis using the epicentre ez < tnektet- <dig> insertion kit  according to the manufacturer’s recommendations.
mutagenized pcr products were inserted into plitmus <dig>  and introduced into e. coli strain genehogs .
after transformation, transformants were selected on agar containing tetracycline .
nucleotide sequences of eight to twelve transformants with transposon insertions at different positions within each repeat region were used to determine the whole sequences of the repeat regions.
sequencing was performed using primers deduced from the marker gene of the ez < tnektet- <dig> insertion kit .
the initial nucleotide sequence of the cp <dig> draft genome was submitted to embl under the accession number he <dig>  <cit> .
as the  <dig> sequencing technique is prone to frameshift errors at homopolymer stretches  <cit> , the genome sequence was verified at  <dig> critical regions.
an updated cp <dig> genome sequence was submitted to genbank in march  <dig>  sequence analyses and alignments were carried out using the accelrys ds gene software package of the accelrys inc. .
putative open reading frames  were suggested using the algorithm of the rast server .
similarity and identity values were calculated using different blast algorithms  at the ncbi homepage  <cit> .
putative rho-independent transcription terminators were identified using transterm  <cit>  and arnold .
RESULTS
morphology and host range of cp21
phage cp <dig> showed the typical morphology of a myovirus  with an isometric head  and a long contractile tail .
thus, it fits well into campylobacter phage group ii whose members have longer tails than group iii phages  <cit> .
as with other campylobacter phages, cp <dig> has a neck without a collar, and a small base plate.
its tail fibers are fragile and could therefore not be measured.
for the determination of the cp <dig> host range,  <dig> strains belonging to six campylobacter species were tested by spot assays.
phage activity was limited to the species c. jejuni and c. coli, of which  <dig> out of  <dig> and four out of  <dig> strains, respectively, were lysed  .
fig.
<dig> morphology and structural proteins of cp <dig>  a electron micrographs of cp <dig> phage particles with a non-contracted and a contracted tail.
the bars correspond to 100 nm.
b sds-page and mass spectrometric analysis of structural proteins
afor the determination of the ibb_ <dig> host range, only c. jejuni and c. coli strains were tested that were lysed by any other phage
phage cp <dig> is closely related to other group ii phages
the genomic sequence of cp <dig> was determined by  <dig> technology  <cit> .
as this technique is prone to frameshift errors at homopolymer stretches  <cit> , more than  <dig> critical regions of the cp <dig> genome showing striking deviations  to nearly identical sequences of other group ii phages were amplified by pcr and resequenced using the sanger method.
after verification, the published cp <dig> nucleotide sequence  <cit>  has been revised at these positions.
due to the circular permutation of the cp <dig> genome and to facilitate comparisons with related phages, the end points of the genome were defined in accordance with the cp <dig> genome  <cit> .
the cp <dig> genome  showed strong overall dna homologies  to the completely sequenced group ii phages cp <dig>  and cpt <dig>  and also to the five contigs of phage ibb_ <dig> .
however, the cp <dig> genome is  <dig>  kb and  <dig>  kb larger than the genomes of cp <dig> and cpt <dig>  respectively .
several dna regions,  <dig>  to 3 kb in size, are missing or relocated in the other group ii phages .
some of them encode homing endonucleases and transposases that may have caused deletions or genomic rearrangements.
this is clearly discernible in the region between the positions  <dig>  kb and  <dig>  kb which in cp <dig> harbors a transposase gene flanked by short dna repeats.
while repeats also exist at a similar position in cp <dig> and cpt <dig>  the transposase gene is located more than 100 kb apart in these two phages.
altogether, cp <dig> contains eight homing endonuclease and transposase genes whereas only three to five of these genes were detected in the other group ii phages  .
another striking difference between the phages is the fact that unlike cp <dig>  cpt <dig> and ibb_ <dig> which contain trna genes for arginine and tyrosine, cp <dig> possesses trna genes for threonine and proline .
myoviridae
myoviridae
myoviridae
myoviridae
c. jejuni, c. coli
c. jejuni, c. coli
c. jejuni, c. coli
c. jejuni, c. coli
sam s-adenosyl-l-methionine, orf open reading frame, pfge pulsed-field gel electrophoresis, n.a.
not available, gps gene products
athe genome characteristics of ibb_ <dig> base on five annotated contigs
baccording to the suggested arrangement of the five contigs, the majority of orfs  are located on the forward strand  <cit>
fig.
<dig> genetic map of the cp <dig> genome.
putative genes are coloured according to the predicted functions of their products.
the position of putative rho-independent transcription terminators, trna genes and repeat regions are indicated
at the protein level,  <dig> out of  <dig>  predicted cp <dig> products are more than 90 % identical to cp <dig> proteins  .
only for  <dig> products, no counterparts could be found in cp <dig>  cpt <dig> and ibb_ <dig>  the function of most of these proteins is unknown.
cp <dig> revealed almost no dna sequence relatedness to group iii phages.
though, a number of proteins, most of them involved in phage assembly and replication, show some similarity to products of group iii phages.
these common proteins of group ii and group iii phages represent modules  that are typically found in t4-type phages and make up the core genome.
a comparison of virion structural modules  demonstrated the distant relationship of cp <dig> and cp <dig> and also of group iii phage cp <dig> to t4-type phages   <cit> .
using mass spectrometry, a number of cp <dig> structural proteins similar to t <dig> head and tail proteins have been identified .
fig.
<dig> group ii phages contain very similar proteins.
circular illustration  of the cp <dig> genome and similarities of predicted products
to proteins of the campylobacter group ii phages cp <dig>  cpt <dig> and ibb_ <dig>  positions of the cp <dig> repeat regions and of putative rho-independent transcription terminators  are indicated
fig.
<dig> arrangement of structural genes in t <dig>  cp <dig> and cp <dig>  genes are denoted according to the nomenclature of t <dig> and functionally assigned.
wac, whisker antigen control gene
cp <dig> and cp220/cpt <dig> possess different genome organizations
while the arrangement and orientation of cp <dig> structural genes bear some resemblance to the modules of t4-type phages, the location and orientation of the corresponding cp <dig> genes is quite diverse .
the same holds true for genes involved in phage replication.
both structural and replication genes are not clustered in cp <dig>  instead, these genes are widely dispersed on the genome .
similar to cp <dig> and cpt <dig>  cp <dig> contains four closely related repeat regions  which divide the genome into four modules , three of which carrying genes for structural and replication proteins.
with the exception of one repeat region in cp <dig> and cpt <dig>  <cit> , the remaining regions are extragenic.
they are similarly located on the three phage genomes and show strong dna homologies to each other.
the dot plot alignment of the phages cp <dig> and cp220/cpt <dig> showed that their genomes are closely related but differently organized .
a closer view at the gene maps disclosed that two  of the four cp <dig> modules are inverted .
as a result many structural genes located in these regions are divergently orientated in cp <dig> and cp220/cpt <dig> .
by contrast, genes situated on module a and d are transcribed in the same direction in all three phages.
fig.
<dig> genome organization and dna homologies of cp <dig>  cp <dig> and cpt <dig>  a modular composition of the genomes.
the modules a, b, c, and
d are separated by long repeat regions .
in cp <dig> module b and c are inverted.
b dot plot alignment of cp <dig> with cpt <dig> and cp <dig>  alignments were performed with the ds gene software package of the accelrys inc. .
the numbers on the axes give the scale for the genomes in kilo bases
the repeat regions of group ii phages are variations of the same theme
the repeat regions rr <dig> to rr <dig> of cp <dig>  cp <dig> and cpt <dig> have a bipartite structure and are composed of numerous repeat units which encompass stretches of unique dna sequences .
some of the enclosed sequences represent genes, namely orf <dig> and the orfs  <dig> to  <dig> within repeat regions rr <dig> and rr <dig>  respectively .
notably, the cp <dig> rr <dig> repeat region, which is much larger than rr <dig> of cp <dig> and cpt <dig> contains two genes for hypothetical proteins that do not exist in the other two phages.
each arm of a repeat region is composed of up to  <dig> units, most of them  <dig> to 86 bp in length .
a core consensus sequence that was found in all cp <dig> units  has a length of  <dig> nucleotides and the sequence 5′-aaacttaaagcaa-3′.
this core sequence was also identified in most cp220/cpt <dig> units.
the comparison of the repeat regions revealed two peculiarities.
firstly, some regions contain direct, some others inverted repeats .
while cp <dig> possesses only one repeat region  comprising direct repeats, both rr <dig> and rr <dig> of cp220/cpt <dig> contain direct repeats.
thus, rr <dig> which is located on the left side of the inverted module b has a different structure in cp <dig> and cp220/cpt <dig>  it is conceivable that this aberration is the result of a recombination event between repeat regions yielding the inversion of the modules b and c. the second striking feature of the repeat regions is the diverse number of units.
this finding pertains to both the total number of units and the numbers of units within the two arms of each region, which, in some cases, vary considerably .
it is noteworthy that even the phages cp <dig> and cpt <dig> which share the same modular composition contain repeat regions revealing significant discrepancies.
this suggests the repeat regions to be targets for recombination.
the number of units can of course also be changed via slipped-strand mis-pairing or secondary structure formation during dna replication  <cit> .
it should be added that the three phages contain additional single units or the core consensus sequence at other positions on their genomes.
in cp <dig> there is e.g., a region  containing several copies of repeat units which, however, show rather extensive sequence polymorphisms .
these sites might be remnants of former recombination or slipped-strand mis-pairing events.
cp <dig> and cpt <dig> possess a similar repeat region, which exhibits a lower complexity than its counterpart in cp <dig> .
fig.
<dig> structure of the repeat regions and repeat units of cp <dig>  cp <dig> and cpt <dig>  a dot plot analyses of the  <dig> repeat regions.
the positions of the repeat regions on each genome are indicated.
b composition of the repeat units
fig.
<dig>
some repeat regions harbour genes.
the size of each repeat region and the number and orientation of the respective repeat units are stated.
empty boxes indicate repeat units that do not contain the complete core sequence.
the inner regions represent non-repetitive sequences, some of which encode proteins
a modular composition is common in group ii phage genomes
to examine whether other yet not characterized group ii phages also possess a modular genome organization, a pcr system was established that enabled us to determine the order of modules.
for this study pcr primers were deduced from conserved sequences flanking the repeat regions of cp <dig>  cp <dig> and cpt <dig>  due to the very low gc content of campylobacter phages and since the long repeat regions probably generate secondary structures that might impede their amplification, primers of up to  <dig> nucleotides in length were designed .
the primers were used in different combinations to identify several modular compositions.
as shown in fig.
8a, single products similar in size as those of the reference phages were obtained in most pcr reactions.
the study disclosed that two phages  possess the same modular arrangement like cp <dig> while three other phages  belong to the subgroup represented by cp220/cpt <dig>  none of the investigated phages showed an individual genome organization.
thus, the hitherto analysed group ii phages belong to two subgroups of which cp <dig> and cp220/cpt <dig> are the prototypes.
fig.
<dig> classification of the investigated phages into two subgroups.
a pcr analysis of the modular composition of the phage genomes.
lane  <dig>  lane  <dig>  lane  <dig>  lane  <dig>  lane  <dig>  lane  <dig> show amplicons obtained with the phage dnas of cp <dig>  cp <dig>  cp <dig>  cp <dig>  cp <dig>  cp <dig>  respectively.
b vspi restriction patterns of the analysed phages
it has already been pointed out that campylobacter phage dnas can efficiently be cleaved by restriction endonucleases recognizing sheer at-sequences  <cit> .
we now compared vspi restriction patterns of group ii phages belonging to different subgroups.
while members of each subgroup exhibited very similar or even identical restriction patterns, the profiles of phages belonging to different subgroups were more divergent .
by in silico analysis of the cp <dig> and cp <dig> genomic sequences it became evident that the differences in vspi restriction patterns of these phages were mainly caused by their diverging modular organization.
thus, restriction analysis might be a simple method to classify a group ii phage into one of the two currently known subgroups or to detect new subgroups.
analysis of phage ibb_35
the technique used to study the genome organization of group ii phages was also applied to ibb_ <dig>  with this phage attempts to close gaps between five contigs were unsuccessful probably due to substances or modifications inhibiting taq and Φ <dig> polymerases and dna restriction  <cit> .
in addition, a striking discrepancy has been observed between the genome sizes calculated by sequencing and pulsed-field gel electrophoresis .
at first we determined the susceptibility of ibb_ <dig> dna to restriction endonucleases which exclusively recognize a/t sequences.
as shown in fig.
9a, dna of this phage was efficiently cleaved by smii and vspi like the dnas of cp <dig>  and cp <dig>  however, based on its restriction patterns, ibb_ <dig> could not be clearly allocated to one of the subgroups.
in the next step, gaps between the ibb_ <dig> contigs were analysed by pcr.
using different combinations of long primers deduced from the ends of the published ibb_ <dig> sequences, we obtained amplicons that disclosed the arrangement of contigs  and demonstrated that ibb_ <dig> possesses the same modular composition as cp <dig> .
the study also confirmed that contig  <dig> and contig  <dig> are linked and showed that the pcr products  were significantly larger than predicted   <cit> .
summing up the sizes of the five contigs and the amplicons, which represent the yet not characterized gaps, a genome size of 180 kb was calculated.
to get some information about the dna regions connecting the contigs, we sequenced the pcr products.
even though we failed to determine the complete sequences of all amplicons, partial sequences were obtained.
the data clearly showed that the ibb_ <dig> contigs are indeed linked by repetitive sequences.
furthermore, the core consensus sequence identified within the repeat regions of the other group ii phages was also found in ibb_ <dig>  in addition, some yet not identified genes probably encoding a head completion protein and other hypothetical proteins were detected in the amplified dna.
these genes are homologous to genes found within the repeat regions rr <dig> and rr <dig> of cp <dig> and cp220/cpt <dig>  thus, ibb_ <dig> possesses some characteristics typical for a group ii phage.
fig.
<dig> analysis of the phage ibb_ <dig> genome.
a smii and vspi restriction patterns.
lane  <dig>  cp <dig>  lane  <dig>  ibb_ <dig>  lane  <dig>  cp <dig>  b order of the five ibb_ <dig> contigs  determined by pcr.
c ibb_ <dig> shows the same modular genome organization as cp220
each phage subgroup exhibited a distinct host range
the previous studies demonstrated that many structural genes of the phages, some of which encoding tail proteins are differently arranged in the two subgroups.
even though the inversion of the modules b and c did not affect the integrity of the respective genes, it cannot be excluded that the genomic rearrangement resulted in a change of phenotypic properties.
to address this issue, we determined the host specificity of members of each subgroup.
for this study,  <dig> campylobacter spp.
strains were investigated in terms of phage susceptibility .
as shown in table  <dig>  phages belonging to the cp220/cpt <dig> subgroup lysed a significant higher number of strains than members of the cp <dig> subgroup.
moreover, phages of each subgroup exhibited similar host ranges.
they mostly lysed the same strains .
thus, there was a striking coincidence between the modular organization and the host specificity of group ii phages.
the only exception was ibb_ <dig> which lysed a high proportion of c. coli strains but, similar to phages of the cp <dig> subgroup, only few c. jejuni strains.
gene product  <dig>  of campylobacter phages has already been identified as a receptor binding protein  <cit> .
the host recognition domains are localized in the conserved c-terminus of gp <dig>  to elucidate whether variations within gp <dig> may account for the observed differences in host specificity, we analysed gp <dig> nucleotide sequences of the group ii phages.
compared to members of the cp <dig> subgroup, all phages belonging to the cp <dig> subgroup revealed a deletion  within gp <dig>  .
in addition, the region encoding the c-terminus of gp <dig> was more similar within each subgroup than to members of the other subgroup .
only gp <dig> of ibb_ <dig> showed some sequence deviations, which might be responsible for the distinct host range of this phage.
the data indicate that the subgroups do not only differ in their genome organization but also in particular genes.
this finding was corroborated by a pcr analysis of trna genes.
as with cp <dig> all members of its subgroup revealed trna genes for threonine and proline, while phages belonging to the cp <dig> subgroup possess trna genes for arginine and tyrosine.
fig.
<dig> similarities of the tail fiber genes.
a schematic illustration of the genes indicating the deletion that was found in the cp <dig> subgroup.
b dendrogram showing the similarities of the tail fiber genes
discussion
over the last decade an increasing number of reports have been published describing the reduction of campylobacter in chicken and on food products by application of lytic phages .
one prerequisite for a commercial exploitation of phages is a profound knowledge on their biology and genetics.
thus far, a number of campylobacter group ii and group iii phages has been genetically characterized  <cit> .
all phages of the same group are closely related to each other while at the nucleotide level, there is only limited similarity between phages belonging to different groups  <cit> .
the genomic analysis of cp <dig> confirmed the strong and more distant relationship to other group ii and group iii phages, respectively.
however, the study also disclosed that group ii phages are not uniform in terms of their gene content and genome organization.
while group iii phages possess numerous homing endonucleases that might be important for reorganization of genes  <cit> , homing endonucleases are less frequently found in group ii phages.
though, group ii phages encode transposases which similarly might be involved in genetic rearrangements.
cp <dig> e.g., codes for five putative transposases, and in cp <dig> four transposase genes have been detected.
in addition, the alignment of the cp <dig> and cp <dig> genomes revealed a number of sites where transposase activities may have caused deletions  <cit> .
besides these small-scale changes, group ii phages have obviously also used shuffling of larger dna fragments resulting in more pronounced genomic variations.
the detailed analysis of the repeat regions of cp <dig> and cp220/cpt <dig> showed that all of them contain the same core consensus sequence that we also found in ibb_ <dig>  moreover, adjacent sequences inside each unit are also related to each other.
therefore, modular shuffling might have occurred both within and between group ii phage genomes.
the striking diversity of the number and orientation of units within each repeat region points to previous recombination events.
notable is the fact that the analysed group ii phages additionally contain single repeat units or the 13 bp core sequence on their genomes.
a massive occurrence of repetitive sequences as is found in cp <dig>  cp <dig> and cpt <dig> is rather uncommon for phages.
in tsukamurella phage tpa <dig> a large number of inverted repeats have been detected but the function of these repeat structures is unknown since neither replication origins nor transposable elements could be identified on the tpa <dig> genome  <cit> .
in this study, a pcr was developed, by use of which the modular organization of the cp <dig> genome has been confirmed.
our pcr and restriction analyses of yet not sequenced group ii phages suggest that these are similarly composed of modules.
this also pertains to phage ibb_ <dig> where previous attempts failed to join five single contigs that correspond well with the modules of cp220/cpt <dig>  <cit> .
it should be emphasized that in contrast to the analysis of phage cp <dig>  <cit> , we did not obtain amplicons of mixed lengths in our pcr experiments.
one important difference of the studies is that some of our primers were approximately twice as long as those used with cp <dig>  due to the very high a + t content of the phage dnas and secondary structures probably formed by the repeat regions, it is likely that the long primers were bound more specifically avoiding the synthesis of by products.
as it can be seen in fig.
7a, some pcr reactions showed a smear but we never observed a ladder of amplicons as it was shown for cp <dig>  <cit> .
hence, it is questionable whether group ii phages actually possess genomes of variable length.
so far, only two subgroups of group ii phages possessing distinct modular arrangements have been identified.
due to the wide distribution of the repeat core sequence on the phage genomes further subgroups might exist.
though, the currently available data suggest that the subgroups did not evolve recently because members of each subgroup do not only possess the same composition of modules but also the same trnas and similar tail fiber genes.
moreover, a sequence analysis of the tail tube gene of the phages revealed a number of positions with subgroup-specific nucleotides .
this points to a more ancient split of group ii phages.
it is of course also possible that other modular compositions may have dramatically consequences since they might affect the transcription of genes which can disrupt the integrity of the phages yielding defective particles.
the inversion of the modules b and c in cp <dig> resulted in a reorganization of structural genes but this variation had apparently no influence on the activity of the phage.
yet, the subgroups differed significantly in their host range.
even though there were some minor divergencies within each subgroup, their members showed very similar lysis patterns.
whether the host specificity of the subgroups is mainly determined by the modular genome organization, by sequence variations within single products like gp <dig>  by both or by other factors has still to be unravelled.
nevertheless, we found a clear link between the host range of particular phages, their modular genome composition and sequence polymorphisms of gp <dig>  the exception to this rule was phage ibb_ <dig>  on the one hand it possesses general properties similar to other group ii phages.
this also refers to the behaviour of the dna during phenol extraction.
partitioning into the interphase, how we observed it with group iii phages  <cit>  did not occur with ibb_ <dig>  on the other hand, however, there was a discrepancy between the modular genome organisation of ibb_ <dig>  which is the same as in phages of the cp <dig> subgroup, and the host range of the phage.
ibb_ <dig> lysed significantly less c. jejuni strains than e.g., cp <dig>  moreover, ibb_ <dig> revealed distinct vspi and smii restriction pattern and its gp <dig> and tail tube gene showed a number of nucleotide exchanges in relation to other phages of the cp <dig> subgroup.
we therefore conclude that ibb_ <dig> represents a more distantly related member of this subgroup.
CONCLUSIONS
in summary the presented data demonstrate that campylobacter group ii phages are diverse regarding their genome organization.
the currently identified subgroups are at least also different in terms of their tail fiber, tail tube and trna genes.
it is conceivable that the subgroups evolved a long time ago and that they do not undergo further changes.
virulence-associated genes have yet not been detected in campylobacter phages.
whether they are genetically stable, which would be another important prerequisite for their exploitation, has still to be elucidated by further experiments focussing on the question which role homing endonucleases and transposases may play for genetic variations, e.g., the acquisition of foreign genes.
sequencing of more group ii phage genomes would additionally be conducive to answer the question, whether these phages are suitable and save.
availability of supporting data
data sets supporting the results of this article are included within the article and its additional files.
the genome sequence determined in this study has been deposited in the national center for biotechnology information  genbank database under accession number he <dig>  access to the data is available upon publication at http://www.ncbi.nlm.nih.gov/nuccore/.
additional file
additional file 1: table s <dig>   strain specificity of campylobacter phages.
table s <dig>  cp <dig> dna regions that are absent or relocated in the other group ii phages.
table s <dig>  cp <dig> orf analysis.
table s <dig>  primers used in this study.
table s <dig>  nucleotide differences within the group ii tail tube genes.
figure s <dig>  additional repeat region on the genomes of cp <dig>  cp <dig> and cpt <dig>  dot plot analyses of the additional repeat region of cp <dig> , cp <dig>  and cpt <dig> .
abbreviations
bfrbundesinstitut für risikobewertung
cnetcampynet
csclcaesium chloride
dsmzgerman collection of microorganisms and cell cultures
gpgene product
ibb_35vb_ccom-ibb_35
nctcnational collection of type cultures
odoptical density
orfopen reading frame
lblysogeny broth
ptppicotiterplate
rrrepeat regions
sm-buffersodium chloride/magnesium sulfate-buffer
temtransmission electron microscopy
wt/volweight per volume
claudia jäckel and jens a. hammerl contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
sh, jah, cj, jr and ak wrote the manuscript, analyzed data and produced tables and figures.
jr analyzed the structure of the phage particles.
all authors edited the manuscript.
all authors read and approved the final manuscript.
acknowledgment
phage ibb_ <dig> was kindly provided by dr. joana azeredo, centre of biological engineering, university of minho, braga, portugal.

 campylobacter is one of the most important foodborne pathogens worldwide. the genus comprises many species of which the thermophilic c. jejuni and its close relative c. coli are reported as the most common causes of acute bacterial enteritis. the bacteria are commensals of the gastrointestinal tract of various mammals and birds and are frequently found in chicken flocks where they may spread rapidly. human infections are caused mainly by the consumption of undercooked meats, especially poultry. to reduce the number of campylobacter along the food chain, various biosecurity measures and post-slaughter decontamination procedures have been tested. as these measures are expensive and not always efficient and since suitable vaccines are not available, phages might be an appropriate means to reduce campylobacter counts in chicken and on chicken products. indeed phage administration in the laboratory reduced c. jejuni colonization of the broiler gut and the contamination on chicken skin by several orders of magnitude. a phage cocktail applied via drinking water also efficiently reduced campylobacter counts in chicken on a commercial broiler farm. phages intended for therapeutic applications or for the control of pathogens in food production have to fulfil a number of requirements to avoid any undesired side effect. amongst others, the phage genomes have to be stable and free from critical genes. therefore, a basic understanding of the biology and genetics of the phages to be used is of great importance. according to their genome size, campylobacter phages described so far are classified into three groups. while group i phages have been rarely isolated and have not been used for applications as yet, members of group ii and group iii are very common. group ii phages might be best suited for the reduction of campylobacter since they can infect both c. jejuni and c. coli strains. though, the successive application of a group iii and a group ii phage reduced the numbers of c. jejuni in chickens most efficiently. up to now three group ii phages have been completely sequenced while vb_ccom-ibb_ is represented, in genbank, in the form of five contigs. the phages revealed strong dna homologies to each other whereas only little dna sequence similarities have been detected to group iii phages. group ii campylobacter phages contain e.g., genes for membrane proteins, transposases, and metabolic enzymes including up to twelve proteins with s-adenosylmethionine domains that are absent in group iii. conversely, group iii phages show an expansion of homing endonucleases, represented by up to nine orfs, that are much less frequently occurring in group ii. however, at the protein level both groups showed some relationship to t4-type phages. a striking feature of cp cpt and cp is that their genomes are composed of large modules separated by long dna repeat regions, which could lead to rearrangements. while the genomes of cp and cpt are collinear, the genome of cp exhibits considerable rearrangement of homologous modules. functional modules are common in temperate and virulent phages. they are shuffled by recombination which may result in new combinations of modules and the emergence of novel phages. the frequency of recombination events largely depends on the phylogenetic distance between the respective hosts. illegitimate recombination events are rather rare and may often lead to defective particles but can take place at nearly random points across the genome. on the other hand, homologous recombinations of modules at specific linker sequences occur more frequently, particularly in closely related phages like those belonging to the lambdoid phage family. in this work, the genome organization of cp was analysed in detail with special focus on the repeat regions which impede the elucidation of the genome structure of group ii phages. on the basis of the available group ii sequences, a pcr typing system was established to study the modular genome arrangement of other yet not characterized group ii phages. the system was also used to unravel the genome organization of phage ibb_ we show that group ii phages can be allocated to at least two subgroups which revealed distinct host ranges. methods bacterial strains and culture conditions most strains originated from the campynet strain collection hosted by the leibniz institute dsmz-german collection of microorganisms and cell cultures, braunschweig, germany,. the remaining strains were taken from the campylobacter strain collection of the national reference laboratory for campylobacter at the federal institute for risk assessment ). if not otherwise indicated, the c. coli strain nctc was used for phage propagation and plaque assays. the cultivation of bacteria was performed as previously described. isolation, propagation and purification of group ii campylobacter phages the phages cp cp and cp were isolated from poultry produced on organic farms in berlin, germany. to recover phages, samples were suspended in sm-buffer and incubated overnight at 45 °c on a stirrer. thereafter, the samples were centrifuged at  × g followed by filtration of the supernatants through  μm nitrocellulose membrane filters. phage activity was determined by spot tests on lawns of the reference strains c. jejuni nctc and c. coli nctc. three previously described group ii phages, obtained from nctc were included in the study. all phages were purified by three consecutive single plaque-assays. high-titre lysates of the phages were obtained by infecting 1 l cultures of the indicator strain with phages at a moi of ~ for 24 h. to remove remaining cells and debris, lysates were centrifuged for 30 min at  × g and then filtrated. phages were concentrated by ultracentrifugation and purified by cscl step gradients. plaque assays, single plaque isolations, and propagation of the phages were performed as previously described. host range determination the host range of the phages was determined by activity assays. 400 μl of the respective indicator strain were mixed with 6 ml prewarmed nzcym soft agar and poured onto a lb agar plate. ten μl of each lysate were spotted onto the overlay agar. plates were incubated overnight at 42 °c under microaerobic conditions. transmission electron microscopy cscl-purified phages were investigated by tem using the negative staining procedure with ammonium molybdate. briefly, drops of phage preparations were applied to pioloform-carbon-coated, 400-mesh copper grids, incubated for 10 min and fixed with  % aqueous glutaraldehyde for 1 min. thereafter, phages were stained with a 3 % aqueous ammonium molybdate solution for 3 min. specimens were examined by tem using a jem- at 80 kv accelerated voltage. mass spectrometrical analysis of structural proteins for the determination of cp structural proteins, cscl-purified phages were disintegrated by boiling in sds buffer for 10 min. proteins were subjected to sds-page and separated at 20 ma on a one-dimensional 15 % gel at 15 °c. bands of interest were excised and prepared for tryptic in gel-digests as previously described. the prepared samples were subjected to tandem matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. mass spectra were analyzed and interpreted using the mascot software followed by ncbi nr database searches. isolation of phage dna, restriction analysis and pcr isolation of phage dna was conducted by incubating purified phage particles in 10 mm tris–hcl, 1 mm edta,  % sds supplemented with 2 μl of proteinase k at 56 °c for 2 h followed by ethanol precipitation. to analyse restriction patterns, phage dnas were digested with restriction endonucleases according to the manufacturer’s recommendations. restriction fragments were separated by agarose gel electrophoresis. in general, pcr analyses were performed in an eppendorf mastercycler ep gradient according to standard protocols as previously described. single reactions were carried out in a final volume of 50 μl by the use of the dreamtaq dna polymerase amplification components. the mastermix of a reaction comprised  μl rnase-free water,  μl 10 × dreamtaq buffer, 5 μl dntp solution,  μl of dreamtaq enzyme, 5 μl of each primer and  μl of template dna. due to the low g + c content of campylobacter phage genomes, conditions of the thermal/time profile of the pcr protocol had to be adapted as stated below. for the determination of the modular structure of the campylobacter group ii phages, the same pcr protocol was used: initial pcr activation and template denaturation at 94 °c for 120 s followed by 35 cycles including denaturation phase at 94 °c for 15 s, annealing at 47 °c for 15 s and elongation for 210 s at 72 °c. in addition, the protocol contained a final elongation step at 72 °c for 1 min before the pcr reactions were stored at 4 °c until further processing. sequence determination and bioinformatic analysis of the cp genome whole-genome sequencing was performed by lgc genomics as previously described. library generation for flx sequencing was carried out according to the manufacturer’s standard protocols. phage dna was sheared by nebulization into fragments ranging in size from to 1000 bp. the fragments were end-polished and the a and b adaptors required for the emulsion pcr and sequencing were added to the ends of the fragments by ligation. the concentration of the resulting fragment library was measured by fluorometry and sequencing was performed on 1/ picotiterplate on the gs flx using roche/ titanium chemistry. a total of sequence reads were assembled using the roche/ newbler software at default settings ). assembly resulted in three independent contigs with an average sequence coverage of more than per consensus base. gaps were closed by pcr and sanger sequencing. to determine the sequences of repeat regions, pcr primers were deduced from their flanking sequences. the repeat regions were amplified by pcr and used as targets for in vitro transposon mutagenesis using the epicentre ez < tnektet- insertion kit according to the manufacturer’s recommendations. mutagenized pcr products were inserted into plitmus and introduced into e. coli strain genehogs. after transformation, transformants were selected on agar containing tetracycline. nucleotide sequences of eight to twelve transformants with transposon insertions at different positions within each repeat region were used to determine the whole sequences of the repeat regions. sequencing was performed using primers deduced from the marker gene of the ez < tnektet- insertion kit. the initial nucleotide sequence of the cp draft genome was submitted to embl under the accession number he. as the sequencing technique is prone to frameshift errors at homopolymer stretches, the genome sequence was verified at critical regions. an updated cp genome sequence was submitted to genbank in march sequence analyses and alignments were carried out using the accelrys ds gene software package of the accelrys inc.. putative open reading frames were suggested using the algorithm of the rast server. similarity and identity values were calculated using different blast algorithms at the ncbi homepage. putative rho-independent transcription terminators were identified using transterm and arnold.  morphology and host range of cp21 phage cp showed the typical morphology of a myovirus with an isometric head and a long contractile tail. thus, it fits well into campylobacter phage group ii whose members have longer tails than group iii phages. as with other campylobacter phages, cp has a neck without a collar, and a small base plate. its tail fibers are fragile and could therefore not be measured. for the determination of the cp host range, strains belonging to six campylobacter species were tested by spot assays. phage activity was limited to the species c. jejuni and c. coli, of which out of and four out of strains, respectively, were lysed. fig. morphology and structural proteins of cp a electron micrographs of cp phage particles with a non-contracted and a contracted tail. the bars correspond to 100 nm. b sds-page and mass spectrometric analysis of structural proteins afor the determination of the ibb_ host range, only c. jejuni and c. coli strains were tested that were lysed by any other phage phage cp is closely related to other group ii phages the genomic sequence of cp was determined by technology. as this technique is prone to frameshift errors at homopolymer stretches, more than critical regions of the cp genome showing striking deviations to nearly identical sequences of other group ii phages were amplified by pcr and resequenced using the sanger method. after verification, the published cp nucleotide sequence has been revised at these positions. due to the circular permutation of the cp genome and to facilitate comparisons with related phages, the end points of the genome were defined in accordance with the cp genome. the cp genome showed strong overall dna homologies to the completely sequenced group ii phages cp and cpt and also to the five contigs of phage ibb_. however, the cp genome is  kb and  kb larger than the genomes of cp and cpt respectively. several dna regions, to 3 kb in size, are missing or relocated in the other group ii phages. some of them encode homing endonucleases and transposases that may have caused deletions or genomic rearrangements. this is clearly discernible in the region between the positions  kb and  kb which in cp harbors a transposase gene flanked by short dna repeats. while repeats also exist at a similar position in cp and cpt the transposase gene is located more than 100 kb apart in these two phages. altogether, cp contains eight homing endonuclease and transposase genes whereas only three to five of these genes were detected in the other group ii phages. another striking difference between the phages is the fact that unlike cp cpt and ibb_ which contain trna genes for arginine and tyrosine, cp possesses trna genes for threonine and proline. myoviridae myoviridae myoviridae myoviridae c. jejuni, c. coli c. jejuni, c. coli c. jejuni, c. coli c. jejuni, c. coli sam s-adenosyl-l-methionine, orf open reading frame, pfge pulsed-field gel electrophoresis, n.a. not available, gps gene products athe genome characteristics of ibb_ base on five annotated contigs baccording to the suggested arrangement of the five contigs, the majority of orfs are located on the forward strand fig. genetic map of the cp genome. putative genes are coloured according to the predicted functions of their products. the position of putative rho-independent transcription terminators, trna genes and repeat regions are indicated at the protein level, out of predicted cp products are more than 90 % identical to cp proteins. only for products, no counterparts could be found in cp cpt and ibb_ the function of most of these proteins is unknown. cp revealed almost no dna sequence relatedness to group iii phages. though, a number of proteins, most of them involved in phage assembly and replication, show some similarity to products of group iii phages. these common proteins of group ii and group iii phages represent modules that are typically found in t4-type phages and make up the core genome. a comparison of virion structural modules demonstrated the distant relationship of cp and cp and also of group iii phage cp to t4-type phages. using mass spectrometry, a number of cp structural proteins similar to t head and tail proteins have been identified. fig. group ii phages contain very similar proteins. circular illustration of the cp genome and similarities of predicted products to proteins of the campylobacter group ii phages cp cpt and ibb_ positions of the cp repeat regions and of putative rho-independent transcription terminators are indicated fig. arrangement of structural genes in t cp and cp genes are denoted according to the nomenclature of t and functionally assigned. wac, whisker antigen control gene cp and cp220/cpt possess different genome organizations while the arrangement and orientation of cp structural genes bear some resemblance to the modules of t4-type phages, the location and orientation of the corresponding cp genes is quite diverse. the same holds true for genes involved in phage replication. both structural and replication genes are not clustered in cp instead, these genes are widely dispersed on the genome. similar to cp and cpt cp contains four closely related repeat regions which divide the genome into four modules, three of which carrying genes for structural and replication proteins. with the exception of one repeat region in cp and cpt, the remaining regions are extragenic. they are similarly located on the three phage genomes and show strong dna homologies to each other. the dot plot alignment of the phages cp and cp220/cpt showed that their genomes are closely related but differently organized. a closer view at the gene maps disclosed that two of the four cp modules are inverted. as a result many structural genes located in these regions are divergently orientated in cp and cp220/cpt. by contrast, genes situated on module a and d are transcribed in the same direction in all three phages. fig. genome organization and dna homologies of cp cp and cpt a modular composition of the genomes. the modules a, b, c, and d are separated by long repeat regions. in cp module b and c are inverted. b dot plot alignment of cp with cpt and cp alignments were performed with the ds gene software package of the accelrys inc.. the numbers on the axes give the scale for the genomes in kilo bases the repeat regions of group ii phages are variations of the same theme the repeat regions rr to rr of cp cp and cpt have a bipartite structure and are composed of numerous repeat units which encompass stretches of unique dna sequences. some of the enclosed sequences represent genes, namely orf and the orfs to within repeat regions rr and rr respectively. notably, the cp rr repeat region, which is much larger than rr of cp and cpt contains two genes for hypothetical proteins that do not exist in the other two phages. each arm of a repeat region is composed of up to units, most of them to 86 bp in length. a core consensus sequence that was found in all cp units has a length of nucleotides and the sequence 5′-aaacttaaagcaa-3′. this core sequence was also identified in most cp220/cpt units. the comparison of the repeat regions revealed two peculiarities. firstly, some regions contain direct, some others inverted repeats. while cp possesses only one repeat region comprising direct repeats, both rr and rr of cp220/cpt contain direct repeats. thus, rr which is located on the left side of the inverted module b has a different structure in cp and cp220/cpt it is conceivable that this aberration is the result of a recombination event between repeat regions yielding the inversion of the modules b and c. the second striking feature of the repeat regions is the diverse number of units. this finding pertains to both the total number of units and the numbers of units within the two arms of each region, which, in some cases, vary considerably. it is noteworthy that even the phages cp and cpt which share the same modular composition contain repeat regions revealing significant discrepancies. this suggests the repeat regions to be targets for recombination. the number of units can of course also be changed via slipped-strand mis-pairing or secondary structure formation during dna replication. it should be added that the three phages contain additional single units or the core consensus sequence at other positions on their genomes. in cp there is e.g., a region containing several copies of repeat units which, however, show rather extensive sequence polymorphisms. these sites might be remnants of former recombination or slipped-strand mis-pairing events. cp and cpt possess a similar repeat region, which exhibits a lower complexity than its counterpart in cp. fig. structure of the repeat regions and repeat units of cp cp and cpt a dot plot analyses of the repeat regions. the positions of the repeat regions on each genome are indicated. b composition of the repeat units fig. some repeat regions harbour genes. the size of each repeat region and the number and orientation of the respective repeat units are stated. empty boxes indicate repeat units that do not contain the complete core sequence. the inner regions represent non-repetitive sequences, some of which encode proteins a modular composition is common in group ii phage genomes to examine whether other yet not characterized group ii phages also possess a modular genome organization, a pcr system was established that enabled us to determine the order of modules. for this study pcr primers were deduced from conserved sequences flanking the repeat regions of cp cp and cpt due to the very low gc content of campylobacter phages and since the long repeat regions probably generate secondary structures that might impede their amplification, primers of up to nucleotides in length were designed. the primers were used in different combinations to identify several modular compositions. as shown in fig. 8a, single products similar in size as those of the reference phages were obtained in most pcr reactions. the study disclosed that two phages possess the same modular arrangement like cp while three other phages belong to the subgroup represented by cp220/cpt none of the investigated phages showed an individual genome organization. thus, the hitherto analysed group ii phages belong to two subgroups of which cp and cp220/cpt are the prototypes. fig. classification of the investigated phages into two subgroups. a pcr analysis of the modular composition of the phage genomes. lane lane lane lane lane lane show amplicons obtained with the phage dnas of cp cp cp cp cp cp respectively. b vspi restriction patterns of the analysed phages it has already been pointed out that campylobacter phage dnas can efficiently be cleaved by restriction endonucleases recognizing sheer at-sequences. we now compared vspi restriction patterns of group ii phages belonging to different subgroups. while members of each subgroup exhibited very similar or even identical restriction patterns, the profiles of phages belonging to different subgroups were more divergent. by in silico analysis of the cp and cp genomic sequences it became evident that the differences in vspi restriction patterns of these phages were mainly caused by their diverging modular organization. thus, restriction analysis might be a simple method to classify a group ii phage into one of the two currently known subgroups or to detect new subgroups. analysis of phage ibb_35 the technique used to study the genome organization of group ii phages was also applied to ibb_ with this phage attempts to close gaps between five contigs were unsuccessful probably due to substances or modifications inhibiting taq and Φ polymerases and dna restriction. in addition, a striking discrepancy has been observed between the genome sizes calculated by sequencing and pulsed-field gel electrophoresis. at first we determined the susceptibility of ibb_ dna to restriction endonucleases which exclusively recognize a/t sequences. as shown in fig. 9a, dna of this phage was efficiently cleaved by smii and vspi like the dnas of cp and cp however, based on its restriction patterns, ibb_ could not be clearly allocated to one of the subgroups. in the next step, gaps between the ibb_ contigs were analysed by pcr. using different combinations of long primers deduced from the ends of the published ibb_ sequences, we obtained amplicons that disclosed the arrangement of contigs and demonstrated that ibb_ possesses the same modular composition as cp. the study also confirmed that contig and contig are linked and showed that the pcr products were significantly larger than predicted. summing up the sizes of the five contigs and the amplicons, which represent the yet not characterized gaps, a genome size of 180 kb was calculated. to get some information about the dna regions connecting the contigs, we sequenced the pcr products. even though we failed to determine the complete sequences of all amplicons, partial sequences were obtained. the data clearly showed that the ibb_ contigs are indeed linked by repetitive sequences. furthermore, the core consensus sequence identified within the repeat regions of the other group ii phages was also found in ibb_ in addition, some yet not identified genes probably encoding a head completion protein and other hypothetical proteins were detected in the amplified dna. these genes are homologous to genes found within the repeat regions rr and rr of cp and cp220/cpt thus, ibb_ possesses some characteristics typical for a group ii phage. fig. analysis of the phage ibb_ genome. a smii and vspi restriction patterns. lane cp lane ibb_ lane cp b order of the five ibb_ contigs determined by pcr. c ibb_ shows the same modular genome organization as cp220 each phage subgroup exhibited a distinct host range the previous studies demonstrated that many structural genes of the phages, some of which encoding tail proteins are differently arranged in the two subgroups. even though the inversion of the modules b and c did not affect the integrity of the respective genes, it cannot be excluded that the genomic rearrangement resulted in a change of phenotypic properties. to address this issue, we determined the host specificity of members of each subgroup. for this study, campylobacter spp. strains were investigated in terms of phage susceptibility. as shown in table  phages belonging to the cp220/cpt subgroup lysed a significant higher number of strains than members of the cp subgroup. moreover, phages of each subgroup exhibited similar host ranges. they mostly lysed the same strains. thus, there was a striking coincidence between the modular organization and the host specificity of group ii phages. the only exception was ibb_ which lysed a high proportion of c. coli strains but, similar to phages of the cp subgroup, only few c. jejuni strains. gene product of campylobacter phages has already been identified as a receptor binding protein. the host recognition domains are localized in the conserved c-terminus of gp to elucidate whether variations within gp may account for the observed differences in host specificity, we analysed gp nucleotide sequences of the group ii phages. compared to members of the cp subgroup, all phages belonging to the cp subgroup revealed a deletion within gp. in addition, the region encoding the c-terminus of gp was more similar within each subgroup than to members of the other subgroup. only gp of ibb_ showed some sequence deviations, which might be responsible for the distinct host range of this phage. the data indicate that the subgroups do not only differ in their genome organization but also in particular genes. this finding was corroborated by a pcr analysis of trna genes. as with cp all members of its subgroup revealed trna genes for threonine and proline, while phages belonging to the cp subgroup possess trna genes for arginine and tyrosine. fig. similarities of the tail fiber genes. a schematic illustration of the genes indicating the deletion that was found in the cp subgroup. b dendrogram showing the similarities of the tail fiber genes discussion over the last decade an increasing number of reports have been published describing the reduction of campylobacter in chicken and on food products by application of lytic phages. one prerequisite for a commercial exploitation of phages is a profound knowledge on their biology and genetics. thus far, a number of campylobacter group ii and group iii phages has been genetically characterized. all phages of the same group are closely related to each other while at the nucleotide level, there is only limited similarity between phages belonging to different groups. the genomic analysis of cp confirmed the strong and more distant relationship to other group ii and group iii phages, respectively. however, the study also disclosed that group ii phages are not uniform in terms of their gene content and genome organization. while group iii phages possess numerous homing endonucleases that might be important for reorganization of genes, homing endonucleases are less frequently found in group ii phages. though, group ii phages encode transposases which similarly might be involved in genetic rearrangements. cp e.g., codes for five putative transposases, and in cp four transposase genes have been detected. in addition, the alignment of the cp and cp genomes revealed a number of sites where transposase activities may have caused deletions. besides these small-scale changes, group ii phages have obviously also used shuffling of larger dna fragments resulting in more pronounced genomic variations. the detailed analysis of the repeat regions of cp and cp220/cpt showed that all of them contain the same core consensus sequence that we also found in ibb_ moreover, adjacent sequences inside each unit are also related to each other. therefore, modular shuffling might have occurred both within and between group ii phage genomes. the striking diversity of the number and orientation of units within each repeat region points to previous recombination events. notable is the fact that the analysed group ii phages additionally contain single repeat units or the 13 bp core sequence on their genomes. a massive occurrence of repetitive sequences as is found in cp cp and cpt is rather uncommon for phages. in tsukamurella phage tpa a large number of inverted repeats have been detected but the function of these repeat structures is unknown since neither replication origins nor transposable elements could be identified on the tpa genome. in this study, a pcr was developed, by use of which the modular organization of the cp genome has been confirmed. our pcr and restriction analyses of yet not sequenced group ii phages suggest that these are similarly composed of modules. this also pertains to phage ibb_ where previous attempts failed to join five single contigs that correspond well with the modules of cp220/cpt. it should be emphasized that in contrast to the analysis of phage cp, we did not obtain amplicons of mixed lengths in our pcr experiments. one important difference of the studies is that some of our primers were approximately twice as long as those used with cp due to the very high a + t content of the phage dnas and secondary structures probably formed by the repeat regions, it is likely that the long primers were bound more specifically avoiding the synthesis of by products. as it can be seen in fig. 7a, some pcr reactions showed a smear but we never observed a ladder of amplicons as it was shown for cp. hence, it is questionable whether group ii phages actually possess genomes of variable length. so far, only two subgroups of group ii phages possessing distinct modular arrangements have been identified. due to the wide distribution of the repeat core sequence on the phage genomes further subgroups might exist. though, the currently available data suggest that the subgroups did not evolve recently because members of each subgroup do not only possess the same composition of modules but also the same trnas and similar tail fiber genes. moreover, a sequence analysis of the tail tube gene of the phages revealed a number of positions with subgroup-specific nucleotides. this points to a more ancient split of group ii phages. it is of course also possible that other modular compositions may have dramatically consequences since they might affect the transcription of genes which can disrupt the integrity of the phages yielding defective particles. the inversion of the modules b and c in cp resulted in a reorganization of structural genes but this variation had apparently no influence on the activity of the phage. yet, the subgroups differed significantly in their host range. even though there were some minor divergencies within each subgroup, their members showed very similar lysis patterns. whether the host specificity of the subgroups is mainly determined by the modular genome organization, by sequence variations within single products like gp by both or by other factors has still to be unravelled. nevertheless, we found a clear link between the host range of particular phages, their modular genome composition and sequence polymorphisms of gp the exception to this rule was phage ibb_ on the one hand it possesses general properties similar to other group ii phages. this also refers to the behaviour of the dna during phenol extraction. partitioning into the interphase, how we observed it with group iii phages did not occur with ibb_ on the other hand, however, there was a discrepancy between the modular genome organisation of ibb_ which is the same as in phages of the cp subgroup, and the host range of the phage. ibb_ lysed significantly less c. jejuni strains than e.g., cp moreover, ibb_ revealed distinct vspi and smii restriction pattern and its gp and tail tube gene showed a number of nucleotide exchanges in relation to other phages of the cp subgroup. we therefore conclude that ibb_ represents a more distantly related member of this subgroup.  in summary the presented data demonstrate that campylobacter group ii phages are diverse regarding their genome organization. the currently identified subgroups are at least also different in terms of their tail fiber, tail tube and trna genes. it is conceivable that the subgroups evolved a long time ago and that they do not undergo further changes. virulence-associated genes have yet not been detected in campylobacter phages. whether they are genetically stable, which would be another important prerequisite for their exploitation, has still to be elucidated by further experiments focussing on the question which role homing endonucleases and transposases may play for genetic variations, e.g., the acquisition of foreign genes. sequencing of more group ii phage genomes would additionally be conducive to answer the question, whether these phages are suitable and save. availability of supporting data data sets supporting the results of this article are included within the article and its additional files. the genome sequence determined in this study has been deposited in the national center for biotechnology information genbank database under accession number he access to the data is available upon publication at http://www.ncbi.nlm.nih.gov/nuccore/. additional file additional file 1: table s strain specificity of campylobacter phages. table s cp dna regions that are absent or relocated in the other group ii phages. table s cp orf analysis. table s primers used in this study. table s nucleotide differences within the group ii tail tube genes. figure s additional repeat region on the genomes of cp cp and cpt dot plot analyses of the additional repeat region of cp, cp and cpt. abbreviations bfrbundesinstitut für risikobewertung cnetcampynet csclcaesium chloride dsmzgerman collection of microorganisms and cell cultures gpgene product ibb_35vb_ccom-ibb_35 nctcnational collection of type cultures odoptical density orfopen reading frame lblysogeny broth ptppicotiterplate rrrepeat regions sm-buffersodium chloride/magnesium sulfate-buffer temtransmission electron microscopy wt/volweight per volume claudia jäckel and jens a. hammerl contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions sh, jah, cj, jr and ak wrote the manuscript, analyzed data and produced tables and figures. jr analyzed the structure of the phage particles. all authors edited the manuscript. all authors read and approved the final manuscript. acknowledgment phage ibb_ was kindly provided by dr. joana azeredo, centre of biological engineering, university of minho, braga, portugal.
thus far, no genes with pathogenic potential have been detected in campylobacter phages. we determined and analysed the genomic sequence of group ii phage cp that is closely related to the already characterized group ii phages cp and cpt the genomes of these phages are comprised of four modules separated by very similar repeat regions, some of which harbouring open reading frames. though, the arrangement of the modules and the location of some orfs on the genomes are different in cp and in cp220/cpt in this work, a pcr system was established to study the modular genome organization of other group ii phages demonstrating that they belong to different subgroups of the cp220-like virus genus, the prototypes of which are cp and cp the subgroups revealed different restriction patterns and, interestingly enough, also distinct host specificities, tail fiber proteins and trna genes. however, preliminary studies suggested that the genomes of group ii phages may be diverse and prone to genomic rearrangements. campylobacter group ii phages are diverse regarding their genome organization. since all hitherto characterized group ii phages contain numerous genes for transposases and homing endonucleases as well as similar repeat regions, it cannot be excluded that these phages are genetically unstable. to answer this question, further experiments and sequencing of more group ii phages should be performed. the application of phages is a promising tool to reduce the number of campylobacter along the food chain. we show that the contigs represent modules linked by long repeat regions enclosing some yet not identified orfs.
 the application of phages is a promising tool to reduce the number of campylobacter along the food chain. besides the efficacy against a broad range of strains, phages have to be safe in terms of their genomes. thus far, no genes with pathogenic potential have been detected in campylobacter phages. however, preliminary studies suggested that the genomes of group ii phages may be diverse and prone to genomic rearrangements.  we determined and analysed the genomic sequence of group ii phage cp that is closely related to the already characterized group ii phages cp and cpt the genomes of these phages are comprised of four modules separated by very similar repeat regions, some of which harbouring open reading frames. though, the arrangement of the modules and the location of some orfs on the genomes are different in cp and in cp220/cpt in this work, a pcr system was established to study the modular genome organization of other group ii phages demonstrating that they belong to different subgroups of the cp220-like virus genus, the prototypes of which are cp and cp the subgroups revealed different restriction patterns and, interestingly enough, also distinct host specificities, tail fiber proteins and trna genes. we additionally analysed the genome of group ii phage vb_ccom-ibb_ for which to date only five individual contigs could be determined. we show that the contigs represent modules linked by long repeat regions enclosing some yet not identified orfs. the data suggest that ibb_ is a member of the cp subgroup.  campylobacter group ii phages are diverse regarding their genome organization. since all hitherto characterized group ii phages contain numerous genes for transposases and homing endonucleases as well as similar repeat regions, it cannot be excluded that these phages are genetically unstable. to answer this question, further experiments and sequencing of more group ii phages should be performed. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords campylobacterphagegenomestabilityrepeatsissue-copyright-statement© the author 2015 