BACKGROUND
campylobacteriosis is the most prevalent form of bacterial acute enteritis worldwide.
in symptomatic cases it is characterized by a prodromal phase with fever, vomiting, and headaches followed by watery or bloody diarrhea and abdominal cramps  <cit> .
in consequence of acute enteritis, extraintestinal post-infectious sequelae, namely, the guillain-barré syndrome, inflammatory bowel disease, and reactive arthritis may occur  <cit> .
the average incidence reported in the european union was  <dig>  per  <dig>  population in  <dig>  <cit> , in the usa  <dig>  cases per  <dig>  population in  <dig>  and in china  <dig> cases per  <dig>  population in urban areas compared to  <dig> cases per  <dig>  population in rural areas  <cit> .
in europe,  <dig>  % were reported to have been caused by campylobacter jejuni and  <dig>  % by campylobacter coli  <cit> .
c. coli is phylogenetically subdivided into three clades  <cit> : clade  <dig> isolates commonly colonize swine but can also be isolated from poultry and humans, although less frequently.
clades  <dig> and  <dig> are typically isolated from environmental waters  <cit> .
at the moment, seven completed c. coli chromosomal genome sequences , several scaffold genomes, and various contigs have been deposited in the ncbi genome database .
the completed genome sequences, range from  <dig>  to  <dig>  mb, have a g + c content of about  <dig> to 32 %, and contain  <dig> –  <dig> predicted genes including  <dig> –  <dig> protein coding orfs .
one of the major epigenetic mechanisms in prokaryotes is dna methylation  <cit> .
dna methylation patterns influence gene expression  <cit> , through silencing of transcription  <cit>  as well as dna replication initiation  <cit>  and mismatch repair  <cit> .
dna methylation also serves as a protection of the host genome against extraneous dna  <cit>  through restriction-modification systems .
rm-systems consist of two components:  a restriction endonuclease that recognizes a specific dna motif and  a cognate dna methyltransferase that methylates the same dna, preventing its cleavage by the restriction endonuclease  <cit> .
the majority of rm-systems can be categorized into four types :
type i rm-systems typically consist of three types of subunits: two restriction endonuclease subunits , which facilitate dna cleavage, one specificity subunit  for recognition of specific dna sequence motifs, and two dna methylase subunits  that catalyse n <dig> adenine methylation  <cit> .
this composition enables type i rm-systems to digest unmethylated dna, whereas hemimethylated dna is further methylated and fully methylated dna is insusceptible to restriction  <cit> .
type ii rm-systems are mostly composed of two homodimeric r subunits and a separated m subunit.
the r and m subunits recognize the same dna motif, which is typically a 4–8 bp palindrome  <cit> .
type iii rm-systems are comprised of two modification  subunits and two restriction  subunits.
type iii rm-systems must bind to two inversely oriented copies of its 5–6 bp asymmetric recognition motif.
cleavage of unmethylated dna typically occurs 25–27 bp away from the binding sites  <cit> .
type iv rm-systems consist of two separate r subunits cleaving dna that contains methylated, hydroxymethylated or glucosyl-hydroxymethylated cytosines.
cleavage typically occurs 30 bp away from one of the binding sites  <cit> .
furthermore, there exist many orphan dna methylases that are not part of a rm-system e.g.
dna adenine methylase  and cell cycle-regulated dna methyltransferase   <cit> .
it must be considered that the genes encoding for particular components of a rm-system are diverse within microbial species i.e.
c. jejuni and c. coli .
in c. jejuni the putative type i rm-system locus  reveals significant diversity regarding gene order, chromosomal location, intervening orfs and gene sequence leading to the classification of the c. jejuni hsd loci into at least three families, namely the iab, ic, and if family.
especially sequence variations in the hsds gene suggest at least  <dig> different target sequence specificities and therewith differences in dna methylation  <cit> .
holt and coworkers demonstrated that cj1051c  encodes an active restriction-modification type iig enzyme in c. jejuni that significantly decreases transformation efficiency with plasmids  <cit> .
additionally it was shown that type iis restriction modification enzyme cj0030/cj <dig> is subject to phase variable gene expression due to mutations in polyc/g tracts .
additionally cj0139/cj <dig> encode a putative 5-methylcytosine-restriction system, cj0690c a type ii rm-system and cj0979c a nuclease that could be part of a rm-system  <cit> .
furthermore it was shown for c. jejuni that gene product of cj <dig> is a n6-adenine-specific dna methyltransferase that is not a dam homologue and not part of a rm-system.
knockout of cj <dig> affects flagellar appearance, motility, adherence, and invasion indicating its role for epigenetic control of proteins involved in these processes  <cit> .
further non-ubiquitous modification/methyltransferase gene loci have been observed in c. jejuni st- <dig> isolates, namely fixl  that showed homology with dna adenine/modification methylases in campylobacter rectus, a homolog of icea1/nlaiii , cjj5070_14910c predicted as ulcer associated adenine-specific dna methyltransferase, which is an ortholog of the catg-specific methyltransferase hpyim of h. pylori, and the orphan dna methyltransferase in orf cjj5070_ <dig>  <cit> .
three strain-specific rm-systems have been reported for c. jejuni st <dig> complex: r. hinpi restriction endonuclease , modification methylase hhal , and r.pab <dig> restriction endonuclease   <cit> .
in contrast to c. coli, type iii rm-systems have been reported for c. jejuni subsp.
jejuni, c. jejuni subsp.
doylei, campylobacter lari, and campylobacter upsaliensis  <cit> .
until to date the methylomes of c. jejuni subsp.
jejuni  and of the related microbial species helicobacter pylori  have been analyzed , but so far no c. coli methylome has been examined.
studies performing isoschizomer digestion assays indicated differences in methylation at gatc sites in genomic dna of c. coli isolates, which suggested host-associated dna modification systems  <cit> .
in this study we analyzed the first methylome of c. coli using smrt dna sequencing.
this approach facilitates analysis of methylation motifs as well as rm-system gene loci in parallel.
for smrt sequencing a c. coli isolate was selected, which was tested positive for gatc site methylation isoschizomer digestion assay screening.
RESULTS
smrt sequencing and annotation
screening of  <dig> c. coli isolates performing 5’-gatc-3’-specific isoschizomer digestion assays revealed six bacterial isolates showing dam activity.
of these six isolates, the isolate bfr-ca- <dig> was selected for smrt sequencing, since it reproducibly tested positive in five biological independent analyses.
furthermore, its origin from broiler hearts sampled at a retail store in berlin, germany, indicates its relevance for food hygiene and thus for the infection of humans.
mlst typing revealed sequence type st- <dig> and clonal complex cc- <dig>  accordingly, it is a potentially human pathogenic isolate of clade  <dig>
susceptibility testing indicated a quinolone resistant phenotype  measured by broth microdilution: 16 μg/ml ciprofloxacin, 32 μg/ml nalidixic acid).
in contrast, the isolate was tested susceptible for macrolides, tetracyclines and aminoglycosides .
sequencing of the c. coli isolate using one pacific bioscience smrt cell resulted in  <dig>  continuous long reads  with an average  length of  <dig>   base pairs .
<dig>  subreads  of high quality and an average length of  <dig>  bp could be extracted from the clrs.
utilizing the pacific bioscience high-quality de novo genome assembly algorithm , we obtained a single polished contig of  <dig> , <dig> base pairs with an average 500-fold coverage and a confidence score of > <dig>  % .
the g + c content of the contig was  <dig>  % and the most closely related genome sequence available in the ncbi genome database was that of c. coli 15– <dig> .
no plasmids were detected in this isolate.
application of the ncbi/rast/prodigal annotation pipelines resulted in 1637/1769/ <dig> predicted coding sequences and 275/366/ <dig>  predicted hypothetical proteins.
.
furthermore,  <dig> rna genes  were predicted by ncbi,  <dig> rnas  by rast, and  <dig> rnas  by prodigal.
the rast subsystem coverage was 57 % , of which amino acid metabolism , protein metabolism , and cofactors, vitamins, prosthetic groups, and pigments  represented the largest groups .fig.
<dig> pie chart representing rast subsystems identified in the bfr-ca- <dig> genome.
the  <dig> most abundant subsystems on the “category” level as identified by rast are represented by a particular color indicated at the right-hand side of the figure
figure 2a shows a comparison of the bfr-ca- <dig> genome sequence to other c. coli genomes  using the artemis tool.
here, homologous regions as identified by blast are indicated by red  or blue line segments .
it can be seen that c. coli  <dig> mainly shows three stretches of inverse homologous regions to bfr-ca- <dig> and lacks similarity in the region of the identified cjie <dig> .
rm <dig> displays a long stretch of close homology with minor gaps, but also inverse homologous segments within the plasmid and the beginning and end of the chromosome.
a similar pattern can be observed for the comparison of bfr-ca- <dig> to c. jejuni nctc  <dig> .
this indicates that c. coli bfr-ca- <dig> is phylogenetically closer to clade  <dig> and therewith to c. jejuni islolates than to clade  <dig> c. coli isolates.fig.
<dig> comparison of c. coli bfr-ca- <dig> genome to those of c. coli rm <dig> and  <dig> a as well as c. jejuni nctc  <dig> b using the artemis comparison tool .
comparison of the bfr-ca- <dig> genome to those of c. coli clade  <dig> strain rm <dig> and c. coli clade  <dig> strain  <dig> as well as to the genome of c. jejuni nctc  <dig> indicates that the clade  <dig> strain bfr-ca- <dig> genome organization is more related to c. coli clade  <dig> and c. jejuni strains than to clade  <dig> strains.
the cjie1-homologue prophage in the bfr-ca- <dig> genome is indicated in green
poly-g/c tracts and the capsular polysaccharid gene locus
analysis of poly-g/c tracts within the contig of bfr-ca- <dig> revealed  <dig> locations with homopolymeric stretches of at least eight g or c nucleotides .
most of these tracts occur in the vicinity of hypothetical proteins, however, two motifs could be identified within the capsular polysaccharid gene locus  close to orfs identified as glycosyltransferases  by rast.
other orfs with neighboring poly-g/c motifs comprise e.g.
transferases and ligases.table  <dig> positions of poly-g tracts in the bfr-ca- <dig> genome
poly-g/c tracts were searched in the bfr-ca- <dig> genome using regular expression describing at least  <dig> consecutive g/c.
the first two columns denote the genome location and length of the expression found.
column  <dig> shows the rast annotation of the orf closest to the homopolymeric stretch.
column  <dig> and  <dig> represent the relative position of the stretch to the orf’s start  and stop codon , with negative numbers representing upstream locations.
columns  <dig> to  <dig> denote the location and orientation of the orf
poly-g/c tracts were searched in the bfr-ca- <dig> genome using regular expression describing at least  <dig> consecutive g/c.
the first two columns denote the genome location and length of the expression found.
column  <dig> shows the rast annotation of the orf closest to the homopolymeric stretch.
column  <dig> and  <dig> represent the relative position of the stretch to the orf’s start  and stop codon , with negative numbers representing upstream locations.
columns  <dig> to  <dig> denote the location and orientation of the orf
the cps locus of bfr-ca- <dig> ranges from  <dig> , <dig> to  <dig> , <dig> .
spanning  <dig>  bp and encoding  <dig> orfs  it is of comparable size to the largest known other campylobacter cps cluster, which measures 38 kb excluding flanking kps regions   <cit> .
the cps loci of different c. coli and c. jejuni strains are very variable in size and gene content and there are only three genes that are comparatively conserved: the capsular polysaccharide export system periplasmic protein gene kpsd, the gdp-mannose  <dig> -dehydratase gene and the capsular polysaccharide biosynthesis/export periplasmic protein wcba/kpsc.
the highest sequence similarities exist to the cps clusters of c. coli strains rm <dig>  and fb <dig> .
compared to the cps locus of strain x, query coverage of 28 %  was observed.
the genes for kpsc, hddc, gmha <dig>  hdda, dmha, fcl- <dig>  fcl- <dig>  hypothetical protein x.
<dig> and kpsf are present in both cps clusters.
n-linked flagellar glycosylation locus
the n-linked flagellar glycosylation locus extends from  <dig> ,815- <dig> , <dig> .
it consist of  <dig> orfs and is 99 % identical to the corresponding locus in other c. coli genomes e.g.
rm <dig>  or  <dig> .
in comparison to other c. jejuni strains , insertion of the lipid carrier udp-n-acetylgalactosaminyltransferase gene and an α- <dig> -n-acetylgalactosamine transferase pglh gene was observed.
directly upstream of the n-linked flagellar glycosylation locus the lipooligosaccharide biosynthesis locus  is located.
the los locus extends from the udp-glucose 4-epimerase gene gale to the d-glycero-d-manno-heptose  <dig> -bisphosphate phosphatase gene rfad.
the los locus is one of the more variable regions in campylobacter genomes.
los loci containing  <dig>  to  <dig>  orfs have been described and depending on gene content and organization  <dig> different los classes  have been defined  <cit> .
the los locus of bfr-ca- <dig> ranges from position  <dig> , <dig> to  <dig> , <dig>  and contains  <dig> orfs.
no sialyltransferase cstii/iii homologue genes and no n-acetylgalactosaminyltransferase neuabc homologue genes are present in this cluster and therefore bfr-ca- <dig> expresses an unsialylated los.
it does not exactly belong to one of the los classes described for c. jejuni but it is closer related to the los classes e, p, o, and h. remarkably, the waam and waav homologue genes are immediate neighbours, therefore the established sequencing approach would have been difficult due to problems resolving repetitive genomic regions  <cit> .
virulence-associated genes
neither dimethylsulfoxide  reductase systems nor gamma glutamyltranspeptidase  gene could be detected in the bfr-ca- <dig> genome, which is typical for a clade  <dig> c. coli isolates, but three genes homologous to the iron transport protein tonb were present in the bfr-ca- <dig> genome, which has been reported as typical for clade  <dig> and  <dig> c. coli isolates  <cit> .
the cytolethal distending toxin  operon contains all three subunits and is therewith complete.
campylobacter jejuni integrated element 1-like element & crispr elements
at position  <dig> ,993- <dig> , <dig> we could identify a  <dig>  bp sized campylobacter jejuni integrated element  <dig> -homologue prophage that shows 96 % identity  to cjie <dig> described in c. jejuni rm <dig>  <cit> .
no crispr elements were found by any method.
methylation motifs and rm-systems in the genome of c. coli bfr-ca-9557
in addition to reconstruction of the genome sequence smrt sequencing allows determination of base modification by analysis of the sequencing kinetics.
using the smrt analysis modification and motif detection, we could identify  <dig>  putatively n-6-methylated adenines ,  <dig> 4-methylated cytosines  and further  <dig>  rather unspecific “modified bases” where the type of modification was not recognized by the software .
methylated bases were arranged within eight different dominant methylation motifs .
all motifs are recognized by n- <dig> adenine-specific methyltransferases.
a small fraction  of m6a bases were not clustered into any of the motifs, and no consensus motif could be identified for either m4c-methylated bases or the majority of other unspecific modified bases.table  <dig> methylation motifs of c. coli bfr-ca-9557
<dig> qv = quality value
methylation motifs were identified using the pacbio smrt analysis software .
column  <dig> shows the sequence consensus of the motif, whereby non-uniform positions are represented by iupac ambiguity codes.
column  <dig> and  <dig> denote the position of the modified base within the motif and the type of methylation.
column  <dig> represents the fraction of a motif’s occurrences in the genome  for which a methylation has been detected .
column  <dig> and  <dig> denote the average modification quality  and average coverage of motifs detected as modified.
the last column shows the partner motif, i.e.
the reverse complement of the motif
fig.
<dig> sequence logos of eight methylation motifs.
the consensus sequences of all eight motifs are depicted as sequence logo as obtained by the weblogo  <dig> server .
the height of each stack indicates the degree of conservation .
the height of the letters represents the relative frequency of the base.
the asterisk under a particular letter indicates the modified/methylated base.
the two motifs in g and in h are partner motifs that are methylated at both strands.
all motifs are recognized by n- <dig> adenine-specific methyltransferases
the motifs g <dig> & g <dig> and h <dig> & h <dig>  are partner motifs and motifs a and b represent palindromic sequences, all of these partners containing methylated bases on both strands.
in contrast, bases of motifs c, d, e, and f were methylated on only one strand.
the major methylation motifs a, b, c, e, g, and h were almost completely methylated across their occurrences within the genome.
the percentage of methylation ranges from  <dig>  % to  <dig>  %.
in contrast, only  <dig>  % of the m6a methylation motif f sequences were methylated.
using the recognition sequence search feature within the restriction enzyme database – rebase  <cit> , only two out of the eight motifs  could be matched to existing recognition sequences of restriction systems .
the remaining motifs represent yet unknown recognition sequences with the longer motifs  most likely being specific to c. coli or to this particular strain.
using the rebase sequence search feature and the ncbi, g2l and prodigal/prokka annotations four orfs encoding subunits of type i, seven orfs encoding subunits of type ii, and three orfs encoding subunits of type iv rm-systems have been identified .
only two of the rebase predicted recognition sequences correspond to a specific motif detected by smrt sequencing, namely: gaattc/raatty  recognized by the dna modification methylase  fokim_ <dig>  and gatc  recognized by the dna modification methyltransferase dpna .
additionally rebase predicts a second candidate for a gatc-specific dna modification methyltransferase in orf # <dig>  in contrast, rast and prodigal predict a 16s rrna -n)-methyltransferase for this orf.table  <dig> putative c. coli bfr-ca- <dig> restriction modification systems
orf #1
22302
22302
1orf # according to the ncbi annotation pipeline for rast and prodigal orf # see additional files  <dig> and 4
2disrupted orf
3predicted sequence does not correspond to any motif detected by smrt sequencing
4according to rebase orf # <dig> encodes a type ii rm-system
5in contrast to rast and prodigal rebase predicts a second candidate for a gatc-specific dna modification methyltransferase for this orf
restriction modification systems have been identified as outlined in section “methods”.
column  <dig> to  <dig> denote the number, strand direction and genome position of the orf as identified by the ncbi annotation pipeline.
column  <dig> contains the description of the orf in terms of aggregated annotations from ncbi, rast and prodigal.
the type and predicted recognition sequence of the motif are shown in columns  <dig> and 6
the two recognition sequences gagnnnnngt and ggatg predicted for the type i restriction-modification system2c dna-methyltransferase subunit m  and modification methylase foki homologue , respectively, do not correspond to any motif detected by smrt sequencing in the genome of c. coli bfr-ca- <dig>
rebase search hits to the remaining ten rm-system subunit genes have not been associated with a specific recognition sequence.
therewith, rebase was not able to predict a corresponding rm-system subunit for the motifs c, d, e, f, g, and h. this is most likely due to the fact that besides motifs a  and b  none of the published c. jejuni and h. pylori methylomes contains one of these motifs  <cit> .
motif b  is present in the methylomes of h. pylori  <dig> and j99-r <dig>  <cit>  but not in any of the publicly available c. jejuni methylomes.
in the two h. pylori genomes orfs hp <dig> and jhp <dig> encoding a rm-system iim subunit have been assigned to this motif  <cit> .
the c. coli bfr-ca- <dig> homologue of hp <dig>  demonstrates a sequence identity of 76 % .
in contrast, the raatty motif  has not been detected in one of the two h. pylori methylomes, but is was present in c. jejuni f <dig>  nctc  <dig> as well as 81– <dig> and in all three isolates a corresponding rm-system subunit has been assigned  <cit> .
the c. coli bfr-ca- <dig> homologue of cj <dig>  demonstrates a sequence identity of 85 % .
the biological implications of this particular subset of rm-systems are difficult to predict, especially since the majority of methylation motifs and methylases in the genome of c. coli bfr-ca- <dig> are quite novel.
from the closely related bacterial species h. pylori it has been described in detail that rm-systems play a crucial role in forming strong barriers to prevent uptake of both plasmid and chromosomal dna  <cit> .
h. pylori bacterial cells are inherently very competent for dna uptake, but this competence for dna uptake varies significantly between specific strains.
it has been observed that strains showing a very low endogenous rm-system activity demonstrate the highest transformation efficiency.
especially the presence of an rm-system homologous to mboi, which has been used for isoschizomer digestion assays in our study, has been described as major component of the h. pylori transformation barrier  <cit> .
this mboi homologous rm-system named hpyiii is highly conserved among h. pylori strains and mediates protection against mboi digestion.
in mboi sensitive strains the hpyiiir gene was found absent but a homologue to c. jejuni cj <dig>  namely hrga, was detected.
hpyiiir negative but hrga positive h. pylori strains have been associated with gastric cancer in asian patients, while the pathogenic mechanism remains unclear  <cit> .
besides this role for dna uptake the high intra-species variety in rm-system configuration and therewith the highly diverse methylation status of h. pylori chromosomal dna was suggested to serve as a new typing system  <cit> .
this dna methylation based typing system may help to discriminate h. pylori isolates and as well isolates of related bacterial species like c. jejuni or c. coli for epidemiological and clinical purposes.
CONCLUSIONS
the objective of the study was to analyze the methylome of a clade  <dig> c. coli isolate using smrt sequencing.
the c. coli isolate bfr- <dig> was tested positive in 5’-gmatc-3’ specific isoschizomer digestion assays and hence chosen for smrt sequencing.
using one smrt cell the complete genome of c. coli bfr-ca- <dig> was sequenced and assembled into a single contig of  <dig>  mbp.
the genome contains a cjie1-like element prophage.
smrt analysis modification and motif detection have identified eleven different dominant methylation motifs, while  <dig> rm-system subunits have been identified using rebase and three different annotation pipelines.
only two of the rebase predicted recognition sequences correspond to a specific motif detected by smrt sequencing.
the remaining  <dig> motifs did not correspond to any database record and are therefore characteristic for c. coli or at least for c. coli bfr-ca- <dig>  however, no comparable motifs have been described in the related microbial species c. jejuni or h. pylori.

 campylobacteriosis is the most prevalent form of bacterial acute enteritis worldwide. in symptomatic cases it is characterized by a prodromal phase with fever, vomiting, and headaches followed by watery or bloody diarrhea and abdominal cramps. in consequence of acute enteritis, extraintestinal post-infectious sequelae, namely, the guillain-barré syndrome, inflammatory bowel disease, and reactive arthritis may occur. the average incidence reported in the european union was per population in, in the usa cases per population in and in china cases per population in urban areas compared to cases per population in rural areas. in europe,  % were reported to have been caused by campylobacter jejuni and  % by campylobacter coli. c. coli is phylogenetically subdivided into three clades: clade isolates commonly colonize swine but can also be isolated from poultry and humans, although less frequently. clades and are typically isolated from environmental waters. at the moment, seven completed c. coli chromosomal genome sequences, several scaffold genomes, and various contigs have been deposited in the ncbi genome database. the completed genome sequences, range from to  mb, have a g + c content of about to 32 %, and contain – predicted genes including – protein coding orfs. one of the major epigenetic mechanisms in prokaryotes is dna methylation. dna methylation patterns influence gene expression, through silencing of transcription as well as dna replication initiation and mismatch repair. dna methylation also serves as a protection of the host genome against extraneous dna through restriction-modification systems. rm-systems consist of two components: a restriction endonuclease that recognizes a specific dna motif and a cognate dna methyltransferase that methylates the same dna, preventing its cleavage by the restriction endonuclease. the majority of rm-systems can be categorized into four types: type i rm-systems typically consist of three types of subunits: two restriction endonuclease subunits, which facilitate dna cleavage, one specificity subunit for recognition of specific dna sequence motifs, and two dna methylase subunits that catalyse n adenine methylation. this composition enables type i rm-systems to digest unmethylated dna, whereas hemimethylated dna is further methylated and fully methylated dna is insusceptible to restriction. type ii rm-systems are mostly composed of two homodimeric r subunits and a separated m subunit. the r and m subunits recognize the same dna motif, which is typically a 4–8 bp palindrome. type iii rm-systems are comprised of two modification subunits and two restriction subunits. type iii rm-systems must bind to two inversely oriented copies of its 5–6 bp asymmetric recognition motif. cleavage of unmethylated dna typically occurs 25–27 bp away from the binding sites. type iv rm-systems consist of two separate r subunits cleaving dna that contains methylated, hydroxymethylated or glucosyl-hydroxymethylated cytosines. cleavage typically occurs 30 bp away from one of the binding sites. furthermore, there exist many orphan dna methylases that are not part of a rm-system e.g. dna adenine methylase and cell cycle-regulated dna methyltransferase. it must be considered that the genes encoding for particular components of a rm-system are diverse within microbial species i.e. c. jejuni and c. coli. in c. jejuni the putative type i rm-system locus reveals significant diversity regarding gene order, chromosomal location, intervening orfs and gene sequence leading to the classification of the c. jejuni hsd loci into at least three families, namely the iab, ic, and if family. especially sequence variations in the hsds gene suggest at least different target sequence specificities and therewith differences in dna methylation. holt and coworkers demonstrated that cj1051c encodes an active restriction-modification type iig enzyme in c. jejuni that significantly decreases transformation efficiency with plasmids. additionally it was shown that type iis restriction modification enzyme cj0030/cj is subject to phase variable gene expression due to mutations in polyc/g tracts. additionally cj0139/cj encode a putative 5-methylcytosine-restriction system, cj0690c a type ii rm-system and cj0979c a nuclease that could be part of a rm-system. furthermore it was shown for c. jejuni that gene product of cj is a n6-adenine-specific dna methyltransferase that is not a dam homologue and not part of a rm-system. knockout of cj affects flagellar appearance, motility, adherence, and invasion indicating its role for epigenetic control of proteins involved in these processes. further non-ubiquitous modification/methyltransferase gene loci have been observed in c. jejuni st- isolates, namely fixl that showed homology with dna adenine/modification methylases in campylobacter rectus, a homolog of icea1/nlaiii, cjj5070_14910c predicted as ulcer associated adenine-specific dna methyltransferase, which is an ortholog of the catg-specific methyltransferase hpyim of h. pylori, and the orphan dna methyltransferase in orf cjj5070_. three strain-specific rm-systems have been reported for c. jejuni st complex: r. hinpi restriction endonuclease, modification methylase hhal, and r.pab restriction endonuclease. in contrast to c. coli, type iii rm-systems have been reported for c. jejuni subsp. jejuni, c. jejuni subsp. doylei, campylobacter lari, and campylobacter upsaliensis. until to date the methylomes of c. jejuni subsp. jejuni and of the related microbial species helicobacter pylori have been analyzed, but so far no c. coli methylome has been examined. studies performing isoschizomer digestion assays indicated differences in methylation at gatc sites in genomic dna of c. coli isolates, which suggested host-associated dna modification systems. in this study we analyzed the first methylome of c. coli using smrt dna sequencing. this approach facilitates analysis of methylation motifs as well as rm-system gene loci in parallel. for smrt sequencing a c. coli isolate was selected, which was tested positive for gatc site methylation isoschizomer digestion assay screening.  smrt sequencing and annotation screening of c. coli isolates performing 5’-gatc-3’-specific isoschizomer digestion assays revealed six bacterial isolates showing dam activity. of these six isolates, the isolate bfr-ca- was selected for smrt sequencing, since it reproducibly tested positive in five biological independent analyses. furthermore, its origin from broiler hearts sampled at a retail store in berlin, germany, indicates its relevance for food hygiene and thus for the infection of humans. mlst typing revealed sequence type st- and clonal complex cc- accordingly, it is a potentially human pathogenic isolate of clade susceptibility testing indicated a quinolone resistant phenotype measured by broth microdilution: 16 μg/ml ciprofloxacin, 32 μg/ml nalidixic acid). in contrast, the isolate was tested susceptible for macrolides, tetracyclines and aminoglycosides. sequencing of the c. coli isolate using one pacific bioscience smrt cell resulted in continuous long reads with an average length of base pairs. subreads of high quality and an average length of  bp could be extracted from the clrs. utilizing the pacific bioscience high-quality de novo genome assembly algorithm, we obtained a single polished contig of, base pairs with an average 500-fold coverage and a confidence score of >  %. the g + c content of the contig was  % and the most closely related genome sequence available in the ncbi genome database was that of c. coli 15–. no plasmids were detected in this isolate. application of the ncbi/rast/prodigal annotation pipelines resulted in 1637/1769/ predicted coding sequences and 275/366/ predicted hypothetical proteins.. furthermore, rna genes were predicted by ncbi, rnas by rast, and rnas by prodigal. the rast subsystem coverage was 57 %, of which amino acid metabolism, protein metabolism, and cofactors, vitamins, prosthetic groups, and pigments represented the largest groups.fig. pie chart representing rast subsystems identified in the bfr-ca- genome. the most abundant subsystems on the “category” level as identified by rast are represented by a particular color indicated at the right-hand side of the figure figure 2a shows a comparison of the bfr-ca- genome sequence to other c. coli genomes using the artemis tool. here, homologous regions as identified by blast are indicated by red or blue line segments. it can be seen that c. coli mainly shows three stretches of inverse homologous regions to bfr-ca- and lacks similarity in the region of the identified cjie. rm displays a long stretch of close homology with minor gaps, but also inverse homologous segments within the plasmid and the beginning and end of the chromosome. a similar pattern can be observed for the comparison of bfr-ca- to c. jejuni nctc. this indicates that c. coli bfr-ca- is phylogenetically closer to clade and therewith to c. jejuni islolates than to clade c. coli isolates.fig. comparison of c. coli bfr-ca- genome to those of c. coli rm and a as well as c. jejuni nctc b using the artemis comparison tool. comparison of the bfr-ca- genome to those of c. coli clade strain rm and c. coli clade strain as well as to the genome of c. jejuni nctc indicates that the clade strain bfr-ca- genome organization is more related to c. coli clade and c. jejuni strains than to clade strains. the cjie1-homologue prophage in the bfr-ca- genome is indicated in green poly-g/c tracts and the capsular polysaccharid gene locus analysis of poly-g/c tracts within the contig of bfr-ca- revealed locations with homopolymeric stretches of at least eight g or c nucleotides. most of these tracts occur in the vicinity of hypothetical proteins, however, two motifs could be identified within the capsular polysaccharid gene locus close to orfs identified as glycosyltransferases by rast. other orfs with neighboring poly-g/c motifs comprise e.g. transferases and ligases.table positions of poly-g tracts in the bfr-ca- genome poly-g/c tracts were searched in the bfr-ca- genome using regular expression describing at least consecutive g/c. the first two columns denote the genome location and length of the expression found. column shows the rast annotation of the orf closest to the homopolymeric stretch. column and represent the relative position of the stretch to the orf’s start and stop codon, with negative numbers representing upstream locations. columns to denote the location and orientation of the orf poly-g/c tracts were searched in the bfr-ca- genome using regular expression describing at least consecutive g/c. the first two columns denote the genome location and length of the expression found. column shows the rast annotation of the orf closest to the homopolymeric stretch. column and represent the relative position of the stretch to the orf’s start and stop codon, with negative numbers representing upstream locations. columns to denote the location and orientation of the orf the cps locus of bfr-ca- ranges from, to,. spanning  bp and encoding orfs it is of comparable size to the largest known other campylobacter cps cluster, which measures 38 kb excluding flanking kps regions. the cps loci of different c. coli and c. jejuni strains are very variable in size and gene content and there are only three genes that are comparatively conserved: the capsular polysaccharide export system periplasmic protein gene kpsd, the gdp-mannose -dehydratase gene and the capsular polysaccharide biosynthesis/export periplasmic protein wcba/kpsc. the highest sequence similarities exist to the cps clusters of c. coli strains rm and fb. compared to the cps locus of strain x, query coverage of 28 % was observed. the genes for kpsc, hddc, gmha hdda, dmha, fcl- fcl- hypothetical protein x. and kpsf are present in both cps clusters. n-linked flagellar glycosylation locus the n-linked flagellar glycosylation locus extends from,815-,. it consist of orfs and is 99 % identical to the corresponding locus in other c. coli genomes e.g. rm or. in comparison to other c. jejuni strains, insertion of the lipid carrier udp-n-acetylgalactosaminyltransferase gene and an α- -n-acetylgalactosamine transferase pglh gene was observed. directly upstream of the n-linked flagellar glycosylation locus the lipooligosaccharide biosynthesis locus is located. the los locus extends from the udp-glucose 4-epimerase gene gale to the d-glycero-d-manno-heptose -bisphosphate phosphatase gene rfad. the los locus is one of the more variable regions in campylobacter genomes. los loci containing to orfs have been described and depending on gene content and organization different los classes have been defined. the los locus of bfr-ca- ranges from position, to, and contains orfs. no sialyltransferase cstii/iii homologue genes and no n-acetylgalactosaminyltransferase neuabc homologue genes are present in this cluster and therefore bfr-ca- expresses an unsialylated los. it does not exactly belong to one of the los classes described for c. jejuni but it is closer related to the los classes e, p, o, and h. remarkably, the waam and waav homologue genes are immediate neighbours, therefore the established sequencing approach would have been difficult due to problems resolving repetitive genomic regions. virulence-associated genes neither dimethylsulfoxide reductase systems nor gamma glutamyltranspeptidase gene could be detected in the bfr-ca- genome, which is typical for a clade c. coli isolates, but three genes homologous to the iron transport protein tonb were present in the bfr-ca- genome, which has been reported as typical for clade and c. coli isolates. the cytolethal distending toxin operon contains all three subunits and is therewith complete. campylobacter jejuni integrated element 1-like element & crispr elements at position,993-, we could identify a  bp sized campylobacter jejuni integrated element -homologue prophage that shows 96 % identity to cjie described in c. jejuni rm. no crispr elements were found by any method. methylation motifs and rm-systems in the genome of c. coli bfr-ca-9557 in addition to reconstruction of the genome sequence smrt sequencing allows determination of base modification by analysis of the sequencing kinetics. using the smrt analysis modification and motif detection, we could identify putatively n-6-methylated adenines, 4-methylated cytosines and further rather unspecific “modified bases” where the type of modification was not recognized by the software. methylated bases were arranged within eight different dominant methylation motifs. all motifs are recognized by n- adenine-specific methyltransferases. a small fraction of m6a bases were not clustered into any of the motifs, and no consensus motif could be identified for either m4c-methylated bases or the majority of other unspecific modified bases.table methylation motifs of c. coli bfr-ca-9557 qv = quality value methylation motifs were identified using the pacbio smrt analysis software. column shows the sequence consensus of the motif, whereby non-uniform positions are represented by iupac ambiguity codes. column and denote the position of the modified base within the motif and the type of methylation. column represents the fraction of a motif’s occurrences in the genome for which a methylation has been detected. column and denote the average modification quality and average coverage of motifs detected as modified. the last column shows the partner motif, i.e. the reverse complement of the motif fig. sequence logos of eight methylation motifs. the consensus sequences of all eight motifs are depicted as sequence logo as obtained by the weblogo server. the height of each stack indicates the degree of conservation. the height of the letters represents the relative frequency of the base. the asterisk under a particular letter indicates the modified/methylated base. the two motifs in g and in h are partner motifs that are methylated at both strands. all motifs are recognized by n- adenine-specific methyltransferases the motifs g & g and h & h are partner motifs and motifs a and b represent palindromic sequences, all of these partners containing methylated bases on both strands. in contrast, bases of motifs c, d, e, and f were methylated on only one strand. the major methylation motifs a, b, c, e, g, and h were almost completely methylated across their occurrences within the genome. the percentage of methylation ranges from  % to  %. in contrast, only  % of the m6a methylation motif f sequences were methylated. using the recognition sequence search feature within the restriction enzyme database – rebase, only two out of the eight motifs could be matched to existing recognition sequences of restriction systems. the remaining motifs represent yet unknown recognition sequences with the longer motifs most likely being specific to c. coli or to this particular strain. using the rebase sequence search feature and the ncbi, g2l and prodigal/prokka annotations four orfs encoding subunits of type i, seven orfs encoding subunits of type ii, and three orfs encoding subunits of type iv rm-systems have been identified. only two of the rebase predicted recognition sequences correspond to a specific motif detected by smrt sequencing, namely: gaattc/raatty recognized by the dna modification methylase fokim_ and gatc recognized by the dna modification methyltransferase dpna. additionally rebase predicts a second candidate for a gatc-specific dna modification methyltransferase in orf # in contrast, rast and prodigal predict a 16s rrna -n)-methyltransferase for this orf.table putative c. coli bfr-ca- restriction modification systems orf #1 22302 22302 1orf # according to the ncbi annotation pipeline for rast and prodigal orf # see additional files and 4 2disrupted orf 3predicted sequence does not correspond to any motif detected by smrt sequencing 4according to rebase orf # encodes a type ii rm-system 5in contrast to rast and prodigal rebase predicts a second candidate for a gatc-specific dna modification methyltransferase for this orf restriction modification systems have been identified as outlined in section “methods”. column to denote the number, strand direction and genome position of the orf as identified by the ncbi annotation pipeline. column contains the description of the orf in terms of aggregated annotations from ncbi, rast and prodigal. the type and predicted recognition sequence of the motif are shown in columns and 6 the two recognition sequences gagnnnnngt and ggatg predicted for the type i restriction-modification system2c dna-methyltransferase subunit m and modification methylase foki homologue, respectively, do not correspond to any motif detected by smrt sequencing in the genome of c. coli bfr-ca- rebase search hits to the remaining ten rm-system subunit genes have not been associated with a specific recognition sequence. therewith, rebase was not able to predict a corresponding rm-system subunit for the motifs c, d, e, f, g, and h. this is most likely due to the fact that besides motifs a and b none of the published c. jejuni and h. pylori methylomes contains one of these motifs. motif b is present in the methylomes of h. pylori and j99-r but not in any of the publicly available c. jejuni methylomes. in the two h. pylori genomes orfs hp and jhp encoding a rm-system iim subunit have been assigned to this motif. the c. coli bfr-ca- homologue of hp demonstrates a sequence identity of 76 %. in contrast, the raatty motif has not been detected in one of the two h. pylori methylomes, but is was present in c. jejuni f nctc as well as 81– and in all three isolates a corresponding rm-system subunit has been assigned. the c. coli bfr-ca- homologue of cj demonstrates a sequence identity of 85 %. the biological implications of this particular subset of rm-systems are difficult to predict, especially since the majority of methylation motifs and methylases in the genome of c. coli bfr-ca- are quite novel. from the closely related bacterial species h. pylori it has been described in detail that rm-systems play a crucial role in forming strong barriers to prevent uptake of both plasmid and chromosomal dna. h. pylori bacterial cells are inherently very competent for dna uptake, but this competence for dna uptake varies significantly between specific strains. it has been observed that strains showing a very low endogenous rm-system activity demonstrate the highest transformation efficiency. especially the presence of an rm-system homologous to mboi, which has been used for isoschizomer digestion assays in our study, has been described as major component of the h. pylori transformation barrier. this mboi homologous rm-system named hpyiii is highly conserved among h. pylori strains and mediates protection against mboi digestion. in mboi sensitive strains the hpyiiir gene was found absent but a homologue to c. jejuni cj namely hrga, was detected. hpyiiir negative but hrga positive h. pylori strains have been associated with gastric cancer in asian patients, while the pathogenic mechanism remains unclear. besides this role for dna uptake the high intra-species variety in rm-system configuration and therewith the highly diverse methylation status of h. pylori chromosomal dna was suggested to serve as a new typing system. this dna methylation based typing system may help to discriminate h. pylori isolates and as well isolates of related bacterial species like c. jejuni or c. coli for epidemiological and clinical purposes.  the objective of the study was to analyze the methylome of a clade c. coli isolate using smrt sequencing. the c. coli isolate bfr- was tested positive in 5’-gmatc-3’ specific isoschizomer digestion assays and hence chosen for smrt sequencing. using one smrt cell the complete genome of c. coli bfr-ca- was sequenced and assembled into a single contig of mbp. the genome contains a cjie1-like element prophage. smrt analysis modification and motif detection have identified eleven different dominant methylation motifs, while rm-system subunits have been identified using rebase and three different annotation pipelines. only two of the rebase predicted recognition sequences correspond to a specific motif detected by smrt sequencing. the remaining motifs did not correspond to any database record and are therefore characteristic for c. coli or at least for c. coli bfr-ca- however, no comparable motifs have been described in the related microbial species c. jejuni or h. pylori. 
c. coli bfr-ca- shares one of these, but four orfs could be assigned to putative type i rm-systems, seven orfs to type ii rm-systems and three orfs to type iv rm-systems. the goal of the study was to analyze the methylome of a c. coli strain susceptible to dpni digestion, to identify its methylation motifs and restriction modification systems, and compare them to related organisms like c. jejuni and helicobacter pylori. the genome contains a cjie1-like element prophage and is phylogenetically closer to c. coli clade isolates than clade 6-methylated adenines that are predominantly arranged in eight different methylation motifs and 4-methylated cytosines have been detected. only five dominant methylation motifs have been identified in c. jejuni, which have been associated with known rm-systems. the remaining methylation motifs are specific for c. coli bfr-ca- and have been neither detected in c. jejuni nor in h. pylori. campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. in contrast to campylobacter jejuni, about 5 % of campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by dpni cutting specifically 5’-gmatc-3’ motifs. the results of this study give us new insights into epigenetics of campylobacteraceae and provide the groundwork to resolve the function of rm-systems in c. coli. in accordance with dpni prescreening rm-system iip, methylation of gatc motifs was detected in c. coli bfr-ca- a homologous iip rm-system has been described for h. pylori. using one smrt cell and the pacbio rs sequencing technology followed by pacbio modification and motif analysis the complete genome of the dpni susceptible strain c. coli bfr-ca- was sequenced to 500-fold coverage and assembled into a single contig of mbp. this indicates significant differences in dna methylation between both microbial species.
 campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. in contrast to campylobacter jejuni, about 5 % of campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by dpni cutting specifically 5’-gmatc-3’ motifs. this indicates significant differences in dna methylation between both microbial species. the goal of the study was to analyze the methylome of a c. coli strain susceptible to dpni digestion, to identify its methylation motifs and restriction modification systems, and compare them to related organisms like c. jejuni and helicobacter pylori.  using one smrt cell and the pacbio rs sequencing technology followed by pacbio modification and motif analysis the complete genome of the dpni susceptible strain c. coli bfr-ca- was sequenced to 500-fold coverage and assembled into a single contig of mbp. the genome contains a cjie1-like element prophage and is phylogenetically closer to c. coli clade isolates than clade 6-methylated adenines that are predominantly arranged in eight different methylation motifs and 4-methylated cytosines have been detected. only two of these motifs correspond to known restriction modification motifs. characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %.  only five dominant methylation motifs have been identified in c. jejuni, which have been associated with known rm-systems. c. coli bfr-ca- shares one of these, but four orfs could be assigned to putative type i rm-systems, seven orfs to type ii rm-systems and three orfs to type iv rm-systems. in accordance with dpni prescreening rm-system iip, methylation of gatc motifs was detected in c. coli bfr-ca- a homologous iip rm-system has been described for h. pylori. the remaining methylation motifs are specific for c. coli bfr-ca- and have been neither detected in c. jejuni nor in h. pylori. the results of this study give us new insights into epigenetics of campylobacteraceae and provide the groundwork to resolve the function of rm-systems in c. coli. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords campylobacter coligenomemethylationmotifsmethylomerestriction modification systemsisoschizomer digestion assaysmrt sequencingpacbioissue-copyright-statement© the author 2015 