BACKGROUND
while our appreciation of microbial diversity has been greatly increased by the exponential increase in the availability of genome sequences and by metagenomic approaches  <cit> , it has also highlighted our relative lack of understanding about what drives variation, and which limitations and constraints control the process of genome variation.
diversity at the level of gene order and genome content can be introduced via the reorganisation of the genome, through combinations of gene inversion, recombination, gene duplication, deletion and horizontal gene transfer  <cit> .
such movement, deletion or introduction of genes or operons can create a problem for the cell, as the reorganisation of the genome may result in disruption of transcriptional circuitry controlling the expression levels of such genes.
however, variability can also be introduced at the gene level, e.g.
by generation of alternative transcription start sites, promoter recognition sequences or alterations in the 5′ untranslated regions affecting folding or stability.
the level of rna in a cell is usually controlled at the transcriptional and post-transcriptional levels.
in bacteria, transcriptional regulation is commonly mediated via control of transcription initiation by rna polymerase  at the promoter  <cit> .
alternatively, post-transcriptional gene regulation is often mediated by the  action of non-coding or antisense rna  <cit> , rna chaperones  <cit>  and the activity of ribonucleases  <cit> .
in the last two years, the use of high-throughput sequencing of cdna  has revealed that the complexity of the microbial transcriptome is much higher than previously predicted  <cit> .
however, the high level of phylogenetic diversity within the bacterial kingdom has so far limited the possibilities for interspecies transcriptome comparison, since the species for which high resolution transcriptome maps are available are either too closely related  or too distantly related to allow meaningful comparisons at the evolutionary level.
the epsilon-subdivision of the proteobacteria  is a lineage which contains both pathogenic and non-pathogenic bacteria.
the best studied examples of the former category are the human pathogens campylobacter jejuni and helicobacter pylori, which belong to the order campylobacterales <cit> .
however, next to these important human pathogens, the epsilonproteobacteria also contain chemolithoautotrophic microorganisms isolated from deep-sea vents  <cit> , as well as the bovine rumen-colonising bacterium wolinella succinogenes <cit> .
despite the differences in ecological niches between the genera, and the genome sizes of epsilonproteobacteria varying between  <dig>  and  <dig>  mbp, genomic comparisons revealed that the epsilonproteobacteria share similar transcription machinery including few sigma factors , metabolic pathways and limitations, and have about half of the predicted genes in the genome in common with other epsilonproteobacteria  <cit> .
however, while these genomes share functionality, genome architecture and often low g + c content, the gene order and genome organisation have diverged significantly.
this raises the question on how the genome and associated transcriptome copes with such large scale reorganisations of the genome when genera and species evolutionary diverge over time.
to address this question, we have mapped the primary transcriptome of c. jejuni at the single nucleotide resolution using differential rna-seq, have compared it with the primary transcriptome map of h. pylori <cit>  and have used genome sequences of  <dig> other taxa of the epsilonproteobacteria to assess conservation and evolution of transcriptional circuitry in this highly variable group of bacteria.
RESULTS
differential rna-seq analysis of the c. jejuni primary transcriptome
the c. jejuni nctc  <dig> genome contains  <dig> annotated coding sequences , with only few stable rna molecules known outside the ribosomal and transfer rna species  <cit> .
a single nucleotide resolution map of the c. jejuni transcriptome was generated by differential rna-sequencing  using a motile variant of c. jejuni strain nctc  <dig>  <cit>  and roche  <dig> sequencing.
to assess whether the drna-seq cdna libraries are a good representation of transcribed sequences of c. jejuni, we compared the rpkm-values obtained for the cdss from the non-enriched   <dig> cdna sequencing with the previously published illumina-based rna-seq data for c. jejuni nctc  <dig>  <cit>  and the signal intensity on a pcr-product based c. jejuni microarray  <cit>  normalised to a genomic dna reference  <cit> .
there was a good correlation between the rpkm values for the two rna-seq experiments and the microarray data , with the best correlation observed between the two rna-seq based approaches.
genome-wide identification of c. jejuni transcription start sites and promoters
the drna-seq data were subsequently used for the identification of transcription start sites  of primary rnas, which are protected from digestion by terminator exonuclease  through their 5′-triphosphate modification  <cit> .
the drna-seq method is based on the comparison of read distribution between the two cdna-library enriched in primary 5′ ends , and the non-enriched cdna library .
read distribution in the -tex library displayed distribution throughout coding sequences, whereas treatment of rna with terminator exonuclease for the + tex library led to a typical cdna read distribution resembling a sawtooth-like profile with an elevated 5′ flank  <cit>  .
tss were annotated as primary, secondary, internal and antisense, based on their genomic location and association with annotated features, according to previously described criteria   <cit> .
we identified a total of  <dig> tss in the c. jejuni transcriptome , which consisted of  <dig> primary and  <dig> secondary tss located in intergenic regions which are associated with an annotated feature .
a total of  <dig> genes are transcribed from two independent promoters , with  <dig> tss located inside coding sequences or pseudogenes, and  <dig> tss located antisense to coding sequences or annotated features.
comparison of the drna-seq tss with  <dig> previously published c. jejuni tss, and  <dig> additional tss determined by 5′ race analysis for this study , showed that 32/ <dig> drna-seq tss were identical, and an additional  <dig> were within  <dig> nt distance  of the previously described tss, a difference which may be caused by the difficulty of  <dig> sequencing to accurately read long homopolymeric stretches  <cit> .
in addition, due to the low number of tss available for strain nctc  <dig>  we used tss from other reference strains and clinical isolates, and hence there may be strain differences in tss as well  <cit> .
the remaining  <dig> tss were previously reported to lack recognisable promoter sequences, and as they were obtained by primer extension probably represent the 5′ end of processed rna species rather than primary rnas.
this percentage match is similar as that described previously for h. pylori <cit>  and salmonella enterica serovar typhimurium  <cit> .
comparison with an independently performed study using illumina sequencing published during preparation of this manuscript  <cit> , showed that  <dig> tss described in additional file 3: table s <dig> match tss described in that study, but also highlights the identification of  <dig> additional tss not described in  <cit> .
there are several possible explanations for this discrepancy, which includes the different sequencing technology used , as well as difference in growth conditions or growth phase of the c. jejuni cultures.
it does highlight that the c. jejuni supergenome described in  <cit>  will undoubtedly be further expanded by future rna-seq based studies with c. jejuni.
analysis of c. jejuni promoter sequences
c. jejuni has three sigma factors for promoter recognition, with σ <dig> and σ <dig> thought to be primarily involved in flagellar biogenesis, and σ <dig> to function as major vegetative sigma factor  <cit> .
this was confirmed by drna-seq analysis, as only 26/ <dig>  of tss were preceded by a putative σ <dig> recognition sequence  and 18/ <dig>  of tss were preceded by a σ <dig> recognition sequence   <cit> .
the remaining 948/ <dig>  of tss were preceded by a gntanaat motif at 4– <dig> nt upstream of the tss , consistent with a − <dig> pribnow box for σ <dig> <cit> .
as previously predicted  <cit> , a − <dig> sequence was not present, but the sequences upstream of the − <dig> box showed a periodic signal centering on the − <dig>  − <dig>  − <dig> and − <dig> residues upstream of the tss .
we further analysed the average profile for  <dig> physico-chemical and geometrical dna properties of the aligned σ <dig> promoters from position + <dig> to − <dig> , including the corresponding  <dig> overlapping dinucleotides  <cit> .
this highlighted the conservation of  <dig> dinucleotides individually and the overall nucleotide, dinucleotide and physical properties conservation in comparison .
furthermore, the overall nucleotide and dinucleotide conservation is quite similar, whereas some properties are partly higher conserved, especially the two measures slide and entropy at positions – <dig> – <dig>  slide is known to be indicative for dna stiffness  <cit> , which is related to the dna entropy.
this indicates that the right dna stiffness at these positions might support promoter functioning.
we also found a significant correlation of two physical properties  of neighboured dinucleotides at positions –31/– <dig> –33/– <dig> .
overall there was a good correlation between the nucleotide sequence and physico-chemical and geometrical dna properties of the aligned σ <dig> promoters.
in addition, there was no difference observed between σ <dig> promoters upstream of internal tss and antisense tss when compared to primary tss and secondary tss in intergenic regions .
genome-wide antisense transcription in c. jejuni
within the  <dig> tss,  <dig> were on the antisense strand of annotated features , which confirmed the presence of genome-wide cis-antisense transcription, as recently described in other microbes  <cit> .
we subsequently confirmed four antisense tss by 5′ race , thus ensuring that the antisense tss identified are not an artifact of the drna-seq technology.
antisense transcripts were often relatively short , and many display a low number of reads, which may indicate spurious or pervasive transcription  <cit> .
the presence of antisense tss was not related to the level of transcription of the gene in either microarray or drna-seq, nor is antisense transcription related to specific functional categories of the genes opposite to the antisense tss .
some genes have multiple antisense tss, and antisense transcription was also detected opposite to transcriptionally active c. jejuni pseudogenes, which may allow for silencing of these pseudogenes via the activity of the double strand-specific ribonuclease iii  <cit>  or block the progress of rna polymerase via transcriptional interference  <cit> .
antisense rna may contribute to downregulation of parts of operons by post-transcriptional modification, without a requirement for transcriptional regulators.
alternatively, since some of the antisense tss were located at the 3′ end of the coding sequence, they may function in transcript termination.
non-coding rna
the annotations of the c. jejuni nctc  <dig> genome sequence  <cit>  suggested the presence of several species of non-coding and stable rnas, such as rrnas, trnas, tmrna, rnase p and the signal recognition particle  rna.
furthermore, the presence of a thiamine pyrophosphate -responsive riboswitch was predicted upstream of the thic gene  <cit> , as well as a possible purine riboswitch upstream of the purd gene  <cit> , but no other ncrna species were predicted or recognised, consistent with the absence of the hfq rna chaperone commonly associated with ncrna-dependent regulation in bacteria  <cit> .
a total of  <dig> putative non-coding and stable rnas  were identified in intergenic regions, scattered over the c. jejuni genome .
we confirmed transcription of eight of these ncrnas using northern hybridisation .
most of the ncrnas detected were relatively short , consistent with the relatively small and densily packed nature of the c. jejuni genome.
transcription of other predicted ncrnas  was confirmed using drna-seq, with the srp rna also being detected by northern hybridisation .
comparison of the c. jejuni srnas recently described by dugar et al.
<cit>  and the earlier c. jejuni rna-seq study by chaudhuri et al.
<cit>  showed a good correlation with the first study with  <dig> new ncrnas described here, but only partial overlap with the second study, as two proposed non-coding rnas matched , with the rest either gene promoters  or absent in our study.
the highest transcribed c. jejuni non-coding rna  is located upstream of the purd  gene, and a shorter version of this sequence was previously proposed as potential purine riboswitch  <cit> .
however, the same region was recently identified in h. pylori to harbor a homolog of the abundant 6s rna, a widespread regulator of rna polymerase that was first described in e. coli <cit> .
investigation of the  <dig> nt transcript  showed that it started further upstream than the previously predicted purine riboswitch, and that it folds in a structure corresponding to that of bacterial 6s rna  <cit> , with a closing stem, central bubble and terminal loops .
the e. coli 6s rna accumulates during exponential growth, and regulates the activity of σ70-containing rnap by mimicking its open complex promoter structure, thus complexing σ70-cofactored rnap  <cit> .
in e. coli, rnap releases itself from 6s rna after a nutritional upshift by the production of a small product rna , originating in the central bubble  <cit> .
our original analysis did not show any such prna, but as our cut-off for cdna reads was < <dig> nt, we also searched the < <dig> nt cdna reads for sequences on the complementary strand of 6s rna, and indeed found a  <dig> nt rna antisense to the 6s rna  at a similar position as detected for one of the two prnas of h. pylori 6s rna  <cit> .
in c. jejuni, 6s rna transcription is not significantly regulated in the different phases of exponential growth, and is not significantly altered upon growth cessation after exposure to ph  <dig>  or  <dig>  , suggesting its role in c. jejuni may be distinct from that reported for e. coli.
leader peptides upstream of amino acid biosynthetic genes
for tss that are > <dig> nt upstream of the annotated translation initiation codon , we searched the putative 5′ untranslated region  for the presence of a small open reading frame  with a potential ribosome binding site  upstream, as was recently described for the mfrx gene upstream of the c. jejuni mfrabe genes  <cit> .
several small orfs were thus identified and the length of the corresponding 5′ utr was corrected.
for three of these short orfs, a functional prediction can be made based on their location upstream of the leucine, tryptophan and methionine amino acid biosynthetic operons in c. jejuni   <cit> .
these three orfs are likely to encode regulatory leader peptides, which couple transcription of amino acid biosynthetic genes to the availability of amino acid-coupled trnas  <cit> , which has not been described for epsilonproteobacteria.
the small orf  upstream of the leuabcd  genes contains  <dig> leu-codons at the c-terminal end of the polypeptide, which are all rare codons for leucine in c. jejuni , which together represent only  <dig> % of the leu codons in c. jejuni.
similarly, the short orf  upstream of the trpedfba  genes does contain a single trp-codon at the c-terminal end of the polypeptide.
finally, a third short orf  is located on a short rna preceding the metba  genes, with  <dig> met-codons .
the rnas encoding these orfs all terminate shortly behind the stopcodon, and we propose that these polypeptides function as leader peptides, which allow transcription termination in the absence of ribosome stalling, and antitermination when the ribosome stalls due to the lack of availibility of trnas charged with the respective amino acid  <cit> .
5′ untranslated regions and leaderless mrnas
the average length of the 5′ untranslated regions  from  <dig> primary tss ranged from  <dig> to  <dig> nt .
a motif search using the meme motif discovery tool identified the sequence of 5′-aagga as conserved rbs motif .
the relatively short 5′ utrs of the other promoters in intergenic regions are consistent with the c. jejuni genome being tightly packed, since >93% of the genome is thought to contain functional regions  <cit> .
with the exception of the annotated tpp riboswitch upstream of the cj <dig>  gene  <cit> , there were no metabolite-sensing riboswitches detected in the collection of 5′ utrs.
this is consistent with a previous study predicting an absence of these structures in c. jejuni and related bacteria  <cit> .
nineteen of the 5′ utrs were < <dig> nt in length, with 12/ <dig> of the tss starting on the first nucleotide of the translation initiation codon, and these 5′ utrs lacked a recognisable shine-dalgarno  sequence, with all the connected genes having an atg startcodon  and are preceded by a tanaat σ <dig> promoter sequence .
such mrnas are known as leaderless mrnas  <cit> , and were previously thought to be rare in bacteria.
leaderless mrnas allow for translation during a range of physiological conditions, without competition for 30s ribosomes  <cit> .
the genes translated from the c. jejuni leaderless mrnas indeed encoded proteins predicted to be involved in stress-responses, like the dna repair systems nth endonuclease iii  and muty , the outer membrane efflux protein cmed  and the predicted multidrug efflux pump cj1257c .
comparison of primary transcriptomes of c. jejuni and h. pylori
the availability of drna-seq datasets for h. pylori <cit>  and c. jejuni  allowed for a direct transcriptome comparison between two relatively distant species within the order campylobacterales.
both species are pathogenic to man, have a similar genome size  and cellular morphology, and colonise mucus layers within the mammalian and avian gastrointestinal tracts.
gene order
we first used blastp to compare the orfs annotated in both genomes, and found they share  <dig> orfs when only counting the highest scoring ortholog.
these are however not ordered similary, as the gene order- based genome synteny was very low , with only  <dig> regions where  <dig> or more orthologs were in the same order.
the longest regions containing a conserved gene order are a ribosomal operon , the operon encoding a putative nadh-ubiquinone oxidoreductase  and the operon containing the f1f <dig> atpase , but the latter two still contain insertions of non-orthologous genes within the region.
other regions, such as the region containing the spot gene  <cit>  are contiguous in the c. jejuni genome, but split over two regions in the h. pylori genome  <cit>  .
we used one of these conserved regions  to directly compare the primary transcriptomes of both organisms .
even though the gene order is conserved, the location of promoters is not.
both sets of genes are transcribed from a σ70-dependent promoter upstream of the pyrh gene, but the location of the internal promoters differs completely between c. jejuni and h. pylori, with the latter having an additional promoter upstream of rpoz, and although both genomes have an internal promoter in the tyrs gene, its location is not conserved .
non-coding rna
similarly, non-coding rnas are not conserved between c. jejuni and h. pylori <cit> , with the exception of the stable rnas like the 6s rna.
when the genomic locations of one h. pylori ncrna  and one c. jejuni ncrna  were compared , this showed that the neighbouring genes are not conserved between the species, which may explain the species-specificity of the ncrnas.
one possible explanation for the uniqueness of the ncrnas identified here, may be that these ncrnas are generated and deleted during genome reorganisations and gene reshuffling.
the exception is the 6s rna, which is in both genomes upstream of the purd gene , although the upstream gene differs, and also there is significant sequence difference between the 6s rna genes of both organisms  <cit> .
antisense transcripts and internal promoters
both the c. jejuni and h. pylori genomes only encode σ <dig>  σ <dig> and σ <dig> sigma factors, with the large majority  of promoters being transcribed by σ <dig> <cit> .
the h. pylori and c. jejuni σ <dig> promoters show a high degree of homology, both with the gntanaat motif as − <dig> box  <cit> , and higher conservation of the t-residues at − <dig>  − <dig>  − <dig> and − <dig> .
to compare for conservation of the transcriptional landscape between c. jejuni and h. pylori, we compared the genes in both genomes which have a) antisense transcription or an internal promoter and b) an ortholog in the comparator genome.
this allowed for the comparison of 383 h. pylori genes with antisense transcription detected  <cit>  with  <dig> c. jejuni genes for which antisense transcription was detected.
of these only  <dig> of these genes displayed antisense transcription in both c. jejuni and h. pylori , however, for only two of these genes  the location of the antisense tss was conserved.
the antisense rna in the clpb  gene is located in a part encoding a conserved sequence in the clpb protein, with the antisense rna being of identical length , with both promoter and asrnas being ~80% identical in dna sequence.
in contrast, 44/ <dig> other asrnas were located in different parts  of the corresponding c. jejuni and h. pylori genes, as is shown in figure 3c for the gyrb  gene, with the h. pylori asrna being located in the first half of the gene, and the c. jejuni asrna being located in the part encoding the c-terminal end of the protein.
the σ <dig> promoter motifs were not conserved in these two genomes, with in both cases the highly conserved −7 t residue being altered, thus probably inactivating the σ <dig> recognition sequence.
similar results were obtained with the  <dig> internal promoters of h. pylori and  <dig> internal promoters of c. jejuni.
only  <dig> of these internal promoters were located in orthologs shared between h. pylori and c. jejuni , and of these  <dig>  only six have the internal promoter in the same position in the gene, thus highlighting the lack of conservation of the transcriptional landscapes between h. pylori and c. jejuni.
predictive comparisons with other epsilonproteobacterial genomes
to investigate whether the lack of conservation of transcriptional landscapes between c. jejuni and h. pylori were dependent on phylogenetic differences, we used comparative genomics and genome synteny analyses with the genomes of  <dig> other epsilonproteobacteria .
these included  <dig> members of the campylobacteraceae ,  <dig> members of the helicobacteraceae , and  <dig> species of other epsilonproteobacteria found in deep-sea hydrothermal vents.
a phylogenetic tree based on 16s rdna sequences  shows the phylogenetic relationships between the investigated species, and shows the subdivision of campylobacter spp into thermophilic  and non-thermophilic species , and within the helicobacter spp the subdivision into gastric helicobacter spp  and enterohepatic helicobacter spp .
gene order
we subsequently used the annotated features to compare genome synteny of all these species with c. jejuni nctc  <dig> and h. pylori  <dig> .
from these analyses it is clear that gene order -based genome synteny is only conserved between closely related species , with genome synteny rapidly lost beyond these closely related species .
this progressive lack of genome synteny may explain the large differences between the experimentally determined c. jejuni and h. pylori transcriptomes, and may also explain the lack of conservation of non-coding rnas within this phylogenetic clade   <cit> .
leaderless mrna
we also assessed the difference between epsilonproteobacteria at the gene level, using the difference between leaderless and leadered mrnas .
an initial comparison of leaderless mrnas between c. jejuni and h. pylori showed clear differences, as only 3/ <dig> genes are leaderless in both organisms, with  <dig> leaderless in c. jejuni only, and  <dig> leaderless in h. pylori only.
one example of such difference is shown in figure 5a, for the c. jejuni cj0153c gene.
this gene is located in a conserved five-gene region , but is leaderless in c. jejuni, effectively splitting the five genes into two separate operons, while there is only a single five-gene operon in h. pylori .
the intergenic regions showed clear differences with the σ <dig> - <dig> sequence  in c. jejuni and an rbs in h. pylori .
as the cj0155c-0151c genes are conserved throughout the epsilonproteobacteria, we compared the intergenic regions between the cj0154c and cj0153c orthologs in all  <dig> genomes, and this showed that the majority of genomes  contain a predicted σ <dig> - <dig> sequence, while 13/ <dig> contain a recognisable rbs sequence, with  <dig> genomes containing neither .
the rbs was only present in helicobacter spp, and interestingly also in c. upsaliensis, which is surprising in view of its close phylogenetic relationship to c. jejuni, and suggests a secondary, independent evolutionary change in c. upsaliensis or the effect of a natural transformation event .
we expanded this search to all  <dig> genes which are leaderless in either c. jejuni or h. pylori in all  <dig> epsilonproteobacterial genomes.
all genomes were searched for orthologs of the c. jejuni and h. pylori genes, and the − <dig> to + <dig> sequences  were searched manually for σ <dig> - <dig> box and atg startcodon   <cit>  and potential ribosome binding site and all three possible startcodons , and were also used for a meme motif search .
from the overview presented in figure 5b, it is clear that the predicted leaderless or leadered mrnas do not strictly follow the 16s rdna-based phylogenetic tree .
while c. jejuni, c. doylei  and c. coli cannot be distinguished in this analysis, this is also mostly true for h. pylori, h. acinonychis and h. cetorum, mirroring the genome synteny analyses .
only a single gene  is a predicted leaderless mrna in all species containing this gene, which is always upstream of the uvrc dna repair gene, again supporting an important role of leaderless mrnas in stress responses  <cit> .
interestingly, the sulfurimonas spp  have more leaderless mrnas in common with the campylobacter spp than with other members of the helicobacteraceae, while there is virtually no conservation of leaderless mrnas within the genus helicobacter .
another surprise was that the enterohepatic helicobacter spp had only one or two predicted leaderless mrnas in common with other epsilonproteobacteria, which suggests that they may contain a completely different set of leaderless mrnas, something which was not followed up for this study.
antisense transcription and internal promoters
next we compared five internal and three antisense promoters conserved between c. jejuni and h. pylori  in the other epsilonproteobacterial genomes.
blast-searches were used to identify the corresponding regions in the respective orthologs, and sequences were searched for potential σ <dig> - <dig> box both manually and using meme .
as with the leaderless mrnas, there was no full conservation of internal or antisense promoters, although for the promoter internal to cj <dig> this can be linked to the genomic organisation: in w. succinogenes the cj <dig> ortholog is fused to the downstream cj <dig> ortholog , whereas in caminibacter mediatlanticus and nautilia profundicola the downstream cj <dig> ortholog is absent .
similarly, there is a good albeit imperfect correlation between the presence of the cj <dig> and cj <dig>  orthologs and the presence of an internal promoter in cj <dig> .
with regard to the antisense promoters, most of the cj0509c  orthologs in the epsilonproteobacteria contain a predicted σ <dig> - <dig> box at the equivalent position , whereas the predicted antisense promoters in the cj <dig>  orthologs are confined to the phylogenetically closely related species, i.e.
the c. jejuni, c. doylei and c. coli group vs the h. pylori, h. acinonychis and h. cetorum group, consistent with evolutionary relationships between these species .
leader peptides
finally, we also searched the epsilonproteobacterial genomes for orthologs of the leucine, tryptophan and methionine amino acid biosynthetic genes, and whether they contained potential leader peptides upstream .
while leul orthologs  were found in most epsilonproteobacterial genomes with leucine biosynthetic genes, trpl and metl orthologs were only found in c. jejuni, c. coli and c. doylei, and were absent from other epsilonproteobacteria with tryptophan or methionine biosynthetic genes .
as with the previous examples, this suggests that there are clear differences between genomes and transcriptomes within the epsilonproteobacteria, and also that changes in genome content and gene order have necessitated the development of differential forms of transcriptional, post-transcriptional and possibly translational regulation of gene and protein expression.
CONCLUSIONS
in this study, we present the primary transcriptome of campylobacter jejuni at single nucleotide resolution, obtained by using differential rna-sequencing analysis using  <dig> sequencing.
our analysis confirms that the original analyses of the c. jejuni genome  <cit>  have indeed underestimated its versatility and complexity, with a wealth of non-coding and antisense rnas, as well as intragenic promoters and leaderless mrnas.
all these features are likely to contribute to the success of c. jejuni as pathogen, allowing it to survive in the food chain and infect different hosts.
our analysis complements and supplements the previously released and reannotated genome sequence and protein interactome maps for c. jejuni <cit> , and rna-seq analyses using illumina sequencing  <cit> .
the large number of transcription start sites found in the relatively small c. jejuni genome supports the findings in other bacteria, where a much larger number of tss have been detected than was expected, e.g.
the > <dig>  tss identified in sinorhizobium meliloti <cit> , which has a much larger genome, megaplasmids and multiple sigma factors when compared to the epsilonproteobacteria.
the availability of the drna-seq datasets for two related members of the epsilonproteobacteria has allowed for the first high resolution comparison of primary transcriptomes at the single-nucleotide level of related, but independent species .
all characterised members of the epsilonproteobacteria have relatively small genomes , and show high levels of variation, probably due to a relative scarcity of dna repair mechanisms and the exchange of dna by natural transformation and horizontal gene transfer  <cit> .
interestingly, despite the variability in both the genome and transcriptomes of these organisms, there were parts which showed high levels of conservation  and others which showed no conservation at all .
with regard to transcriptome organisation, there were the large scale differences already predicted by the comparison of genome sequences   <cit> , but also very subtle differences with respect to coupling and uncoupling of transcriptional networks, for instance by the appearance and disappearance of promoters coupled to leaderless mrnas , and generation and absence of internal and antisense promoters .
overall, there was very low synteny between the regulatory features of the c. jejuni and h. pylori transcriptomes with respect to the position and sequence of internal promoters, antisense rnas and non-coding rnas, with the exception of the ancestral 6s rna.
orthologous sequences to the c. jejuni ncrnas and asrnas were only found in other c. jejuni strains and partially in closely related species , and similarly conservation of h. pylori features was limited to other h. pylori strains, and partially with h. acinonychis and h. cetorum <cit> .
all this suggests that many of these regulatory features of the transcriptomes of epsilonproteobacteria will have developed after genera have evolutionary split from a common ancestor, and are likely to be in constant flux depending on their ecological niches and its influence on genome reorganisation, mutation frequency and horizontal gene transfer.
the large differences observed between c. jejuni and h. pylori, and even the differences observed between c. jejuni strains  <cit>  promises that future rna-seq experiments with other epsilonproteobacteria can be expected to show up many new and exciting features.

 while our appreciation of microbial diversity has been greatly increased by the exponential increase in the availability of genome sequences and by metagenomic approaches, it has also highlighted our relative lack of understanding about what drives variation, and which limitations and constraints control the process of genome variation. diversity at the level of gene order and genome content can be introduced via the reorganisation of the genome, through combinations of gene inversion, recombination, gene duplication, deletion and horizontal gene transfer. such movement, deletion or introduction of genes or operons can create a problem for the cell, as the reorganisation of the genome may result in disruption of transcriptional circuitry controlling the expression levels of such genes. however, variability can also be introduced at the gene level, e.g. by generation of alternative transcription start sites, promoter recognition sequences or alterations in the 5′ untranslated regions affecting folding or stability. the level of rna in a cell is usually controlled at the transcriptional and post-transcriptional levels. in bacteria, transcriptional regulation is commonly mediated via control of transcription initiation by rna polymerase at the promoter. alternatively, post-transcriptional gene regulation is often mediated by the action of non-coding or antisense rna, rna chaperones and the activity of ribonucleases. in the last two years, the use of high-throughput sequencing of cdna has revealed that the complexity of the microbial transcriptome is much higher than previously predicted. however, the high level of phylogenetic diversity within the bacterial kingdom has so far limited the possibilities for interspecies transcriptome comparison, since the species for which high resolution transcriptome maps are available are either too closely related or too distantly related to allow meaningful comparisons at the evolutionary level. the epsilon-subdivision of the proteobacteria is a lineage which contains both pathogenic and non-pathogenic bacteria. the best studied examples of the former category are the human pathogens campylobacter jejuni and helicobacter pylori, which belong to the order campylobacterales. however, next to these important human pathogens, the epsilonproteobacteria also contain chemolithoautotrophic microorganisms isolated from deep-sea vents, as well as the bovine rumen-colonising bacterium wolinella succinogenes. despite the differences in ecological niches between the genera, and the genome sizes of epsilonproteobacteria varying between and mbp, genomic comparisons revealed that the epsilonproteobacteria share similar transcription machinery including few sigma factors, metabolic pathways and limitations, and have about half of the predicted genes in the genome in common with other epsilonproteobacteria. however, while these genomes share functionality, genome architecture and often low g + c content, the gene order and genome organisation have diverged significantly. this raises the question on how the genome and associated transcriptome copes with such large scale reorganisations of the genome when genera and species evolutionary diverge over time. to address this question, we have mapped the primary transcriptome of c. jejuni at the single nucleotide resolution using differential rna-seq, have compared it with the primary transcriptome map of h. pylori and have used genome sequences of other taxa of the epsilonproteobacteria to assess conservation and evolution of transcriptional circuitry in this highly variable group of bacteria.  differential rna-seq analysis of the c. jejuni primary transcriptome the c. jejuni nctc genome contains annotated coding sequences, with only few stable rna molecules known outside the ribosomal and transfer rna species. a single nucleotide resolution map of the c. jejuni transcriptome was generated by differential rna-sequencing using a motile variant of c. jejuni strain nctc and roche sequencing. to assess whether the drna-seq cdna libraries are a good representation of transcribed sequences of c. jejuni, we compared the rpkm-values obtained for the cdss from the non-enriched cdna sequencing with the previously published illumina-based rna-seq data for c. jejuni nctc and the signal intensity on a pcr-product based c. jejuni microarray normalised to a genomic dna reference. there was a good correlation between the rpkm values for the two rna-seq experiments and the microarray data, with the best correlation observed between the two rna-seq based approaches. genome-wide identification of c. jejuni transcription start sites and promoters the drna-seq data were subsequently used for the identification of transcription start sites of primary rnas, which are protected from digestion by terminator exonuclease through their 5′-triphosphate modification. the drna-seq method is based on the comparison of read distribution between the two cdna-library enriched in primary 5′ ends, and the non-enriched cdna library. read distribution in the -tex library displayed distribution throughout coding sequences, whereas treatment of rna with terminator exonuclease for the + tex library led to a typical cdna read distribution resembling a sawtooth-like profile with an elevated 5′ flank. tss were annotated as primary, secondary, internal and antisense, based on their genomic location and association with annotated features, according to previously described criteria. we identified a total of tss in the c. jejuni transcriptome, which consisted of primary and secondary tss located in intergenic regions which are associated with an annotated feature. a total of genes are transcribed from two independent promoters, with tss located inside coding sequences or pseudogenes, and tss located antisense to coding sequences or annotated features. comparison of the drna-seq tss with previously published c. jejuni tss, and additional tss determined by 5′ race analysis for this study, showed that 32/ drna-seq tss were identical, and an additional were within nt distance of the previously described tss, a difference which may be caused by the difficulty of sequencing to accurately read long homopolymeric stretches. in addition, due to the low number of tss available for strain nctc we used tss from other reference strains and clinical isolates, and hence there may be strain differences in tss as well. the remaining tss were previously reported to lack recognisable promoter sequences, and as they were obtained by primer extension probably represent the 5′ end of processed rna species rather than primary rnas. this percentage match is similar as that described previously for h. pylori and salmonella enterica serovar typhimurium. comparison with an independently performed study using illumina sequencing published during preparation of this manuscript, showed that tss described in additional file 3: table s match tss described in that study, but also highlights the identification of additional tss not described in. there are several possible explanations for this discrepancy, which includes the different sequencing technology used, as well as difference in growth conditions or growth phase of the c. jejuni cultures. it does highlight that the c. jejuni supergenome described in will undoubtedly be further expanded by future rna-seq based studies with c. jejuni. analysis of c. jejuni promoter sequences c. jejuni has three sigma factors for promoter recognition, with σ and σ thought to be primarily involved in flagellar biogenesis, and σ to function as major vegetative sigma factor. this was confirmed by drna-seq analysis, as only 26/ of tss were preceded by a putative σ recognition sequence and 18/ of tss were preceded by a σ recognition sequence. the remaining 948/ of tss were preceded by a gntanaat motif at 4– nt upstream of the tss, consistent with a − pribnow box for σ. as previously predicted, a − sequence was not present, but the sequences upstream of the − box showed a periodic signal centering on the − − − and − residues upstream of the tss. we further analysed the average profile for physico-chemical and geometrical dna properties of the aligned σ promoters from position + to −, including the corresponding overlapping dinucleotides. this highlighted the conservation of dinucleotides individually and the overall nucleotide, dinucleotide and physical properties conservation in comparison. furthermore, the overall nucleotide and dinucleotide conservation is quite similar, whereas some properties are partly higher conserved, especially the two measures slide and entropy at positions – – slide is known to be indicative for dna stiffness, which is related to the dna entropy. this indicates that the right dna stiffness at these positions might support promoter functioning. we also found a significant correlation of two physical properties of neighboured dinucleotides at positions –31/– –33/–. overall there was a good correlation between the nucleotide sequence and physico-chemical and geometrical dna properties of the aligned σ promoters. in addition, there was no difference observed between σ promoters upstream of internal tss and antisense tss when compared to primary tss and secondary tss in intergenic regions. genome-wide antisense transcription in c. jejuni within the tss, were on the antisense strand of annotated features, which confirmed the presence of genome-wide cis-antisense transcription, as recently described in other microbes. we subsequently confirmed four antisense tss by 5′ race, thus ensuring that the antisense tss identified are not an artifact of the drna-seq technology. antisense transcripts were often relatively short, and many display a low number of reads, which may indicate spurious or pervasive transcription. the presence of antisense tss was not related to the level of transcription of the gene in either microarray or drna-seq, nor is antisense transcription related to specific functional categories of the genes opposite to the antisense tss. some genes have multiple antisense tss, and antisense transcription was also detected opposite to transcriptionally active c. jejuni pseudogenes, which may allow for silencing of these pseudogenes via the activity of the double strand-specific ribonuclease iii or block the progress of rna polymerase via transcriptional interference. antisense rna may contribute to downregulation of parts of operons by post-transcriptional modification, without a requirement for transcriptional regulators. alternatively, since some of the antisense tss were located at the 3′ end of the coding sequence, they may function in transcript termination. non-coding rna the annotations of the c. jejuni nctc genome sequence suggested the presence of several species of non-coding and stable rnas, such as rrnas, trnas, tmrna, rnase p and the signal recognition particle rna. furthermore, the presence of a thiamine pyrophosphate -responsive riboswitch was predicted upstream of the thic gene, as well as a possible purine riboswitch upstream of the purd gene, but no other ncrna species were predicted or recognised, consistent with the absence of the hfq rna chaperone commonly associated with ncrna-dependent regulation in bacteria. a total of putative non-coding and stable rnas were identified in intergenic regions, scattered over the c. jejuni genome. we confirmed transcription of eight of these ncrnas using northern hybridisation. most of the ncrnas detected were relatively short, consistent with the relatively small and densily packed nature of the c. jejuni genome. transcription of other predicted ncrnas was confirmed using drna-seq, with the srp rna also being detected by northern hybridisation. comparison of the c. jejuni srnas recently described by dugar et al. and the earlier c. jejuni rna-seq study by chaudhuri et al. showed a good correlation with the first study with new ncrnas described here, but only partial overlap with the second study, as two proposed non-coding rnas matched, with the rest either gene promoters or absent in our study. the highest transcribed c. jejuni non-coding rna is located upstream of the purd gene, and a shorter version of this sequence was previously proposed as potential purine riboswitch. however, the same region was recently identified in h. pylori to harbor a homolog of the abundant 6s rna, a widespread regulator of rna polymerase that was first described in e. coli. investigation of the nt transcript showed that it started further upstream than the previously predicted purine riboswitch, and that it folds in a structure corresponding to that of bacterial 6s rna, with a closing stem, central bubble and terminal loops. the e. coli 6s rna accumulates during exponential growth, and regulates the activity of σ70-containing rnap by mimicking its open complex promoter structure, thus complexing σ70-cofactored rnap. in e. coli, rnap releases itself from 6s rna after a nutritional upshift by the production of a small product rna, originating in the central bubble. our original analysis did not show any such prna, but as our cut-off for cdna reads was < nt, we also searched the < nt cdna reads for sequences on the complementary strand of 6s rna, and indeed found a nt rna antisense to the 6s rna at a similar position as detected for one of the two prnas of h. pylori 6s rna. in c. jejuni, 6s rna transcription is not significantly regulated in the different phases of exponential growth, and is not significantly altered upon growth cessation after exposure to ph  or, suggesting its role in c. jejuni may be distinct from that reported for e. coli. leader peptides upstream of amino acid biosynthetic genes for tss that are > nt upstream of the annotated translation initiation codon, we searched the putative 5′ untranslated region for the presence of a small open reading frame with a potential ribosome binding site upstream, as was recently described for the mfrx gene upstream of the c. jejuni mfrabe genes. several small orfs were thus identified and the length of the corresponding 5′ utr was corrected. for three of these short orfs, a functional prediction can be made based on their location upstream of the leucine, tryptophan and methionine amino acid biosynthetic operons in c. jejuni. these three orfs are likely to encode regulatory leader peptides, which couple transcription of amino acid biosynthetic genes to the availability of amino acid-coupled trnas, which has not been described for epsilonproteobacteria. the small orf upstream of the leuabcd genes contains leu-codons at the c-terminal end of the polypeptide, which are all rare codons for leucine in c. jejuni, which together represent only % of the leu codons in c. jejuni. similarly, the short orf upstream of the trpedfba genes does contain a single trp-codon at the c-terminal end of the polypeptide. finally, a third short orf is located on a short rna preceding the metba genes, with met-codons. the rnas encoding these orfs all terminate shortly behind the stopcodon, and we propose that these polypeptides function as leader peptides, which allow transcription termination in the absence of ribosome stalling, and antitermination when the ribosome stalls due to the lack of availibility of trnas charged with the respective amino acid. 5′ untranslated regions and leaderless mrnas the average length of the 5′ untranslated regions from primary tss ranged from to nt. a motif search using the meme motif discovery tool identified the sequence of 5′-aagga as conserved rbs motif. the relatively short 5′ utrs of the other promoters in intergenic regions are consistent with the c. jejuni genome being tightly packed, since >93% of the genome is thought to contain functional regions. with the exception of the annotated tpp riboswitch upstream of the cj gene, there were no metabolite-sensing riboswitches detected in the collection of 5′ utrs. this is consistent with a previous study predicting an absence of these structures in c. jejuni and related bacteria. nineteen of the 5′ utrs were < nt in length, with 12/ of the tss starting on the first nucleotide of the translation initiation codon, and these 5′ utrs lacked a recognisable shine-dalgarno sequence, with all the connected genes having an atg startcodon and are preceded by a tanaat σ promoter sequence. such mrnas are known as leaderless mrnas, and were previously thought to be rare in bacteria. leaderless mrnas allow for translation during a range of physiological conditions, without competition for 30s ribosomes. the genes translated from the c. jejuni leaderless mrnas indeed encoded proteins predicted to be involved in stress-responses, like the dna repair systems nth endonuclease iii and muty, the outer membrane efflux protein cmed and the predicted multidrug efflux pump cj1257c. comparison of primary transcriptomes of c. jejuni and h. pylori the availability of drna-seq datasets for h. pylori and c. jejuni allowed for a direct transcriptome comparison between two relatively distant species within the order campylobacterales. both species are pathogenic to man, have a similar genome size and cellular morphology, and colonise mucus layers within the mammalian and avian gastrointestinal tracts. gene order we first used blastp to compare the orfs annotated in both genomes, and found they share orfs when only counting the highest scoring ortholog. these are however not ordered similary, as the gene order- based genome synteny was very low, with only regions where or more orthologs were in the same order. the longest regions containing a conserved gene order are a ribosomal operon, the operon encoding a putative nadh-ubiquinone oxidoreductase and the operon containing the f1f atpase, but the latter two still contain insertions of non-orthologous genes within the region. other regions, such as the region containing the spot gene are contiguous in the c. jejuni genome, but split over two regions in the h. pylori genome. we used one of these conserved regions to directly compare the primary transcriptomes of both organisms. even though the gene order is conserved, the location of promoters is not. both sets of genes are transcribed from a σ70-dependent promoter upstream of the pyrh gene, but the location of the internal promoters differs completely between c. jejuni and h. pylori, with the latter having an additional promoter upstream of rpoz, and although both genomes have an internal promoter in the tyrs gene, its location is not conserved. non-coding rna similarly, non-coding rnas are not conserved between c. jejuni and h. pylori, with the exception of the stable rnas like the 6s rna. when the genomic locations of one h. pylori ncrna and one c. jejuni ncrna were compared, this showed that the neighbouring genes are not conserved between the species, which may explain the species-specificity of the ncrnas. one possible explanation for the uniqueness of the ncrnas identified here, may be that these ncrnas are generated and deleted during genome reorganisations and gene reshuffling. the exception is the 6s rna, which is in both genomes upstream of the purd gene, although the upstream gene differs, and also there is significant sequence difference between the 6s rna genes of both organisms. antisense transcripts and internal promoters both the c. jejuni and h. pylori genomes only encode σ σ and σ sigma factors, with the large majority of promoters being transcribed by σ. the h. pylori and c. jejuni σ promoters show a high degree of homology, both with the gntanaat motif as − box, and higher conservation of the t-residues at − − − and −. to compare for conservation of the transcriptional landscape between c. jejuni and h. pylori, we compared the genes in both genomes which have a) antisense transcription or an internal promoter and b) an ortholog in the comparator genome. this allowed for the comparison of 383 h. pylori genes with antisense transcription detected with c. jejuni genes for which antisense transcription was detected. of these only of these genes displayed antisense transcription in both c. jejuni and h. pylori, however, for only two of these genes the location of the antisense tss was conserved. the antisense rna in the clpb gene is located in a part encoding a conserved sequence in the clpb protein, with the antisense rna being of identical length, with both promoter and asrnas being ~80% identical in dna sequence. in contrast, 44/ other asrnas were located in different parts of the corresponding c. jejuni and h. pylori genes, as is shown in figure 3c for the gyrb gene, with the h. pylori asrna being located in the first half of the gene, and the c. jejuni asrna being located in the part encoding the c-terminal end of the protein. the σ promoter motifs were not conserved in these two genomes, with in both cases the highly conserved −7 t residue being altered, thus probably inactivating the σ recognition sequence. similar results were obtained with the internal promoters of h. pylori and internal promoters of c. jejuni. only of these internal promoters were located in orthologs shared between h. pylori and c. jejuni, and of these only six have the internal promoter in the same position in the gene, thus highlighting the lack of conservation of the transcriptional landscapes between h. pylori and c. jejuni. predictive comparisons with other epsilonproteobacterial genomes to investigate whether the lack of conservation of transcriptional landscapes between c. jejuni and h. pylori were dependent on phylogenetic differences, we used comparative genomics and genome synteny analyses with the genomes of other epsilonproteobacteria. these included members of the campylobacteraceae, members of the helicobacteraceae, and species of other epsilonproteobacteria found in deep-sea hydrothermal vents. a phylogenetic tree based on 16s rdna sequences shows the phylogenetic relationships between the investigated species, and shows the subdivision of campylobacter spp into thermophilic and non-thermophilic species, and within the helicobacter spp the subdivision into gastric helicobacter spp and enterohepatic helicobacter spp. gene order we subsequently used the annotated features to compare genome synteny of all these species with c. jejuni nctc and h. pylori. from these analyses it is clear that gene order -based genome synteny is only conserved between closely related species, with genome synteny rapidly lost beyond these closely related species. this progressive lack of genome synteny may explain the large differences between the experimentally determined c. jejuni and h. pylori transcriptomes, and may also explain the lack of conservation of non-coding rnas within this phylogenetic clade. leaderless mrna we also assessed the difference between epsilonproteobacteria at the gene level, using the difference between leaderless and leadered mrnas. an initial comparison of leaderless mrnas between c. jejuni and h. pylori showed clear differences, as only 3/ genes are leaderless in both organisms, with leaderless in c. jejuni only, and leaderless in h. pylori only. one example of such difference is shown in figure 5a, for the c. jejuni cj0153c gene. this gene is located in a conserved five-gene region, but is leaderless in c. jejuni, effectively splitting the five genes into two separate operons, while there is only a single five-gene operon in h. pylori. the intergenic regions showed clear differences with the σ - sequence in c. jejuni and an rbs in h. pylori. as the cj0155c-0151c genes are conserved throughout the epsilonproteobacteria, we compared the intergenic regions between the cj0154c and cj0153c orthologs in all genomes, and this showed that the majority of genomes contain a predicted σ - sequence, while 13/ contain a recognisable rbs sequence, with genomes containing neither. the rbs was only present in helicobacter spp, and interestingly also in c. upsaliensis, which is surprising in view of its close phylogenetic relationship to c. jejuni, and suggests a secondary, independent evolutionary change in c. upsaliensis or the effect of a natural transformation event. we expanded this search to all genes which are leaderless in either c. jejuni or h. pylori in all epsilonproteobacterial genomes. all genomes were searched for orthologs of the c. jejuni and h. pylori genes, and the − to + sequences were searched manually for σ - box and atg startcodon and potential ribosome binding site and all three possible startcodons, and were also used for a meme motif search. from the overview presented in figure 5b, it is clear that the predicted leaderless or leadered mrnas do not strictly follow the 16s rdna-based phylogenetic tree. while c. jejuni, c. doylei and c. coli cannot be distinguished in this analysis, this is also mostly true for h. pylori, h. acinonychis and h. cetorum, mirroring the genome synteny analyses. only a single gene is a predicted leaderless mrna in all species containing this gene, which is always upstream of the uvrc dna repair gene, again supporting an important role of leaderless mrnas in stress responses. interestingly, the sulfurimonas spp have more leaderless mrnas in common with the campylobacter spp than with other members of the helicobacteraceae, while there is virtually no conservation of leaderless mrnas within the genus helicobacter. another surprise was that the enterohepatic helicobacter spp had only one or two predicted leaderless mrnas in common with other epsilonproteobacteria, which suggests that they may contain a completely different set of leaderless mrnas, something which was not followed up for this study. antisense transcription and internal promoters next we compared five internal and three antisense promoters conserved between c. jejuni and h. pylori in the other epsilonproteobacterial genomes. blast-searches were used to identify the corresponding regions in the respective orthologs, and sequences were searched for potential σ - box both manually and using meme. as with the leaderless mrnas, there was no full conservation of internal or antisense promoters, although for the promoter internal to cj this can be linked to the genomic organisation: in w. succinogenes the cj ortholog is fused to the downstream cj ortholog, whereas in caminibacter mediatlanticus and nautilia profundicola the downstream cj ortholog is absent. similarly, there is a good albeit imperfect correlation between the presence of the cj and cj orthologs and the presence of an internal promoter in cj. with regard to the antisense promoters, most of the cj0509c orthologs in the epsilonproteobacteria contain a predicted σ - box at the equivalent position, whereas the predicted antisense promoters in the cj orthologs are confined to the phylogenetically closely related species, i.e. the c. jejuni, c. doylei and c. coli group vs the h. pylori, h. acinonychis and h. cetorum group, consistent with evolutionary relationships between these species. leader peptides finally, we also searched the epsilonproteobacterial genomes for orthologs of the leucine, tryptophan and methionine amino acid biosynthetic genes, and whether they contained potential leader peptides upstream. while leul orthologs were found in most epsilonproteobacterial genomes with leucine biosynthetic genes, trpl and metl orthologs were only found in c. jejuni, c. coli and c. doylei, and were absent from other epsilonproteobacteria with tryptophan or methionine biosynthetic genes. as with the previous examples, this suggests that there are clear differences between genomes and transcriptomes within the epsilonproteobacteria, and also that changes in genome content and gene order have necessitated the development of differential forms of transcriptional, post-transcriptional and possibly translational regulation of gene and protein expression.  in this study, we present the primary transcriptome of campylobacter jejuni at single nucleotide resolution, obtained by using differential rna-sequencing analysis using sequencing. our analysis confirms that the original analyses of the c. jejuni genome have indeed underestimated its versatility and complexity, with a wealth of non-coding and antisense rnas, as well as intragenic promoters and leaderless mrnas. all these features are likely to contribute to the success of c. jejuni as pathogen, allowing it to survive in the food chain and infect different hosts. our analysis complements and supplements the previously released and reannotated genome sequence and protein interactome maps for c. jejuni, and rna-seq analyses using illumina sequencing. the large number of transcription start sites found in the relatively small c. jejuni genome supports the findings in other bacteria, where a much larger number of tss have been detected than was expected, e.g. the > tss identified in sinorhizobium meliloti, which has a much larger genome, megaplasmids and multiple sigma factors when compared to the epsilonproteobacteria. the availability of the drna-seq datasets for two related members of the epsilonproteobacteria has allowed for the first high resolution comparison of primary transcriptomes at the single-nucleotide level of related, but independent species. all characterised members of the epsilonproteobacteria have relatively small genomes, and show high levels of variation, probably due to a relative scarcity of dna repair mechanisms and the exchange of dna by natural transformation and horizontal gene transfer. interestingly, despite the variability in both the genome and transcriptomes of these organisms, there were parts which showed high levels of conservation and others which showed no conservation at all. with regard to transcriptome organisation, there were the large scale differences already predicted by the comparison of genome sequences, but also very subtle differences with respect to coupling and uncoupling of transcriptional networks, for instance by the appearance and disappearance of promoters coupled to leaderless mrnas, and generation and absence of internal and antisense promoters. overall, there was very low synteny between the regulatory features of the c. jejuni and h. pylori transcriptomes with respect to the position and sequence of internal promoters, antisense rnas and non-coding rnas, with the exception of the ancestral 6s rna. orthologous sequences to the c. jejuni ncrnas and asrnas were only found in other c. jejuni strains and partially in closely related species, and similarly conservation of h. pylori features was limited to other h. pylori strains, and partially with h. acinonychis and h. cetorum. all this suggests that many of these regulatory features of the transcriptomes of epsilonproteobacteria will have developed after genera have evolutionary split from a common ancestor, and are likely to be in constant flux depending on their ecological niches and its influence on genome reorganisation, mutation frequency and horizontal gene transfer. the large differences observed between c. jejuni and h. pylori, and even the differences observed between c. jejuni strains promises that future rna-seq experiments with other epsilonproteobacteria can be expected to show up many new and exciting features. 
gene reshuffling, point mutations and horizontal gene transfer contribute to bacterial genome variation, but require the genome to rewire its transcriptional circuitry to ensure that inserted, mutated or reshuffled genes are transcribed at appropriate levels. regulatory features may have evolved after these species split from a common ancestor, with transcriptome rewiring compensating for changes introduced by genomic reshuffling and horizontal gene transfer. several previously unknown features were identified in the c. jejuni transcriptional landscape, like leaderless mrnas and potential leader peptides upstream of amino acid biosynthesis genes. predictive comparisons using other epsilonproteobacterial genomes suggests that this lack of conservation of transcriptional features is common to all epsilonproteobacterial genomes, and is associated with the absence of genome synteny in this subdivision of the proteobacteria. a cross-species comparison of the primary transcriptomes of c. jejuni and the related epsilonproteobacterium helicobacter pylori highlighted a lack of conservation of operon organisation, position of intragenic and antisense promoters or leaderless mrnas. differential rna-sequencing using sequencing technology was used to determine the primary transcriptome of c. jejuni nctc which consists of transcription start sites, which included putative non-coding and stable rnas, intragenic tss, and antisense tss. the genomes of epsilonproteobacteria display very low synteny, due to high levels of reshuffling and reorganisation of gene order, but still share a significant number of gene orthologs allowing comparison. here we present the primary transcriptome of the pathogenic epsilonproteobacterium campylobacter jejuni, and have used this for comparative and predictive transcriptomics in the epsilonproteobacteria. both the genomes and transcriptomes of epsilonproteobacteria are highly variable, both at the genome level by combining and division of multicistronic operons, but also on the gene level by generation or deletion of promoter sequences and 5′ untranslated regions.
 gene reshuffling, point mutations and horizontal gene transfer contribute to bacterial genome variation, but require the genome to rewire its transcriptional circuitry to ensure that inserted, mutated or reshuffled genes are transcribed at appropriate levels. the genomes of epsilonproteobacteria display very low synteny, due to high levels of reshuffling and reorganisation of gene order, but still share a significant number of gene orthologs allowing comparison. here we present the primary transcriptome of the pathogenic epsilonproteobacterium campylobacter jejuni, and have used this for comparative and predictive transcriptomics in the epsilonproteobacteria.  differential rna-sequencing using sequencing technology was used to determine the primary transcriptome of c. jejuni nctc which consists of transcription start sites, which included putative non-coding and stable rnas, intragenic tss, and antisense tss. several previously unknown features were identified in the c. jejuni transcriptional landscape, like leaderless mrnas and potential leader peptides upstream of amino acid biosynthesis genes. a cross-species comparison of the primary transcriptomes of c. jejuni and the related epsilonproteobacterium helicobacter pylori highlighted a lack of conservation of operon organisation, position of intragenic and antisense promoters or leaderless mrnas. predictive comparisons using other epsilonproteobacterial genomes suggests that this lack of conservation of transcriptional features is common to all epsilonproteobacterial genomes, and is associated with the absence of genome synteny in this subdivision of the proteobacteria.  both the genomes and transcriptomes of epsilonproteobacteria are highly variable, both at the genome level by combining and division of multicistronic operons, but also on the gene level by generation or deletion of promoter sequences and 5′ untranslated regions. regulatory features may have evolved after these species split from a common ancestor, with transcriptome rewiring compensating for changes introduced by genomic reshuffling and horizontal gene transfer. 