BACKGROUND
corynebacterium glutamicum is a non-pathogenic and non-sporulating gram-positive soil bacterium which belongs to the order actinomycetales.
this microorganism has a long history of applications in the production of various amino acids and other industrially relevant compounds  <cit> .
furthermore, it serves as a model organism for close relatives with medical significance such as c. diphtheriae or mycobacterium tuberculosis.
the genome sequence established a decade ago  <cit>  comprises a circular chromosome with a length of almost  <dig>  mb and harbors more than  <dig> annotated protein-coding sequences .
based on the complete genome sequence, transcriptional regulation in c. glutamicum has been studied extensively  <cit>  and revealed a complex regulatory network including  <dig> transcriptional regulator proteins with so far  <dig> regulatory interactions  <cit> .
however, only very little is known about small rnas  and their potential regulatory actions in this organism.
information on rna species beside ribosomal rna  or transfer rna  is absent from the current genome annotation.
it can only be deduced from the genome sequence that c. glutamicum lacks a sequence homologue of the rna chaperone hfq, similar to other actinomycetales <cit> .
so far, the only experimentally defined srna in c. glutamicum  was detected upstream of the gntr-regulator cg <dig> and is located in antisense direction  <cit> .
recently, regulatory rnas have been detected in all three domains of life with unexpectedly large numbers, in the range of hundreds per bacterial and thousands per eukaryotic genome.
in most cases, these transcripts do not encode proteins and so the term non-coding rna  is often applied synonymously.
all hitherto identified rna families are collected in the rna-families  online database  <cit> , frnadb  <cit> , and srnadb  <cit> .
beyond trans-encoded srna genes, these databases also include rna motifs from mrna leader transcripts of protein-coding genes, some of which regulate translation initiation or cause transcriptional attenuation.
elements such as rna thermometers are structures sensitive to temperature shifts and control the accessibility of the shine-dalgarno sequence of the mrna leader .
the classes of attenuation mechanisms are diverse and include amongst others small molecule-mediated riboswitches  as well as classical attenuators regulated by translation of a small leader peptide.
the length of bacterial srnas is generally between  <dig> and  <dig> nt  <cit>  and can be up to  <dig> nt  <cit> .
in addition to rnas with housekeeping function, in-depth analyses of several srnas led to the discovery of various novel regulatory functions.
these functions modulate a wide range of responses to stresses and other environmental stimuli  including rna processing and rna degradation as well as translation control.
different mechanisms of action have been described, the majority representing interactions through basepairing between sense rna and regulating antisense rna.
direct transcriptional regulation through srnas seems to occur rarely, and was first discovered for 6s rna of e. coli <cit> .
the much more frequent post-transcriptional regulation by trans-encoded srnas works through imperfect basepairing with target mrnas .
these srnas show stable secondary structures and their genes are generally located in the “intergenic regions” between protein-coding sequences.
in contrast, cis-antisense srnas  genes are located directly in the antisense direction with respect to their target genes and thus show full complementarity .
to date, different strategies have been applied for the systematic genome-wide search for srnas.
in the enterobacterium e. coli, a number of srnas have been predicted by computational methods .
such in silico analyses are usually based on common features of srnas such as thermodynamic stability, structure conservation, or sequence similarity between species  <cit> , as well as the existence of rho-independent terminators at their 3′-ends  <cit> .
the rfam database provides srna predictions for organisms with known genome sequences calculated from sequence covariance models.
in c. glutamicum, four srnas are predicted by rfam, including 6c rna and the housekeeping rnas tmrna, rnase p, and srp/ <dig> s rna.
experimental strategies for the discovery of srnas in bacteria started with systematic genome-wide screens by shotgun cloning and sequencing of cdna  <cit>  or by using tiling microarrays  and detected large numbers of srnas in all tested organisms.
undoubtedly, new high-throughput sequencing techniques enable the most detailed view on a cellular transcriptome.
thus, rna-sequencing has emerged as a powerful tool for the detection of bacterial srnas .
the creation of rna-sequencing libraries can vary between different platforms in high throughput sequencing  <cit>  but there are similarities between the procedures.
an important step to increase the coverage of mrna or srna in transcriptome sequencing data is the depletion of highly abundant ribosomal rnas.
another is to ensure that the strand-information of the rna is kept in the cdna sequence.
this can be done by using adapters of known sequence to be ligated to the rna before cdna synthesis.
in addition, various specific enzymatic treatments of the rna samples can be used for mapping of transcriptional starts  <cit>  or for detection of processing sites  <cit> .
here, we present the first deep sequencing study of srnas in c. glutamicum.
sequencing libraries were created by the “differential” rna-sequencing  approach  <cit>  with rna samples from exponential growth phase and stress conditions such as heat and cold shock, salt stress, h2o <dig> and diamide stress to gain a broad spectrum of transcription of potential srna genes in response to these conditions.
supported by promoter searches, rna-seq data were analyzed and led to the detection of novel srna genes in c. glutamicum atcc  <dig>  in addition, srna genes were classified and compared with bioinformatic srna predictions based on secondary structure stability and sequence conservation.
RESULTS
detection of potential srna genes in c. glutamicum atcc  <dig> by transcriptome sequencing and read-mapping
transcription of srnas in bacteria is highly variable under different environmental conditions  <cit> .
hence, for a comprehensive survey of srnas in c. glutamicum, we isolated total rna from c. glutamicum cells grown to exponential phase and from cells after a variety of stress treatments and pooled the total rna samples .
the stress treatments were heat shock , cold shock , oxidative stress , diamide stress , and salt stress .
to enrich small rna for a transcriptome sequencing , the pool of total rnas was size-selected for transcripts smaller than  <dig> nucleotides  by precipitation and further depleted of ribosomal rnas  using a hybridization procedure that selectively binds rrna species with biotinylated probes .
the probe:rrna hybrids were then captured by magnetic beads and removed using a magnet.
after this step, the srna fraction was split into two samples as in the differential rna sequencing  approach  <cit> .
hereby, one sample was treated with a 5′-monophosphate-specific exonuclease which degrades specifically transcripts that are processed or undergoing degradation, thus leaving primary transcripts with native 5′-triphosphate ends.
the second sample was left untreated as a representation of the whole small transcriptome of the cell.
the small rna samples were then separately committed to strand-specific sequencing library preparation using the standard illumina truseq small rna kit.
both cdna libraries were sequenced on an illumina ga iix sequencer, obtaining  <dig> bases long single reads from their 5′-ends.
reads were mapped to the chromosome sequence of the c. glutamicum atcc  <dig> wild-type strain  <cit>  using the saruman algorithm implemented in cuda programming language and run on computer graphics cards  <cit>  allowing for up two mismatches per read.
in total,  <dig> , <dig> reads were uniquely mapped for the primary transcripts sample  and  <dig> , <dig> reads were uniquely mapped in case of the total small rna sample .
the rrna was found to be more efficiently depleted in the enzyme-treated library  <dig>  yielding a proportion of only 3% of total reads mapping to ribosomal rna genes.
it also became apparent that the enzyme treatment had depleted residual mrna as seen by the lower fraction of reads attributable to the sense direction of cds.
the remaining set of reads were mapped either cis-antisense to cds or to regions with no annotated genome features .
the cis-antisense reads made up 3% and less in both libraries.
interestingly, the majority of read mappings belong to regions with no annotated features, particularly in the library enriched for primary transcripts , indicating a high number of potential srnas in c. glutamicum.figure  <dig>
workflow for a small rna-seq approach in
c. glutamicum
atcc  <dig>  before cdna library preparation, the small rna fraction was split into two samples for creation of two different sequencing libraries.
the first sample was treated with a 5′-monophosphate-specific exonuclease to degrade transcripts that are processed or undergoing degradation.
the second sample was left untreated and represents the whole of srna transcripts within the cell.
after cdna library preparation, both samples were then separately committed to strand-specific high-throughput sequencing.
distribution of mapped reads to annotated features in the
c. glutamicum
<dig> genome in two different sequencing libraries
asrna primary transcripts were obtained using a 5′-monophosphate-specific exonuclease which degrades specifically transcripts that are either processed or undergoing degradation.
characterization of potential srna genes with the help of bioinformatic promoter analysis
after filtering of mappings to trna and rrna genes and to putative mrnas , the potential srna fraction from the library  <dig>  was utilized for the definition of srna transcript starts .
a number of reads that start at a distinct genomic position normalized to the previous position was defined as read stack and a transcriptional start site  was assumed at the 5′-position at each of these stacks .
for the experiment performed here, the number of read starts used in stringent filtering was determined to be  <dig>  this analysis yielded a number of  <dig> stacks .
as a further filtering step, the 5′-upstream sequences of the assumed tss were analyzed for promoters.
using the tool improbizer  <cit> , we searched for matches to the consensus promoter sequences recognized by the primary housekeeping sigma factor siga  <cit>  or the stress-related ecf-family sigma factor sigh  <cit> , which is known to play a major role under oxidative stress  <cit>  and heat stress conditions  <cit> .
thus,  <dig> putative tss were found to exhibit an upstream siga-recognized promoter sequence  .
the search for sigh promoter sequences was successful in case of  <dig> tss .
for  <dig> transcripts, promoters of both types were detected, indicating srna variants with different 5′-ends.figure  <dig>
definition of start and stop positions of potential srna genes.
workflow for characterization of srna genes with the help of bioinformatic promoter analysis at transcriptional start sites  in library  <dig> .
all tss without promoters were removed and the 3′-ends of potential srna genes were determined with sequence data from library  <dig>   library  <dig>  was used for definition of transcriptional start sites at 5′-ends of read stacks.
the 5′-end of a read stack is defined as a number of read starts that exceed the number of read starts at the previous position by a factor of  <dig>   weblogo  <cit>  presentations of the consensus sequences of - <dig> and - <dig> core regions.
in detail,  <dig> siga and  <dig> sigh promoter sites were detected by the improbizer tool.
the percentage of occurrence of a nucleotide at a particular position is represented by the size of the nucleotide symbol .
to determine the 3′-ends of srnas, we mapped reads from the two libraries and followed each of the initial stacks up to a point where the number of read starts fell below the chosen cut-off of  <dig> reads and defined 3′-ends from these data .
in addition, we searched for rho-independent transcription terminators since these have been reported for numerous of bacterial srnas  <cit> .
this search was performed with the tool transtermhp  <cit>  targeting thymine-rich stretches of dna following a hairpin loop within  <dig> nt around the assumed 3′ends.
thereby, rho-independent terminators were found at  <dig> of these srnas .
in  <dig> cases, the predicted tss were within close distance to each other, indicating multiple promoters.
proposed multiple starts located within 100 bp at the 5′-end and with the same 3′-end were merged to a single region and annotated as such.
classification of potential srna genes by their positions relative to annotated protein-coding sequences
in order to identify putative untranslated regions  of mrnas that are included in the set of potential srna genes, these were then grouped according to their position and direction relative to an adjacent cds .
in total,  <dig> transcripts that had a downstream cds in less than  <dig> nt distance from their 3′-ends were designated as “mrna leader”, .
by comparison with the rna-families database  we could additionally assign the well conserved mraw motif , the cspa motif , and nine predicted riboswitches which are part of 5′-utrs longer than  <dig> nt .
this way we validated the rfam-predicted thiamine pyrophosphate -dependent riboswitches upstream of genes from the thiamine biosynthesis pathway, thic , thim , and thie .
two tpp-riboswitches were found upstream of cg <dig>  and cg <dig> .
one flavin mononucleotide -dependent riboswitch was detected at the putative nicotinamide mononucleotide uptake permease  and one s-adenosyl methionine -dependent type iv riboswitch upstream of cg <dig> .
together with the two riboswitch related rna-motifs   <cit>  and seven additional transcripts putatively encoding small proteins, we ended up with  <dig> regions in the class “mrna leader”.figure  <dig>
classification of potential srna genes by their positions relative to annotated protein-coding sequences.
transcripts with a downstream cds in less than  <dig> nt distance from their 3′-ends were designated as “mrna leader”.
the class also includes riboswitches and orf containing transcripts already predicted and stored in the rfam database  <cit> .
the class “antisense transcripts” comprises three sub-types  cis-antisense rnas  that start within a cds,  transcripts antisense to a 5′-utr, starting within  <dig> nt from the 5′-end of a cds  and  transcripts antisense to a 3′-utr, starting within  <dig> nt from the 3′-end of a cds .
all remaining intergenic transcripts were categorized as trans-encoded srnas.
the class “antisense transcripts” comprises  <dig> regions  and includes three sub-types  cis-antisense rnas  that start in antisense orientation within an opposing cds,  transcripts antisense to a 5′-utr, starting within  <dig> nt from the 5′-end of an opposing cds  and  transcripts antisense to a 3′-utr, starting within  <dig> nt from the 3′-end of an opposing cds .
this analysis defined  <dig> srna regions as asrnas,  <dig> as as5′-utrs, and  <dig> as as3′-utrs.
eight as5′-utrs were also counted as3′-utrs and  <dig> mrna leader were also counted as5′-utrs due to special arrangements of cds.
all remaining  <dig> regions were designated as trans-encoded srnas .
for preparation of an updated c. glutamicum genome annotation, only antisense transcripts and trans-encoded rnas were assigned with locus-tags.
in the new nomenclature the locus tag of each cds will be extended by a trailing zero .
the last digit of the number is used to number novel features in between of old features.
detection of small c. glutamicum genes encoding small proteins
some of the srnas might actually represent mrnas and encode small proteins.
we applied the “orffinder” online tool to extract orfs from sequences in multiple fasta format and subsequently searched for ribosomal binding sites  upstream of the extracted orf sequences with rbsfinder  <cit>  using a window size of 15 bp and the standard rbs settings.
only orfs with a minimum length of  <dig> amino acid residues showing either a rbs  or leaderless transcripts  were taken into account, and in total eight small mrnas were retrieved .
next, we searched for conservation of these peptide sequences in other bacterial genomes using the tblastx algorithm at the ncbi web portal  and found conservation in seven cases .
similar small proteins with e-values less than 10- <dig> were found in other corynebacterium species and also outside corynebacteria.
more widely conserved proteins comprise the well conserved peptide-tag encoded by the tmrna that was identified with  <dig> amino acids length  in c. glutamicum.
beside the peptide encoded by tmrna, only one other peptide, cgb_ <dig> , was detected to be conserved in species beyond corynebacterium.
the smallest of all conserved peptides with  <dig> amino acids length was found to be cgb_ <dig> .
this peptide is already known as valine-containing leader peptide in front of the ilvbnc operon  <cit> .
further putative leader peptides of attenuator structures were detected at cgb_ <dig>  located upstream of trpe of the tryptophan operon, at cgb_ <dig>  in front of the leua gene , encoding isopropylmalate synthase, the first step in leucine biosynthesis, and at arof .
supporting their functional assignment is the occurrence of three consecutive tryptophan residues in the putative leader peptide upstream of the tryptophan operon, four consecutive leucine codons in the presumed leua leader peptide and the amino acids phenylalanine-tyrosine-phenylalanine in the case of the arof leader peptide.
analysis of cis-antisense rna genes, located within protein-coding genes
in our study, more than half of the srna regions  fall into the class “antisense transcripts” .
for the sub-type of asrnas , which are located directly opposite to a cds, the mean length was calculated to be  <dig> nt.
this very small size particularly for asrnas is shown in a box plot diagram .
the asrnas are distributed to  <dig> different cdss with  <dig> cds having more than one antisense transcript.
it is noteworthy that antisense transcription is not only observed at the 5′-ends of the corresponding cds, but often also at the 3′-ends or in the middle of a coding region.
to correlate the functions of encoded proteins with observed asrnas, the respective proteins were classified according to the eggnog functional classification system  <cit> .
thereby,  <dig> of  <dig> asrnas match to cds with eggnog classification .
in case of the remaining  <dig> asrna, either no category or categories with poor characterization were retrieved.
a normal distribution of  <dig> asrnas over all currently annotated cds would result in about 15% of the genes of each eggnog class to contain a asrna.
we considered classes containing a asrna in more than 20% or less than 10% of the members of the class as over- or underrepresented.
the lowest proportions of asrnas were observed for genes within the classes’ cell cycle control/cell division , coenzyme transport and metabolism , transcription , and inorganic transport and metabolism .
on the other hand, we observed a higher proportion of asrnas within genes from the functional class cell envelope biogenesis .
moreover, a higher proportion of genes which are involved in secretion processes  seem to have transcription in antisense direction .
among the genes involved in protein secretion there were both protein secretion mechanisms represented, with secy  encoding a preprotein translocase subunit of the sec secretion system and tatc  encoding a twin-arginine  secretion translocase subunit.
the tatc antisense rna  is apparently transcribed from a siga-dependent promoter, whereat the secy asrna  seems to be transcribed from a sigh-controlled promoter.figure  <dig>
functional classification of
c. glutamicum
genes having
cis
-antisense rnas  according to eggnog.
occurrences and frequencies of asrnas at genes with assigned eggnog  <cit>  classes  are shown.
classes with cis-antisense rna frequencies between 10% and 20% reflect a normal distribution.
red lines represent limits for classes that are underrepresented  or overrepresented .
in this context, we tested also the proportions of asrnas at genes encoding transmembrane helices or signal peptides for secretion.
by bioinformatic search  <dig> of  <dig> asrnas  were detected at the corresponding genes.
statistically, this proportion is not significantly different from a normal distribution since  <dig> genes  encode a signal peptide for secretion or at least one transmembrane helix.
the occurrence of asrnas includes also two of the  <dig> two-component regulatory systems in c. glutamicum, namely the sensory histidine kinase genes cgts <dig>  and cgts <dig>  that sense a specific environmental stimulus at the membrane and the corresponding response regulators genes cgtr <dig>  and cgtr <dig>  that mediate the transcriptional regulation by binding to operators  <cit> .
the cgtsr <dig> genes are involved in phosphate starvation  <cit>  and cgtr <dig> seems to be essential  <cit> .
further asrnas are located opposite to transcriptional regulator genes  such as cysr  and sufr  which are involved in assimilatory sulphate reduction  <cit> and thiol-oxidative stress defense  <cit> , respectively.
beside this, we detected asrnas at the acnr gene   <cit>  and other members of the tetr family .
the following transcriptional regulators were also identified to have antisense transcription, sugr  as regulator of the pep:sugar phosphotransferase system genes  <cit> , ndrr  the regulator of deoxyribonucleotide reduction  <cit> , pcao  the transcriptional activator of the ketoadipate metabolism genes  <cit> , farr  a transcriptional regulator involved in nitrogen metabolism  <cit> , and two members of the hth_3-family .
bioinformatic analysis of sequence and structural conservation of trans-encoded srnas in c. glutamicum atcc 13032
in bacteria srnas often have characteristic structures that are conserved stronger in evolution than their primary sequences.
hence, structure conservation analysis is integrated in a number of srna prediction tools.
in our approach, we used the rnaz tool  <cit>  to detect secondary structure conservation in a multiple genome alignment between the closely related species of c. glutamicum atcc  <dig>  c. efficiens ys- <dig> and c. diphtheriae nctc  <dig>  rnaz predictions made under stringent conditions  overlap with  <dig> trans-encoded srna genes detected with rna-seq .
moreover, the sequencing results were compared with a less stringent set of rnaz predictions  which resulted in  <dig> of  <dig> trans-encoded srnas matching to loci predicted by rnaz  .
hereby, all three housekeeping rnas, tmrna , m <dig> rna , and  <dig> s rna  were predicted at positions very similar to those of the rfam database entries that were calculated by covariance models  <cit> .
this was the case also for 6c rna , which is known to be present in many actinomycetales genera  <cit> .
the 6c rna was named from its two stem-loops, each typically containing six cytosine  residues.
interestingly, in c. glutamicum the 6c rna has two stretches of eight cytosines.
however, the function of these cytosine homopolymers is not known and therefore the relevance of this difference is unclear.table  <dig>
trans
-encoded srna genes with overlapping rnaz- prediction  and their prediction details
cgb_00105
cg0010/cg0012
cgb_00925
cg0092/cg0095
cgb_03505
cg0350/cg0352
a
cgb_03605
cg0360/cg0362
cgb_03995
cg0399/cg0400
cgb_05085
cg0508/cg0510
cgb_05716
cg0571/cg0572
cgb_05756
cg0575/cg0576
cgb_08496
cg0849/cg0850
cgb_08785
cg0878/cg0879
cgb_09095
cg0909/cg0910
cgb_09097
cg0909/cg0910
b
cgb_09185
cg0918/cg0919
cgb_09483
cg0948/cg0949
cgb_13305
cg1330/cg1332
cgb_14495
cg1449/cg1451
cgb_17355
cg1735/cg1736
cgb_17735
cg1773/cg1774
cgb_17805
cg1780/cg1781
cgb_18405
cg1840/cg1841
cgb_18415
cg1841/cg1842
cgb_21516
cg2151/cg2152
cgb_21673
cg2167/cg2168
cgb_22185
cg2218/cg2221
cgb_22215
cg2221/cg2222
cgb_22285
cg2228/cg2229
cgb_22405
cg2240/cg2241
cgb_23783
cg2378/cg2380
cgb_24455
cg2445/cg2446
c
cgb_24535
cg2453/cg2455
cgb_24775
cg2477/cg2478
cgb_25636
cg2563/cg2564
cgb_25955
cg2595/cg2597
cgb_26475
cg2647/cg2648
cgb_28315
cg2831/cg2833
cgb_28685
cg2868/cg2869
cgb_29606
cg2960/cg2962
cgb_30116
cg3011/cg3012
cgb_30685
cg3068/cg3069
cgb_31375
cg3137/cg3138
cgb_31785
cg3178/cg3179
cgb_33045
cg3304/cg3306
cgb_33325
cg3332/cg3334
cgb_34325
cg3432/cg3434
arf01066; 6c.
brf00023; tmrna.
crf00010; rnasep.
experimental validation of three trans-encoded srnas by northern blotting of stress-specific rna samples and detailed structure analysis
for the total set of detected trans-encoded srnas we retrieved a mean length of  <dig> nt .
to validate the sequencing approach, three srnas were selected and subsequently analyzed by northern blotting: the highly conserved 6c rna   and two srnas with high read counts at their tss .
we chose cgb_ <dig>  as an example of a srna with strong secondary structure conservation as shown above  and cgb_ <dig>  as an example lacking secondary structure conservation .
secondary structures of all presented srnas were determined by minimum free energy folding and rna shape analysis  <cit>  which achieved high shape probabilities  in all cases, respectively.
here, the total rnas obtained from different growth conditions were analyzed separately to monitor stress-specific transcription patterns.
the sizes of these srnas determined by northern blot are as follows: ~ <dig> nt for 6c rna, ~ <dig> nt for cgb_ <dig>  ~ <dig> nt and ~ <dig> nt for cgb_ <dig>  in each case, the northern blot signal of the longest srna correlated well with the length determined by sequencing.
compared with the rfam prediction, the 6c rna had a  <dig> nt 5′-extension .
this extended 5′-region seems to be conserved in the corynebacterium species, since a longer 5′-region for 6c rna was also predicted by our rnaz approach .figure  <dig>
secondary structure prediction and experimental validation by northern hybridizations of three selected srnas.
for each of the three srna genes, the left column shows the sequence coverage profile derived from library  <dig> .
the y- and x-axis represent coverage and sequence localization.
grey arrows represent flanking genes, white arrows represent the srnas and brown stem-loop structures represent rho-independent terminators.
the middle column displays northern blot results of all tested conditions at time point of harvesting of c. glutamicum cells, respectively.
exp = exponential phase , unstressed.
further conditions were heat, cold, diamide, nacl, and h2o <dig> stresses, all applied for 15 minutes at an od <dig>  <dig>  the right column presents the secondary structure with lowest minimum free energy .
structure, mfe and shape probabilities determined with rnashapes  <cit> .
sequence code: blue, a; green, c; red, g; yellow, u. validated intergenic srnas by northern hybridizations are
cgb_ <dig> ,
cgb_ <dig> and
cgb_ <dig>
the 6c rna showed no change in transcript abundance in the northern blots .
probably due to a regulatory mechanism, cgb_ <dig> appeared to be absent under heat stress conditions .
the srna cgb_ <dig> also does not appear to be differentially transcribed at a tested condition.
interestingly, the northern blot revealed a shorter second band of at ~ <dig> nt, indicating a second rna species possibly generated by rna processing, since no additional promoter was observed in this region.
discussion
rna sequencing is a novel approach to characterize transcriptomes of bacteria comprehensively.
this technique is especially useful for detection of novel srnas.
here, we present the first small rna-seq approach for c. glutamicum, a member of the genus corynebacterium, which represents also a model organism for the closely related genera within the corynebacterineae, e.g.
mycobacterium, nocardia etc.
in comparison to the knowledge of regulatory srnas in the class of gamma-proteobacteria, especially in e. coli and salmonella species, information about srnas is marginal in actinobacteria.
until now, deep sequencing of transcriptomes in this class has only been reported for mycobacteria <cit>  and streptomyces species  <cit> .
since srnas might be differentially transcribed under stress, a mixed sample of various conditions should ensure the transcription of as many srnas as possible.
the isolation of small rnas, however, yielded not only “true” srnas but also a lot of rnas that are processed or in the process of degradation.
therefore, a number of filtering steps were performed on the cdna reads achieved.
besides using a chosen cut-off for the increase in the number of read starts relative to the preceding position for calling a transcript start, the 5′-ends were validated by promoter searches, and from these validated 5′-ends, 3′-ends of transcripts were determined by another chosen cut-off of ten reads.
it has to be stated that these cut-offs were arbitrary and adjusted to the size of the data set.
as the next step, transcripts were classified by their relative positions to annotated protein-coding sequences .
the length of 5′-utrs of coding sequences is variable, zero for leaderless transcripts and especially long for genes regulated by cis-regulative elements such as riboswitches.
the difficulty of utr length definition was also reported in other studies  <cit> .
interestingly, we observed a short transcript length particularly for asrnas.
as expected, the class of leader mrnas represents the longest transcripts, resulting from transcription into the adjacent cds.
this difference was not obtained for the different types of srnas in sinorhizobium meliloti <cit> .
generally, a shorter average size of srnas compared to srnas from other bacteria was also reported from streptomyces coelicolor <cit> .
cis-antisense srnas are abundant in c. glutamicum and located in ~15% of all annotated protein-coding genes.
high-resolution tiling arrays and rna sequencing led to the discovery of extensive antisense transcription in several other bacteria .
in these previous studies, the reported percentage of genes within a genome which are targeted by asrna varies up and is >46% in helicobacter <cit> .
the first asrna in c. glutamicum was detected upstream of cg <dig> <cit> , thereby overlapping the mrna of the transcriptional regulator of the gntr family in antisense direction.
in our study, we detected four more asrnas which are located opposite to already known transcriptional regulatory genes and further five asrnas at putative regulatory genes.
so far, it has been investigated that asrnas can modulate the level of transcriptional regulators, metabolic, toxic and virulence proteins or repress transposases .
for c. glutamicum, our analysis of antisense transcripts based on the eggnog classification system revealed that asrnas seem to occur frequently at genes encoding proteins with functions in cell envelope biogenesis and protein secretion processes.
however, there are hundreds of short transcripts in antisense direction of coding sequences in c. glutamicum the functions of which remain to be elucidated.
in general, antisense transcripts often influence rna stability of their target mrna either by promoting or blocking ribonucleases .
furthermore, asrnas can induce a structural change in their target mrna that effects transcription attenuation  <cit> .
other studies showed that asrna can also hinder rna polymerase extending the transcript encoded in the opposite strand by transcription interference  or can affect translation of the target gene by regulation of ribosome binding  <cit> .
further classification, especially of trans-encoded srnas, can be done by sequence and structural analysis.
the comparison of candidates predicted by the rnaz tool  <cit>  and by the rfam database  <cit>  with sequencing results, allowed us to detect srnas that are conserved in all bacteria , in actinomycetales  or within the closely related species c. efficiens and c. diphtheriae.
however, more than the half of srnas seems to be specific for c. glutamicum.
at this point we want to note that our study did not detect two widely conserved elements known to be involved in bacterial srna: 6s rna  <cit>  and short palindromic repeat  loci .
interestingly, at least one crispr locus has been identified in the genomes of almost all other corynebacterium species   <cit> .
many bioinformatic prediction tools were developed for srna research during the last decade.
the comparison of the actual in vivo expression of srnas with bioinformatic prediction results often revealed only little correspondence  <cit> .
apart from the rnaz program  <cit>  which was utilized in this study, the srnapredict algorithms  <cit>  are prominent bioinformatic tools which have been used in various bacterial srna studies.
as implemented in srnapredict, the analysis on rho-independent terminators is often integrated in tools for srna detection.
however, more than 75% of the trans-encoded srnas detected by our sequencing approach are not followed by a rho-independent terminator and especially the number of asrnas with rho-independent terminators is marginal.
a similar observation was obtained within in a search for srnas in vibrio splendidus <cit> .
the correct termination of one srna  at a site without an obvious terminator structure was proven by northern blot analysis.
rna-seq analyses deliver an unmatched single nucleotide resolution.
however, confirmatory methods are required, such as northern blotting and are used in the present study, to look at stress-specific transcription.
an example is presented with cgb_00105: under heat shock we detected no transcription of this srna whereas cold shock and chemically induced stresses had no influence on the amount of transcript.
heat shock condition was also observed to trigger a different transcription start site for arna cis-antisense rna  <cit> .
in the case of cgb_ <dig>  two transcripts of different length are detected in each case.
here, rather srna maturation or degradation by endo- or exoribonucleases is likely.
for 6c rna no change by one of the chosen stress treatments was observed.
at this point, there is no hint for the function of 6c rna in c. glutamicum.
currently, the 6c rna was reported to be involved in the glxr regulatory network in c. glutamicum <cit> .
glxr is known as a global regulator of carbon source metabolism and energy conversion.
in streptomyces coelicolor, 6c rna showed an increased transcription during sporulation  <cit> .
in bacteria there is an additional group of transcripts, comprising rnas that act as both, regulatory rnas and mrnas.
rna with dual properties is exemplified by tmrna, which combines the features of a trna and an mrna.
this housekeeping rna recycles stalled ribosomes by adding a proteolysis-inducing tag to unfinished polypeptides  <cit> .
our results show that the tmrna peptide-tag in c. glutamicum corresponds well to known sequences of a wide phylogenetic spectrum  <cit> .
short peptides encoded within 5′-utrs of mrna sequences are known as characteristic feature in a mechanism called transcriptional attenuation.
in our study, we detected attenuator transcripts at different genes and operons involved in amino acid synthesis, each encoding a suitable leader peptide.
such rnas are also included as cis-regulatory motives in the rfam database.
transcriptional attenuation was first described for the tryptophan  operon in e. coli <cit>  where terminator formation is associated to a leader sequence and is influenced by the availability of trnatrp .
accordingly, it has been observed for the ilvbnc operon in c. glutamicum <cit>  and different amino acid operons in other microorganisms , encoding isopropylmalate synthase, the first step in leucine biosynthesis, and at arof , encoding one of the two dahp synthases  <cit>  in c. glutamicum, responsible for the first step of shikimate pathway in the biosynthesis of aromatic amino acids.
since c. glutamicum is a well-known industrial producer of amino acids, these findings might become relevant for future engineering of amino acid producer strains.
CONCLUSIONS
our present study represents the first comprehensive screening for small rnas in corynebacteriaceae, a family that comprises important bacteria of industrial and medical relevance.
high-throughput sequencing techniques are often applied for the search and investigation of srnas in bacterial genomes.
similar to srna studies in other bacteria, we detected hundreds of srna genes in c. glutamicum atcc  <dig>  in c. glutamicum, more than half of all small rnas genes was classified as antisense transcripts.
cis-antisense srna genes were detected at cds with various functions.
however, cds specifying proteins from the functional classes 'cell envelope biogenesis’ and 'secretion processes’ appear to be overrepresented.
trans-encoded srna genes were found distributed over the entire genome and showed secondary structure conservation among corynebacteria in about 30%.
the 6c rna, already known from other actinomycetales genera showed strong transcription at unstressed exponential growth and all tested stress conditions.
the 6s rna, highly conserved in bacteria, was not found in c. glutamicum.
additionally, we detected riboswitches, transcriptional attenuators and other cis-regulatory motives, demonstrating the potential of our study for unraveling novel regulatory processes by small rnas in c. glutamicum.

 corynebacterium glutamicum is a non-pathogenic and non-sporulating gram-positive soil bacterium which belongs to the order actinomycetales. this microorganism has a long history of applications in the production of various amino acids and other industrially relevant compounds. furthermore, it serves as a model organism for close relatives with medical significance such as c. diphtheriae or mycobacterium tuberculosis. the genome sequence established a decade ago comprises a circular chromosome with a length of almost  mb and harbors more than annotated protein-coding sequences. based on the complete genome sequence, transcriptional regulation in c. glutamicum has been studied extensively and revealed a complex regulatory network including transcriptional regulator proteins with so far regulatory interactions. however, only very little is known about small rnas and their potential regulatory actions in this organism. information on rna species beside ribosomal rna or transfer rna is absent from the current genome annotation. it can only be deduced from the genome sequence that c. glutamicum lacks a sequence homologue of the rna chaperone hfq, similar to other actinomycetales. so far, the only experimentally defined srna in c. glutamicum was detected upstream of the gntr-regulator cg and is located in antisense direction. recently, regulatory rnas have been detected in all three domains of life with unexpectedly large numbers, in the range of hundreds per bacterial and thousands per eukaryotic genome. in most cases, these transcripts do not encode proteins and so the term non-coding rna is often applied synonymously. all hitherto identified rna families are collected in the rna-families online database, frnadb, and srnadb. beyond trans-encoded srna genes, these databases also include rna motifs from mrna leader transcripts of protein-coding genes, some of which regulate translation initiation or cause transcriptional attenuation. elements such as rna thermometers are structures sensitive to temperature shifts and control the accessibility of the shine-dalgarno sequence of the mrna leader. the classes of attenuation mechanisms are diverse and include amongst others small molecule-mediated riboswitches as well as classical attenuators regulated by translation of a small leader peptide. the length of bacterial srnas is generally between and nt and can be up to nt. in addition to rnas with housekeeping function, in-depth analyses of several srnas led to the discovery of various novel regulatory functions. these functions modulate a wide range of responses to stresses and other environmental stimuli including rna processing and rna degradation as well as translation control. different mechanisms of action have been described, the majority representing interactions through basepairing between sense rna and regulating antisense rna. direct transcriptional regulation through srnas seems to occur rarely, and was first discovered for 6s rna of e. coli. the much more frequent post-transcriptional regulation by trans-encoded srnas works through imperfect basepairing with target mrnas. these srnas show stable secondary structures and their genes are generally located in the “intergenic regions” between protein-coding sequences. in contrast, cis-antisense srnas genes are located directly in the antisense direction with respect to their target genes and thus show full complementarity. to date, different strategies have been applied for the systematic genome-wide search for srnas. in the enterobacterium e. coli, a number of srnas have been predicted by computational methods. such in silico analyses are usually based on common features of srnas such as thermodynamic stability, structure conservation, or sequence similarity between species, as well as the existence of rho-independent terminators at their 3′-ends. the rfam database provides srna predictions for organisms with known genome sequences calculated from sequence covariance models. in c. glutamicum, four srnas are predicted by rfam, including 6c rna and the housekeeping rnas tmrna, rnase p, and srp/ s rna. experimental strategies for the discovery of srnas in bacteria started with systematic genome-wide screens by shotgun cloning and sequencing of cdna or by using tiling microarrays and detected large numbers of srnas in all tested organisms. undoubtedly, new high-throughput sequencing techniques enable the most detailed view on a cellular transcriptome. thus, rna-sequencing has emerged as a powerful tool for the detection of bacterial srnas. the creation of rna-sequencing libraries can vary between different platforms in high throughput sequencing but there are similarities between the procedures. an important step to increase the coverage of mrna or srna in transcriptome sequencing data is the depletion of highly abundant ribosomal rnas. another is to ensure that the strand-information of the rna is kept in the cdna sequence. this can be done by using adapters of known sequence to be ligated to the rna before cdna synthesis. in addition, various specific enzymatic treatments of the rna samples can be used for mapping of transcriptional starts or for detection of processing sites. here, we present the first deep sequencing study of srnas in c. glutamicum. sequencing libraries were created by the “differential” rna-sequencing approach with rna samples from exponential growth phase and stress conditions such as heat and cold shock, salt stress, h2o and diamide stress to gain a broad spectrum of transcription of potential srna genes in response to these conditions. supported by promoter searches, rna-seq data were analyzed and led to the detection of novel srna genes in c. glutamicum atcc in addition, srna genes were classified and compared with bioinformatic srna predictions based on secondary structure stability and sequence conservation.  detection of potential srna genes in c. glutamicum atcc by transcriptome sequencing and read-mapping transcription of srnas in bacteria is highly variable under different environmental conditions. hence, for a comprehensive survey of srnas in c. glutamicum, we isolated total rna from c. glutamicum cells grown to exponential phase and from cells after a variety of stress treatments and pooled the total rna samples. the stress treatments were heat shock, cold shock, oxidative stress, diamide stress, and salt stress. to enrich small rna for a transcriptome sequencing, the pool of total rnas was size-selected for transcripts smaller than nucleotides by precipitation and further depleted of ribosomal rnas using a hybridization procedure that selectively binds rrna species with biotinylated probes. the probe:rrna hybrids were then captured by magnetic beads and removed using a magnet. after this step, the srna fraction was split into two samples as in the differential rna sequencing approach. hereby, one sample was treated with a 5′-monophosphate-specific exonuclease which degrades specifically transcripts that are processed or undergoing degradation, thus leaving primary transcripts with native 5′-triphosphate ends. the second sample was left untreated as a representation of the whole small transcriptome of the cell. the small rna samples were then separately committed to strand-specific sequencing library preparation using the standard illumina truseq small rna kit. both cdna libraries were sequenced on an illumina ga iix sequencer, obtaining bases long single reads from their 5′-ends. reads were mapped to the chromosome sequence of the c. glutamicum atcc wild-type strain using the saruman algorithm implemented in cuda programming language and run on computer graphics cards allowing for up two mismatches per read. in total,, reads were uniquely mapped for the primary transcripts sample and, reads were uniquely mapped in case of the total small rna sample. the rrna was found to be more efficiently depleted in the enzyme-treated library yielding a proportion of only 3% of total reads mapping to ribosomal rna genes. it also became apparent that the enzyme treatment had depleted residual mrna as seen by the lower fraction of reads attributable to the sense direction of cds. the remaining set of reads were mapped either cis-antisense to cds or to regions with no annotated genome features. the cis-antisense reads made up 3% and less in both libraries. interestingly, the majority of read mappings belong to regions with no annotated features, particularly in the library enriched for primary transcripts, indicating a high number of potential srnas in c. glutamicum.figure workflow for a small rna-seq approach in c. glutamicum atcc before cdna library preparation, the small rna fraction was split into two samples for creation of two different sequencing libraries. the first sample was treated with a 5′-monophosphate-specific exonuclease to degrade transcripts that are processed or undergoing degradation. the second sample was left untreated and represents the whole of srna transcripts within the cell. after cdna library preparation, both samples were then separately committed to strand-specific high-throughput sequencing. distribution of mapped reads to annotated features in the c. glutamicum genome in two different sequencing libraries asrna primary transcripts were obtained using a 5′-monophosphate-specific exonuclease which degrades specifically transcripts that are either processed or undergoing degradation. characterization of potential srna genes with the help of bioinformatic promoter analysis after filtering of mappings to trna and rrna genes and to putative mrnas, the potential srna fraction from the library was utilized for the definition of srna transcript starts. a number of reads that start at a distinct genomic position normalized to the previous position was defined as read stack and a transcriptional start site was assumed at the 5′-position at each of these stacks. for the experiment performed here, the number of read starts used in stringent filtering was determined to be this analysis yielded a number of stacks. as a further filtering step, the 5′-upstream sequences of the assumed tss were analyzed for promoters. using the tool improbizer, we searched for matches to the consensus promoter sequences recognized by the primary housekeeping sigma factor siga or the stress-related ecf-family sigma factor sigh, which is known to play a major role under oxidative stress and heat stress conditions. thus, putative tss were found to exhibit an upstream siga-recognized promoter sequence. the search for sigh promoter sequences was successful in case of tss. for transcripts, promoters of both types were detected, indicating srna variants with different 5′-ends.figure definition of start and stop positions of potential srna genes. workflow for characterization of srna genes with the help of bioinformatic promoter analysis at transcriptional start sites in library. all tss without promoters were removed and the 3′-ends of potential srna genes were determined with sequence data from library library was used for definition of transcriptional start sites at 5′-ends of read stacks. the 5′-end of a read stack is defined as a number of read starts that exceed the number of read starts at the previous position by a factor of weblogo presentations of the consensus sequences of - and - core regions. in detail, siga and sigh promoter sites were detected by the improbizer tool. the percentage of occurrence of a nucleotide at a particular position is represented by the size of the nucleotide symbol. to determine the 3′-ends of srnas, we mapped reads from the two libraries and followed each of the initial stacks up to a point where the number of read starts fell below the chosen cut-off of reads and defined 3′-ends from these data. in addition, we searched for rho-independent transcription terminators since these have been reported for numerous of bacterial srnas. this search was performed with the tool transtermhp targeting thymine-rich stretches of dna following a hairpin loop within nt around the assumed 3′ends. thereby, rho-independent terminators were found at of these srnas. in cases, the predicted tss were within close distance to each other, indicating multiple promoters. proposed multiple starts located within 100 bp at the 5′-end and with the same 3′-end were merged to a single region and annotated as such. classification of potential srna genes by their positions relative to annotated protein-coding sequences in order to identify putative untranslated regions of mrnas that are included in the set of potential srna genes, these were then grouped according to their position and direction relative to an adjacent cds. in total, transcripts that had a downstream cds in less than nt distance from their 3′-ends were designated as “mrna leader”,. by comparison with the rna-families database we could additionally assign the well conserved mraw motif, the cspa motif, and nine predicted riboswitches which are part of 5′-utrs longer than nt. this way we validated the rfam-predicted thiamine pyrophosphate -dependent riboswitches upstream of genes from the thiamine biosynthesis pathway, thic, thim, and thie. two tpp-riboswitches were found upstream of cg and cg. one flavin mononucleotide -dependent riboswitch was detected at the putative nicotinamide mononucleotide uptake permease and one s-adenosyl methionine -dependent type iv riboswitch upstream of cg. together with the two riboswitch related rna-motifs and seven additional transcripts putatively encoding small proteins, we ended up with regions in the class “mrna leader”.figure classification of potential srna genes by their positions relative to annotated protein-coding sequences. transcripts with a downstream cds in less than nt distance from their 3′-ends were designated as “mrna leader”. the class also includes riboswitches and orf containing transcripts already predicted and stored in the rfam database. the class “antisense transcripts” comprises three sub-types cis-antisense rnas that start within a cds, transcripts antisense to a 5′-utr, starting within nt from the 5′-end of a cds and transcripts antisense to a 3′-utr, starting within nt from the 3′-end of a cds. all remaining intergenic transcripts were categorized as trans-encoded srnas. the class “antisense transcripts” comprises regions and includes three sub-types cis-antisense rnas that start in antisense orientation within an opposing cds, transcripts antisense to a 5′-utr, starting within nt from the 5′-end of an opposing cds and transcripts antisense to a 3′-utr, starting within nt from the 3′-end of an opposing cds. this analysis defined srna regions as asrnas, as as5′-utrs, and as as3′-utrs. eight as5′-utrs were also counted as3′-utrs and mrna leader were also counted as5′-utrs due to special arrangements of cds. all remaining regions were designated as trans-encoded srnas. for preparation of an updated c. glutamicum genome annotation, only antisense transcripts and trans-encoded rnas were assigned with locus-tags. in the new nomenclature the locus tag of each cds will be extended by a trailing zero. the last digit of the number is used to number novel features in between of old features. detection of small c. glutamicum genes encoding small proteins some of the srnas might actually represent mrnas and encode small proteins. we applied the “orffinder” online tool to extract orfs from sequences in multiple fasta format and subsequently searched for ribosomal binding sites upstream of the extracted orf sequences with rbsfinder using a window size of 15 bp and the standard rbs settings. only orfs with a minimum length of amino acid residues showing either a rbs or leaderless transcripts were taken into account, and in total eight small mrnas were retrieved. next, we searched for conservation of these peptide sequences in other bacterial genomes using the tblastx algorithm at the ncbi web portal and found conservation in seven cases. similar small proteins with e-values less than 10- were found in other corynebacterium species and also outside corynebacteria. more widely conserved proteins comprise the well conserved peptide-tag encoded by the tmrna that was identified with amino acids length in c. glutamicum. beside the peptide encoded by tmrna, only one other peptide, cgb_, was detected to be conserved in species beyond corynebacterium. the smallest of all conserved peptides with amino acids length was found to be cgb_. this peptide is already known as valine-containing leader peptide in front of the ilvbnc operon. further putative leader peptides of attenuator structures were detected at cgb_ located upstream of trpe of the tryptophan operon, at cgb_ in front of the leua gene, encoding isopropylmalate synthase, the first step in leucine biosynthesis, and at arof. supporting their functional assignment is the occurrence of three consecutive tryptophan residues in the putative leader peptide upstream of the tryptophan operon, four consecutive leucine codons in the presumed leua leader peptide and the amino acids phenylalanine-tyrosine-phenylalanine in the case of the arof leader peptide. analysis of cis-antisense rna genes, located within protein-coding genes in our study, more than half of the srna regions fall into the class “antisense transcripts”. for the sub-type of asrnas, which are located directly opposite to a cds, the mean length was calculated to be nt. this very small size particularly for asrnas is shown in a box plot diagram. the asrnas are distributed to different cdss with cds having more than one antisense transcript. it is noteworthy that antisense transcription is not only observed at the 5′-ends of the corresponding cds, but often also at the 3′-ends or in the middle of a coding region. to correlate the functions of encoded proteins with observed asrnas, the respective proteins were classified according to the eggnog functional classification system. thereby, of asrnas match to cds with eggnog classification. in case of the remaining asrna, either no category or categories with poor characterization were retrieved. a normal distribution of asrnas over all currently annotated cds would result in about 15% of the genes of each eggnog class to contain a asrna. we considered classes containing a asrna in more than 20% or less than 10% of the members of the class as over- or underrepresented. the lowest proportions of asrnas were observed for genes within the classes’ cell cycle control/cell division, coenzyme transport and metabolism, transcription, and inorganic transport and metabolism. on the other hand, we observed a higher proportion of asrnas within genes from the functional class cell envelope biogenesis. moreover, a higher proportion of genes which are involved in secretion processes seem to have transcription in antisense direction. among the genes involved in protein secretion there were both protein secretion mechanisms represented, with secy encoding a preprotein translocase subunit of the sec secretion system and tatc encoding a twin-arginine secretion translocase subunit. the tatc antisense rna is apparently transcribed from a siga-dependent promoter, whereat the secy asrna seems to be transcribed from a sigh-controlled promoter.figure functional classification of c. glutamicum genes having cis -antisense rnas according to eggnog. occurrences and frequencies of asrnas at genes with assigned eggnog classes are shown. classes with cis-antisense rna frequencies between 10% and 20% reflect a normal distribution. red lines represent limits for classes that are underrepresented or overrepresented. in this context, we tested also the proportions of asrnas at genes encoding transmembrane helices or signal peptides for secretion. by bioinformatic search of asrnas were detected at the corresponding genes. statistically, this proportion is not significantly different from a normal distribution since genes encode a signal peptide for secretion or at least one transmembrane helix. the occurrence of asrnas includes also two of the two-component regulatory systems in c. glutamicum, namely the sensory histidine kinase genes cgts and cgts that sense a specific environmental stimulus at the membrane and the corresponding response regulators genes cgtr and cgtr that mediate the transcriptional regulation by binding to operators. the cgtsr genes are involved in phosphate starvation and cgtr seems to be essential. further asrnas are located opposite to transcriptional regulator genes such as cysr and sufr which are involved in assimilatory sulphate reduction and thiol-oxidative stress defense, respectively. beside this, we detected asrnas at the acnr gene and other members of the tetr family. the following transcriptional regulators were also identified to have antisense transcription, sugr as regulator of the pep:sugar phosphotransferase system genes, ndrr the regulator of deoxyribonucleotide reduction, pcao the transcriptional activator of the ketoadipate metabolism genes, farr a transcriptional regulator involved in nitrogen metabolism, and two members of the hth_3-family. bioinformatic analysis of sequence and structural conservation of trans-encoded srnas in c. glutamicum atcc 13032 in bacteria srnas often have characteristic structures that are conserved stronger in evolution than their primary sequences. hence, structure conservation analysis is integrated in a number of srna prediction tools. in our approach, we used the rnaz tool to detect secondary structure conservation in a multiple genome alignment between the closely related species of c. glutamicum atcc c. efficiens ys- and c. diphtheriae nctc rnaz predictions made under stringent conditions overlap with trans-encoded srna genes detected with rna-seq. moreover, the sequencing results were compared with a less stringent set of rnaz predictions which resulted in of trans-encoded srnas matching to loci predicted by rnaz. hereby, all three housekeeping rnas, tmrna, m rna, and s rna were predicted at positions very similar to those of the rfam database entries that were calculated by covariance models. this was the case also for 6c rna, which is known to be present in many actinomycetales genera. the 6c rna was named from its two stem-loops, each typically containing six cytosine residues. interestingly, in c. glutamicum the 6c rna has two stretches of eight cytosines. however, the function of these cytosine homopolymers is not known and therefore the relevance of this difference is unclear.table trans -encoded srna genes with overlapping rnaz- prediction and their prediction details cgb_00105 cg0010/cg0012 cgb_00925 cg0092/cg0095 cgb_03505 cg0350/cg0352 a cgb_03605 cg0360/cg0362 cgb_03995 cg0399/cg0400 cgb_05085 cg0508/cg0510 cgb_05716 cg0571/cg0572 cgb_05756 cg0575/cg0576 cgb_08496 cg0849/cg0850 cgb_08785 cg0878/cg0879 cgb_09095 cg0909/cg0910 cgb_09097 cg0909/cg0910 b cgb_09185 cg0918/cg0919 cgb_09483 cg0948/cg0949 cgb_13305 cg1330/cg1332 cgb_14495 cg1449/cg1451 cgb_17355 cg1735/cg1736 cgb_17735 cg1773/cg1774 cgb_17805 cg1780/cg1781 cgb_18405 cg1840/cg1841 cgb_18415 cg1841/cg1842 cgb_21516 cg2151/cg2152 cgb_21673 cg2167/cg2168 cgb_22185 cg2218/cg2221 cgb_22215 cg2221/cg2222 cgb_22285 cg2228/cg2229 cgb_22405 cg2240/cg2241 cgb_23783 cg2378/cg2380 cgb_24455 cg2445/cg2446 c cgb_24535 cg2453/cg2455 cgb_24775 cg2477/cg2478 cgb_25636 cg2563/cg2564 cgb_25955 cg2595/cg2597 cgb_26475 cg2647/cg2648 cgb_28315 cg2831/cg2833 cgb_28685 cg2868/cg2869 cgb_29606 cg2960/cg2962 cgb_30116 cg3011/cg3012 cgb_30685 cg3068/cg3069 cgb_31375 cg3137/cg3138 cgb_31785 cg3178/cg3179 cgb_33045 cg3304/cg3306 cgb_33325 cg3332/cg3334 cgb_34325 cg3432/cg3434 arf01066; 6c. brf00023; tmrna. crf00010; rnasep. experimental validation of three trans-encoded srnas by northern blotting of stress-specific rna samples and detailed structure analysis for the total set of detected trans-encoded srnas we retrieved a mean length of nt. to validate the sequencing approach, three srnas were selected and subsequently analyzed by northern blotting: the highly conserved 6c rna and two srnas with high read counts at their tss. we chose cgb_ as an example of a srna with strong secondary structure conservation as shown above and cgb_ as an example lacking secondary structure conservation. secondary structures of all presented srnas were determined by minimum free energy folding and rna shape analysis which achieved high shape probabilities in all cases, respectively. here, the total rnas obtained from different growth conditions were analyzed separately to monitor stress-specific transcription patterns. the sizes of these srnas determined by northern blot are as follows: ~ nt for 6c rna, ~ nt for cgb_ ~ nt and ~ nt for cgb_ in each case, the northern blot signal of the longest srna correlated well with the length determined by sequencing. compared with the rfam prediction, the 6c rna had a nt 5′-extension. this extended 5′-region seems to be conserved in the corynebacterium species, since a longer 5′-region for 6c rna was also predicted by our rnaz approach.figure secondary structure prediction and experimental validation by northern hybridizations of three selected srnas. for each of the three srna genes, the left column shows the sequence coverage profile derived from library. the y- and x-axis represent coverage and sequence localization. grey arrows represent flanking genes, white arrows represent the srnas and brown stem-loop structures represent rho-independent terminators. the middle column displays northern blot results of all tested conditions at time point of harvesting of c. glutamicum cells, respectively. exp = exponential phase, unstressed. further conditions were heat, cold, diamide, nacl, and h2o stresses, all applied for 15 minutes at an od the right column presents the secondary structure with lowest minimum free energy. structure, mfe and shape probabilities determined with rnashapes. sequence code: blue, a; green, c; red, g; yellow, u. validated intergenic srnas by northern hybridizations are cgb_, cgb_ and cgb_ the 6c rna showed no change in transcript abundance in the northern blots. probably due to a regulatory mechanism, cgb_ appeared to be absent under heat stress conditions. the srna cgb_ also does not appear to be differentially transcribed at a tested condition. interestingly, the northern blot revealed a shorter second band of at ~ nt, indicating a second rna species possibly generated by rna processing, since no additional promoter was observed in this region. discussion rna sequencing is a novel approach to characterize transcriptomes of bacteria comprehensively. this technique is especially useful for detection of novel srnas. here, we present the first small rna-seq approach for c. glutamicum, a member of the genus corynebacterium, which represents also a model organism for the closely related genera within the corynebacterineae, e.g. mycobacterium, nocardia etc. in comparison to the knowledge of regulatory srnas in the class of gamma-proteobacteria, especially in e. coli and salmonella species, information about srnas is marginal in actinobacteria. until now, deep sequencing of transcriptomes in this class has only been reported for mycobacteria and streptomyces species. since srnas might be differentially transcribed under stress, a mixed sample of various conditions should ensure the transcription of as many srnas as possible. the isolation of small rnas, however, yielded not only “true” srnas but also a lot of rnas that are processed or in the process of degradation. therefore, a number of filtering steps were performed on the cdna reads achieved. besides using a chosen cut-off for the increase in the number of read starts relative to the preceding position for calling a transcript start, the 5′-ends were validated by promoter searches, and from these validated 5′-ends, 3′-ends of transcripts were determined by another chosen cut-off of ten reads. it has to be stated that these cut-offs were arbitrary and adjusted to the size of the data set. as the next step, transcripts were classified by their relative positions to annotated protein-coding sequences. the length of 5′-utrs of coding sequences is variable, zero for leaderless transcripts and especially long for genes regulated by cis-regulative elements such as riboswitches. the difficulty of utr length definition was also reported in other studies. interestingly, we observed a short transcript length particularly for asrnas. as expected, the class of leader mrnas represents the longest transcripts, resulting from transcription into the adjacent cds. this difference was not obtained for the different types of srnas in sinorhizobium meliloti. generally, a shorter average size of srnas compared to srnas from other bacteria was also reported from streptomyces coelicolor. cis-antisense srnas are abundant in c. glutamicum and located in ~15% of all annotated protein-coding genes. high-resolution tiling arrays and rna sequencing led to the discovery of extensive antisense transcription in several other bacteria. in these previous studies, the reported percentage of genes within a genome which are targeted by asrna varies up and is >46% in helicobacter. the first asrna in c. glutamicum was detected upstream of cg, thereby overlapping the mrna of the transcriptional regulator of the gntr family in antisense direction. in our study, we detected four more asrnas which are located opposite to already known transcriptional regulatory genes and further five asrnas at putative regulatory genes. so far, it has been investigated that asrnas can modulate the level of transcriptional regulators, metabolic, toxic and virulence proteins or repress transposases. for c. glutamicum, our analysis of antisense transcripts based on the eggnog classification system revealed that asrnas seem to occur frequently at genes encoding proteins with functions in cell envelope biogenesis and protein secretion processes. however, there are hundreds of short transcripts in antisense direction of coding sequences in c. glutamicum the functions of which remain to be elucidated. in general, antisense transcripts often influence rna stability of their target mrna either by promoting or blocking ribonucleases. furthermore, asrnas can induce a structural change in their target mrna that effects transcription attenuation. other studies showed that asrna can also hinder rna polymerase extending the transcript encoded in the opposite strand by transcription interference or can affect translation of the target gene by regulation of ribosome binding. further classification, especially of trans-encoded srnas, can be done by sequence and structural analysis. the comparison of candidates predicted by the rnaz tool and by the rfam database with sequencing results, allowed us to detect srnas that are conserved in all bacteria, in actinomycetales or within the closely related species c. efficiens and c. diphtheriae. however, more than the half of srnas seems to be specific for c. glutamicum. at this point we want to note that our study did not detect two widely conserved elements known to be involved in bacterial srna: 6s rna and short palindromic repeat loci. interestingly, at least one crispr locus has been identified in the genomes of almost all other corynebacterium species. many bioinformatic prediction tools were developed for srna research during the last decade. the comparison of the actual in vivo expression of srnas with bioinformatic prediction results often revealed only little correspondence. apart from the rnaz program which was utilized in this study, the srnapredict algorithms are prominent bioinformatic tools which have been used in various bacterial srna studies. as implemented in srnapredict, the analysis on rho-independent terminators is often integrated in tools for srna detection. however, more than 75% of the trans-encoded srnas detected by our sequencing approach are not followed by a rho-independent terminator and especially the number of asrnas with rho-independent terminators is marginal. a similar observation was obtained within in a search for srnas in vibrio splendidus. the correct termination of one srna at a site without an obvious terminator structure was proven by northern blot analysis. rna-seq analyses deliver an unmatched single nucleotide resolution. however, confirmatory methods are required, such as northern blotting and are used in the present study, to look at stress-specific transcription. an example is presented with cgb_00105: under heat shock we detected no transcription of this srna whereas cold shock and chemically induced stresses had no influence on the amount of transcript. heat shock condition was also observed to trigger a different transcription start site for arna cis-antisense rna. in the case of cgb_ two transcripts of different length are detected in each case. here, rather srna maturation or degradation by endo- or exoribonucleases is likely. for 6c rna no change by one of the chosen stress treatments was observed. at this point, there is no hint for the function of 6c rna in c. glutamicum. currently, the 6c rna was reported to be involved in the glxr regulatory network in c. glutamicum. glxr is known as a global regulator of carbon source metabolism and energy conversion. in streptomyces coelicolor, 6c rna showed an increased transcription during sporulation. in bacteria there is an additional group of transcripts, comprising rnas that act as both, regulatory rnas and mrnas. rna with dual properties is exemplified by tmrna, which combines the features of a trna and an mrna. this housekeeping rna recycles stalled ribosomes by adding a proteolysis-inducing tag to unfinished polypeptides. our results show that the tmrna peptide-tag in c. glutamicum corresponds well to known sequences of a wide phylogenetic spectrum. short peptides encoded within 5′-utrs of mrna sequences are known as characteristic feature in a mechanism called transcriptional attenuation. in our study, we detected attenuator transcripts at different genes and operons involved in amino acid synthesis, each encoding a suitable leader peptide. such rnas are also included as cis-regulatory motives in the rfam database. transcriptional attenuation was first described for the tryptophan operon in e. coli where terminator formation is associated to a leader sequence and is influenced by the availability of trnatrp. accordingly, it has been observed for the ilvbnc operon in c. glutamicum and different amino acid operons in other microorganisms, encoding isopropylmalate synthase, the first step in leucine biosynthesis, and at arof, encoding one of the two dahp synthases in c. glutamicum, responsible for the first step of shikimate pathway in the biosynthesis of aromatic amino acids. since c. glutamicum is a well-known industrial producer of amino acids, these findings might become relevant for future engineering of amino acid producer strains.  our present study represents the first comprehensive screening for small rnas in corynebacteriaceae, a family that comprises important bacteria of industrial and medical relevance. high-throughput sequencing techniques are often applied for the search and investigation of srnas in bacterial genomes. similar to srna studies in other bacteria, we detected hundreds of srna genes in c. glutamicum atcc in c. glutamicum, more than half of all small rnas genes was classified as antisense transcripts. cis-antisense srna genes were detected at cds with various functions. however, cds specifying proteins from the functional classes'cell envelope biogenesis’ and'secretion processes’ appear to be overrepresented. trans-encoded srna genes were found distributed over the entire genome and showed secondary structure conservation among corynebacteria in about 30%. the 6c rna, already known from other actinomycetales genera showed strong transcription at unstressed exponential growth and all tested stress conditions. the 6s rna, highly conserved in bacteria, was not found in c. glutamicum. additionally, we detected riboswitches, transcriptional attenuators and other cis-regulatory motives, demonstrating the potential of our study for unraveling novel regulatory processes by small rnas in c. glutamicum. 
here, we describe an rna-seq approach for comprehensive detection and characterization of small rnas from corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important corynebacterianeae, such as c. diphtheriae and mycobacterium tuberculosis. in our rna-seq approach, total rna from c. glutamicum atcc was prepared from cultures grown in minimal medium at exponential growth or challenged by physical or by chemical stresses at this time point. high throughput short read sequencing and mapping yielded over srna genes. total rna samples were pooled and sequencing libraries were prepared from the isolated small rna fraction. for of trans-encoded srnas significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species c. efficiens ys- and c. diphtheriae nctc three selected trans-encoded srnas were characterized by northern blot analysis and stress-specific transcript patterns were found. in detail, our results give deep insight into the comprehensive equipment of srnas in c. glutamicum and provide a sound basis for further studies concerning the functions of these srnas. by determining their 5′- and 3′-ends and inspection of their locations, these potential srna genes were classified into utrs of mrnas, cis-antisense srnas, and trans-encoded srnas. recent discoveries on bacterial transcriptomes gave evidence that small rnas have important regulatory roles in prokaryotic cells. whole transcriptome data obtained by rna-seq can be used to detect and characterize all transcript species, including small rnas.
 recent discoveries on bacterial transcriptomes gave evidence that small rnas have important regulatory roles in prokaryotic cells. modern high-throughput sequencing approaches enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. whole transcriptome data obtained by rna-seq can be used to detect and characterize all transcript species, including small rnas. here, we describe an rna-seq approach for comprehensive detection and characterization of small rnas from corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important corynebacterianeae, such as c. diphtheriae and mycobacterium tuberculosis.  in our rna-seq approach, total rna from c. glutamicum atcc was prepared from cultures grown in minimal medium at exponential growth or challenged by physical or by chemical stresses at this time point. total rna samples were pooled and sequencing libraries were prepared from the isolated small rna fraction. high throughput short read sequencing and mapping yielded over srna genes. by determining their 5′- and 3′-ends and inspection of their locations, these potential srna genes were classified into utrs of mrnas, cis-antisense srnas, and trans-encoded srnas. for of trans-encoded srnas significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species c. efficiens ys- and c. diphtheriae nctc three selected trans-encoded srnas were characterized by northern blot analysis and stress-specific transcript patterns were found.  the study showed comparable numbers of srnas known from genome-wide surveys in other bacteria. in detail, our results give deep insight into the comprehensive equipment of srnas in c. glutamicum and provide a sound basis for further studies concerning the functions of these srnas. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bacterianon-coding rnahigh-throughput sequencingrna regulationissue-copyright-statement© biomed central ltd 2013 