BACKGROUND
corynebacterium glutamicum is a non-pathogenic, non-sporulating, gram-positive soil bacterium that belongs to the order actinomycetales.
this microorganism is widely used for the production of various amino acids and other industrially relevant compounds  <cit> .
furthermore, the availability of genetic engineering methods, an easy cultivation and a generally-regarded-as-safe status has helped to make it a model organism for systems biology investigations in the corynebacterineae, comprising important pathogens such as corynebacterium diphtheriae and mycobacterium tuberculosis <cit> .
the genome sequence of the  <dig>  mb circular chromosome was established a decade ago  <cit>  and contains 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> .
in addition, c. glutamicum possesses seven sigma factors regulating transcription on a global scale, recognizing specific promoter signals  <cit> .
although the promoters of about  <dig> genes have been identified in the last two decades, this leaves the majority of genes without known transcription signals.
recently, small rnas  were studied in c. glutamicum on a global scale, demonstrating the usefulness of the recently developed method of high-throughput sequencing of cdna   <cit> .
the understanding and deciphering of transcriptome complexity of an organism and the underlying functionalities have become a major focus for post-genome research in recent years  <cit> .
beside the classical approaches for profiling transcripts like northern blots, reverse-transcriptase pcr, race , and microarrays, the recent development of rnaseq has revolutionized transcriptomics.
this allows to analyze transcriptomes not only in a completely comprehensive way but also with single-nucleotide resolution  <cit> .
the features of rnaseq that are unmatched by the classical approaches, i.e.
no background or saturation effects as in fluorescence-based detection, no cross-hybridization, and therefore an almost unrestricted dynamic range of detection, make rnaseq an attractive approach to analyze the entire transcriptome also quantitatively  <cit> .
this novel sequencing approach has been successfully applied for studying whole-genome transcription for various prokaryotes and eukaryotes  <cit>  and revealed an unexpected complexity of these transcriptomes, e.g.
widespread antisense transcription and an enormous amount of small and novel rnas in bacterial genomes  <cit> .
additionally, this method offers the opportunity to improve genome annotation for prokaryotes and eukaryotes, in the latter especially regarding exon identification and alternative splicing effects  <cit> .
beside the use of rnaseq results mapping of complete transcripts, detection of transcription start sites and the analysis of the respective promoters, the transcriptome data can be further analyzed to characterize rbss, providing important information also on translational processes.
quite often 5′-utrs have sizes up to several hundred bases, indicating more complex transcriptional and translational functions such as riboswitches, rna thermometers or binding sites for regulatory rnas  <cit> .
in contrast, rnaseq analyses have demonstrated that some transcripts are leaderless, imposing a different translation mechanism  <cit> .
further important information obtainable from rnaseq data is the arrangement of genes in operons.
the classical operon has multiple genes which are transcribed from a single promoter  <cit> .
operons typically contain genes that are functionally connected, e.g.
in a metabolic pathway  <cit> .
hence, this feature might be helpful in prediction of gene function.
moreover, recent rnaseq data showed that various operons have to be divided into sub-operons due to internal transcription start sites which often respond to different conditions  <cit> .
thus, operon structures are not always simple, but can have rather complex architectures that can be fully resolved by rnaseq analysis.
the rnaseq workflow is complex and a number of technical obstacles have to be overcome.
first, the majority of total rna in a bacterial cell consists of ribosomal rna .
this rrna has to be removed efficiently either by hybridization-based rrna depletion or enzyme-based degradation of processed transcripts   <cit> .
second, it is important to maintain the strand information to be able to discriminate between sense and antisense transcripts.
methods allowing to obtain this information have been developed for eukaryotes  <cit> , and were also adapted for prokaryotes  <cit> .
for bacterial rnaseq studies illumina,  <dig> and solid sequencing platforms have been used  <cit> .
for rnaseq analyses of organisms with known genome sequence, a high number of short reads  is preferable to a small number of long reads, at least for microbial genomes.
in this study we describe an improved rnaseq method that provides a strand-specific characterization of entire transcriptomes at a whole genome level using high-throughput sequencing.
furthermore, we developed two rnaseq library preparation protocols that allow for analyzing the primary transcriptome and the total transcriptome of an organism separately.
additionally, we applied these two rnaseq protocols successfully to the transcriptome of c. glutamicum.
by sequencing the primary transcriptome, we utilized rna samples from exponential growth phase to analyze tsss recognized by the housekeeping sigma factor σa.
the investigation of the obtained rnaseq data delivered more than  <dig>  tsss which helped to correct more than  <dig> gene starts and the detection of a quite high number of leaderless transcripts .
for sequencing the whole transcriptome we used rna samples from nine different conditions  to obtain a broad range of transcripts.
the analysis of this data leads to the identification of operon structures and the detection of novel transcripts in c. glutamicum.
RESULTS
development of native 5′-end and whole transcript rnaseq protocols
to analyze the whole transcriptome as well as the native transcription start sites of c. glutamicum, i.e.
those that originate from initiation of transcription by rna polymerase, a whole transcriptome rnaseq protocol  and a native 5′-end rnaseq protocol were developed , adapting the differential rna-seq approach  <cit> .
key differences of our protocol compared to the differential rna-seq method include:
i) depletion of stable rnas using the ribo-zero rrna removal kit, as preliminary tests indicated that rrna constitutes up to 99% of the total rna in c. glutamicum , necessitating an efficient removal in both libraries.
ii) rna fragmentation by metal hydrolysis to allow for more efficient ligation of the 5′-end rna adapter and better accessibility for the rna loop adapter.
this should result in an increase of completely reverse transcribed cdnas carrying both adapter sequences needed for high-throughput sequencing.
in case of the native 5′-end protocol, this also causes only fragments containing either a 5′-triphosphate  or an unphosphorylated 5′-end  to be retained upon terminator 5′-phosphate-dependent exonuclease.
iii) repair of the 5′-ends of the rna fragments to enable ligation of the 5′-end rna adapter.
both protocols apply an rna 5′-polyphosphatase treatment converting the 5′-triphosphates of primary transcript ends to monophosphates to allow ligation.
thus in case of the native 5′-end protocol, only fragments derived from a 5′-end of a primary transcript will retain a 5′-monophosphate.
in case of the whole transcriptome protocol, an additional treatment with t <dig> polynucleotide kinase is performed, phosphorylating all 5′-ends lacking a phosphate group and thus enhancing adapter ligation efficiency.
iv) for cdna synthesis, a loop adapter is used which hybridizes via a 3′-nnnnnnn-tail preferentially to the 3′-end of the rna fragments as the binding there is stabilized by the stacking energy of the formed dna-rna duplex.
this approach saves a preparation step for the reverse transcription  and allows the direct accessibility of the 3′-fragment ends as the sequence used in the stem of the loop adapter is identical to the illumina paired-read primer  <dig>
v) several size selection steps are performed to remove adapter dimers.
vi) sequencing is done using illumina technology, allowing for a much deeper sequencing and the possibility to obtain paired-end information
the whole transcriptome rnaseq protocol was developed to determine operon structures, so far unknown transcripts as well as transcript ends.
during this procedure the primary transcripts are not enriched, enabling the sequencing of processed and native transcripts.
moreover, the whole transcriptome protocol enables read coverage of entire transcripts, including their 3′-ends.
as this approach should deliver expression of as many transcripts as possible, the rna samples for this data set originated from mixed c. glutamicum cell samples grown under different cultivation conditions .
the objective of generating the native 5′-end data set was to identify as many tsss as possible and to localize promoters, rbss and 5′-utrs of c. glutamicum in a comprehensive way.
we were mainly interested in tsss recognized by the housekeeping sigma factor σa.
therefore the rna samples for this data set originated from mixed c. glutamicum cell samples grown in minimal and in complex media.
the mixing of cells from both conditions should ensure transcription of most housekeeping genes, including those involved in anabolism and in catabolism.
data generation by illumina sequencing and mapping of dna sequence reads to the corynebacterium glutamicum atcc <dig> genome
in order to characterize the primary and the whole transcriptome of c. glutamicum using rnaseq, the two described rnaseq libraries were sequenced on a genome analyzer iix .
a total of 2 ×  <dig>  and  <dig>  million reads were generated from the whole and the primary transcriptome library, respectively.
these reads of  <dig> nt in length  were mapped to the c. glutamicum genome.
after mapping of reads and removal of duplicate mappings ,  <dig>  and  <dig>  million reads were mapped uniquely to the genome from the whole and the primary transcriptome library, respectively .
ain million.
bin case of the whole transcriptome data: sum of combined read pairs, forward reads without reverse mates, and reverse reads without forward mates that map to the genome sequence of c. glutamicum; in case of primary 5′-transcript ends data: forward reads that map to the genome sequence of c. glutamicum.
ccpr: combined pair of reads.
comprehensive identification of transcription start sites from the native 5′-end data set
the analysis of the native 5′-end data set resulted in the identification of  <dig>  tsss.
this number also contains alternative tsss that originate from one promoter and such that belong to rrna and trna genes.
the total number of tsss was reduced by merging alternative tsss  and removing tsss that belong to rrna and trna genes  or false-positive tsss .
an alternative transcription start was assumed to be in a range of ≤  <dig> base.
the + <dig> position with the highest number of read starts within this range was selected as transcription start.
after the elimination of alternative, redundant rrna and trna, and false-positive signals,  <dig>  tsss remain .
these tsss can be classified into two groups: tsss which belong to annotated genes and tsss that are assigned to novel transcripts.
tsss belonging to annotated genes were further categorized into  <dig>  single tsss  and  <dig> multiple tsss , the latter occurring at  <dig> genes.
tss numbers per gene could be as high as six, e.g.
as found for cmt <dig>  which encodes a trehalose corynomycolyl transferase .
tsss belonging to novel transcripts were classified into three categories: antisense tsss – the respective transcript is allocated in antisense orientation to an annotated gene; intragenic tsss – these tsss are located within annotated genes in sense orientation; intergenic tsss – these tsss are located between annotated genes .
in total,  <dig>  tss were assigned to annotated genes, whereby  <dig>  of  <dig>  belong to genes encoding proteins and the remaining seven were assigned to non-coding rna .
in summary, for  <dig>  of the  <dig>   actually annotated genes in c. glutamicum <cit>  a tss was found.
additionally,  <dig> tsss could not be assigned to annotated genes, but to novel, so far unknown transcripts.
overall,  <dig> of these  <dig> tsss belong to antisense transcripts,  <dig> relate to intragenic transcripts and  <dig> are assigned to new intergenic transcripts .
identification of σa-dependent promoters
promoter motifs are sites on the dna to which the rna polymerase attaches in order to start transcription  <cit> .
in bacteria, different species of sigma factors have been identified that are components of the rna polymerase holoenzyme, each recognizing a different promoter motif and thus contributing to transcription of a particular set of genes  <cit> .
the major sigma factor, σ <dig>  has been found in all known bacterial species and is responsible for the transcription of housekeeping genes  <cit> .
σ <dig> binds to specific promoter elements, called the - <dig> and a - <dig> region  <cit> .
thus, using the identified tsss, it is possible to search for promoter motifs.
for this search the web program improbizer <cit>  was used to scan  <dig> bases upstream of each of the  <dig>  identified tsss.
the conserved - <dig> motif “tannnt” was found in about 97%  of the upstream sequences of the identified tsss with a spacer  of  <dig> -  <dig> nt .
the motif as well as the distance of the motif to the tss are similar to the published σa consensus promoter of c. glutamicum <cit> .
then, a - <dig> region was searched using the  <dig>  upstream sequences with identified - <dig> motifs.
as a further requirement for the identification of the - <dig> motif the position of the found motif was taken into account.
therefore, only a spacer length  of  <dig> -  <dig> nt was allowed  <cit> .
regarding these requirements  <dig> motifs could be determined showing a weakly conserved “ttgnca” motif .
this motif fits the previously published data for c. glutamicum <cit> .
re-annotation of coding sequences
the rnaseq data containing the tsss turned out to be very useful for the correction of translational starts of coding sequence.
especially leaderless mrnas were conducive for re-annotation.
in total, the translational start codon positions of  <dig> genes were corrected.
as a result of this re-annotation,  <dig> genes now encode leaderless mrnas and the remaining  <dig> are genes containing 5′-utrs .
the tsss of these leadered genes mapped within the particular gene but not on a start codon that is in-frame to the annotated stop codon, so that a leaderless mrna for the appropriate gene could be excluded and a new, corrected translational start codon was searched downstream of the tss.
for this search, dna sequences from the tss position to the annotated stop codon of each of the  <dig> genes were scanned using the web program orf finder <cit>  with atg, gtg, ctg, and ttg as start codons and tga, taa, and tag as stop codons.
the identified and most meaningful gene starts for the  <dig> genes containing 5′-utrs are listed in additional file 1: table s <dig>
for further rnaseq analyses, especially for the determination of 5′-utrs, analysis of rbss, identification of operons structures, and detection of novel transcripts, these re-annotated gene starts were used.
characteristics of 5′-utrs and leaderless mrnas
the  <dig>  tss that were assigned to annotated, protein coding sequences were used for an analysis of the 5′-utrs, the part of a transcript reaching from the tss to the start codon.
quite surprisingly, ~33%  of the mrnas in c. glutamicum were found to have no 5′-utr .
these mrnas were classified as leaderless in this study.
this high fraction of leaderless mrna do not show any significant preference to functional eggnog <cit>  categories .
the analysis of initiator codons of such mrnas in c. glutamicum revealed that all leaderless mrnas have an aug  or gug  start codon.
beside the many leaderless genes, there is a further accumulation of 5′-utrs with lengths between  <dig> -  <dig> nt .
these short leaders might only harbor a ribosome-binding site for translation.
analysis of the distribution of the 5′-utr lengths revealed that in general the number of 5′-utrs within a certain bin decreased exponentially with increasing length .
however, an interesting exception is represented by  <dig> 5′-utrs with a length of  <dig> -  <dig> nt.
these 5′-utrs do not provide enough sequence for a rbs and spacing to the initiation codon.
hence, the mechanism of translation initiation for such mrnas and whether such transcripts result in functional proteins remains unclear.
a relatively high number of mrnas contain 5′-utrs longer than  <dig> nt .
it can be assumed that these long 5′-utrs play a role in the translational regulation of their respective mrnas through folding into secondary structures.
such cis-regulatory mechanisms of 5′-utrs were previously described for many bacteria and can harbor sequences encoding leader peptides, riboswitches, rna thermometers or binding sites for trans-encoded rnas  <cit> .
to detect putative cis-regulatory 5′-utr candidates, rfam <cit>  database predicted regulatory regions within 5′-utrs in c. glutamicum were compared with the rnaseq data obtained.
altogether,  <dig> regulatory regions were predicted for c. glutamicum and  <dig> of them matched the rnaseq data .
supporting evidence for these riboswitches was provided by the annotation of the associated protein-coding regions.
for example, the gene cg <dig> encodes a predicted mononucleotide transporter and shows a putative flavin mononucleotide  riboswitch within the 5′-utr for sensing flavin mono- and/or dinucleotides.
furthermore, the products of the genes cg <dig> , cg <dig> , and cg <dig>  are involved in thiamine metabolism and these genes contain putative thiamine pyrophosphate -sensing riboswitches within their 5′-utrs.
it is attractive to speculate that the two other genes with a predicted and validated tpp riboswitch, cg <dig> encoding a putative short-chain oxidoreductase and cg <dig>  encoding part of an abc transporter, are also involved in thiamine-dependent processes.
additionally, one rna thermometer, the cspa mrna 5′-utr, was observed within the 5′-utr of cg <dig> that encodes a predicted cold-shock protein.
rfam
predictions for regulatory regions in
c. glutamicum
compared to rnaseq data
rfam
aname, id, coordinates, and bit score of predicted regulatory 5′-utrs for c. glutamicum were taken from the rfam database  <cit> .
brefers to detected tsss and rnaseq ends relate to the last position of the 5′-utr.
the secondary structures of the verified regulatory regions are shown in additional file 3: figure s <dig>
to identify possible secondary structures within the regulatory 5′-utrs predicted by rfam <cit>  and validated by rnaseq, the web server program rnafold <cit>  was utilized.
the sequences of the putative regulatory 5′-utrs including  <dig> bases of the coding region were used for the structure predictions.
in all  <dig> cases, the 5′-utrs can fold into stable stem-loop structures, where the rbs is  sequestered .
it should also be mentioned here that small leader peptides within 5′-utrs of genes and operons involved in amino acid synthesis  were detected in c. glutamicum by rnaseq analysis  <cit> .
these leader peptides are possibly involved in transcription attenuation and control the expression of the appropriated genes dependent on the amino acid level  <cit> .
analysis of ribosome binding sites
based on the 5′-utrs of protein-coding genes, it is possible to analyze rbss.
therefore, the frequency of purines  compared to that of pyrimidines  of each nucleotide within the  <dig> nt upstream of the start codon was computed  and showed an accumulation of purines  in the region between  <dig> to 18 bp upstream of the start codon with a peak around - <dig> relative to the first base of the start codon  indicating a rbs within this region.
a scan for a sequence motif within this region  using the web program improbizer <cit>  confirmed this indication by detecting the conserved motif aggag in about 92% of the 5′-utr sequences .
next, the distance distribution between the rbs and the initiator codon was calculated by binning the length of the spacers.
the distribution for all detected rbs revealed a spacing of  <dig> -  <dig> nt as being the most common , with  <dig>  ±  <dig>  nt as mean spacing .
here, we analyzed only ribosome sites that are located in the 5′-utr of transcripts.
rbss within intercistronic regions were excluded from this analysis and will be addressed in more detail in future studies.
however, the identified rbs motif aggag can also be found within those intercistronic regions .
identification of operon structures based on the whole transcriptome data set
due to the usage of the developed whole transcriptome protocol and the paired-end sequencing, as well as the primary 5′-end data, we were able to identify operon structures in c. glutamicum and could assign genes to monocistronic transcripts, primary operons, and sub-operons.
genes were assigned to a primary operon, if  <dig> or more combined read pairs connect neighboring genes.
thus, a primary operon builds a chain of co-transcribed genes.
all remaining genes that could not be assigned to primary operons were categorizes as monocistronic transcripts.
as rnaseq data from the whole transcriptome as well as data from the primary 5′-ends were available and can be combined, in several cases we found that polycistronic operons have internal transcription starts and posterior genes might also form alternative sub-operons  <cit>  within the larger primary operon .
altogether,  <dig>  annotated genes could be assigned to  <dig> primary operons, including  <dig> sub-operon structures .
furthermore, this analysis showed that two-thirds of the estimated ~ <dig>  genes in c. glutamicum are transcribed as operons and one-third monocistronically .
additionally, the non-coding rnas 6c rna, and  <dig> s rna were identified as monocistronic transcripts, and the m <dig> rna was assigned to a primary operon as it was found co-transcribed with annotated protein-coding genes.
furthermore,  <dig> trnas were also found to be co-transcribed with protein-coding genes .
the remaining  <dig> genes that could not be allocated to primary operons or monocistronic transcripts had very low transcript levels .
generally, the quantity of operons decreases with increasing number of genes .
thus, only  <dig> primary operons contain  <dig> or more genes , whereas  <dig> primary operons contain  <dig> genes.
the largest primary operon identified here contains  <dig> genes coding for proteins with diverse cellular functions .
upon manual inspection, the gene functions of this large transcription unit comprise riboflavin biosynthesis, protein modifying enzymes and the important s-adenosylmethionine  synthase, responsible for the synthesis of the main methyl group donor of the cell, sam.
it was apparent, that also the proteins of other cofactor biosyntheses are organized in long operons.
this was the case for heme, cytochrome, folate and nicotinamide adenine dinucleotide .
other long operons contain functions in protein turnover .
eggnog
aaccording to the first gene in operon; detected within the primary 5′-transcript ends data.
bfunctional classes based on manual inspection are included in brackets.
cgenes within one primary operon are classified to more than  <dig> different eggnog <cit>  functions.
description and classification of novel transcripts
besides the validation of already known genes, the obtained transcriptome data was mined to identify new transcripts that were so far unknown for c. glutamicum.
for description of the start of a new transcript the primary 5′-end as well as the whole transcriptome data was used.
for the determination of the 3′-end of a new transcript the whole transcriptome data was applied.
due to the constraints used for the construction of the cdna library, the data mostly contain transcripts that are larger than  <dig> nt.
the identification and characterization of small rnas in c. glutamicum using rnaseq was studied in detail separately  <cit> .
altogether, the newly found transcripts were classified into three categories: intergenic transcripts that are located between annotated genes, antisense transcripts that are located on the opposite strand of a transcripts and transcripts that start within a cds.
in total, our data revealed  <dig> novel transcripts for c. glutamicum.
of these,  <dig> were identified as intergenic transcripts,  <dig> as antisense transcripts and  <dig> as intragenic transcripts .
for  <dig> of the  <dig> intergenic transcripts, a tss was identified from the primary 5′-end data set.
as intergenic transcripts might encode proteins, the sequences of these intergenic transcripts were scanned for open reading frames using the web program orf finder <cit> , resulting in the identification of at least one open reading frame for  <dig> transcripts.
in total,  <dig> of  <dig> identified open reading frames had at least one homologous protein in another species.
furthermore, to obtain additional evidence for encoded proteins within the new intergenic transcripts, a rbs was searched upstream of the longest predicted open reading frame.
while one transcript was predicted to be leaderless, a potential rbs was identified for  <dig> of the  <dig> remaining transcripts .
in total,  <dig> new transcripts were classified as antisense transcripts.
of these,  <dig> are located in antisense orientation to one or more annotated protein-coding sequences.
the remaining four antisense transcripts are located on the opposite strand of other transcripts .
yet, only for  <dig> of those antisense transcripts a clear tss could be identified.
the remaining transcripts probably arise from σa-independent transcription events occurring only under stress conditions since they were found in the total transcriptome library but not in the 5′-enriched data set.
transcript ends and predicted rho-independent terminators
in addition to the analysis of transcription start sites and operon structures, we also examined the 3′-transcript ends and compared those to rho-independent terminators predicted by transtermhp <cit> .
therefore, we extracted the transtermhp predictions for all monocistronic transcripts and the last gene within the defined primary operons.
overall, we obtained  <dig>  predicted intrinsic terminators and for only  <dig>  of these a clear transcript end could be determined, based on the following criteria: the mean coverage of the last  <dig> bases before the first terminator stem must be at least  <dig> and the ratio of this mean coverage and the coverage of a given base within the terminator must be ≥  <dig>  of the remaining  <dig>  predicted terminators,  <dig>  transcript ends were insufficiently covered and  <dig>  displayed only a gradual decrease in coverage.
for the  <dig> predicted rho-independent terminators we found that the majority of transcript ends were located within stem  <dig> of the predicted rho-independent terminator and the minority within the loop, stem  <dig>  or t-tail .
it was surprising that only a low number of 3′-ends were located in the t-tail.
discussion
this study presents the first comprehensive transcriptome analysis of corynebacterium glutamicum.
the complete genome sequence of c. glutamicum atcc  <dig> and its annotation were published in  <dig>  <cit> , but knowledge on transcriptional organization, promoter elements, and non-coding transcripts was known only for a few selected examples.
the use of rnaseq to analyze and characterize the transcriptomes in prokaryotic as well as in eukaryotic cells has been exploited in recent times  <cit> .
while commercial rnaseq kits were used in most of the above–mentioned studies, here, we introduce two improved rnaseq library preparation protocols which provide to the possibility to customize each step as required.
the described approach can easily be adapted for other species.
for the description of the transcriptome profile of c. glutamicum, we combined the results of two rnaseq protocols, one that addresses the primary and the other the whole transcriptome.
combining both data sets, we were able to report defined 5′- and 3′-ends of annotated transcripts, novel transcripts, and operon structures.
the sequencing of a library that contained enriched primary transcript ends enabled a mapping of  <dig>  tsss that represented the basis for the analysis of promoter motifs, 5′-utrs, rbss and operon structures.
σa-dependent promoters in c. glutamicum
the search for recurring motifs at the  <dig>  tsss revealed a - <dig> region  and a less conserved - <dig> region  of the sigma factor σa-dependent promoter in c. glutamicum.
this primary sigma factor is present in all known eubacteria  <cit>  and in c. glutamicum its reported recognition sequence consists of two hexamers, ttgnca and tanant, located around the positions - <dig> and - <dig> relative to the tss  <cit>  both of which closely match our results.
it was previously observed that c. glutamicum promoters do not have well conserved - <dig> regions  <cit> .
the weak conservation of the - <dig> motif in c. glutamicum can be explained by the fact that there is a high number of promoters with an extended - <dig> promoter element, tgntataat  <cit> , able to specify the full functionality for sigma factor recognition.
in addition to the house-keeping σa and the primary-like σb, c. glutamicum possesses five additional sigma factors of the ecf family , each responsible for recognizing promoters of genes involved in specific functions and stress responses  <cit> .
these sigma factors might be responsible for recognition of promoters under stress conditions that were analyzed only in the whole transcriptome sample.
since the rna sample for 5′-enriched library was generated from unstressed conditions, promoter analyses were only performed for putative housekeeping promoters.
additional analyses of these promoters and their motifs will be performed in future studies .
the identification of tsss also revealed that  <dig> genes contain multiple tsss  that result in different lengths of the 5′-utr.
the occurrence of multiple tsss can be explained by the presence of multiple promoters.
the use of multiple promoters might be helpful for adaption to nutritional signals, with one promoter ensuring a constitutive gene expression and the others increased transcription in presence of specific stimuli  <cit> .
characterization of 5′-utrs indicate novel regulatory elements
our analysis confirmed the existence of  <dig> 5′-utrs that have been predicted by the rfam database  <cit> , among them several riboswitches .
riboswitches control gene expression at the mrna level by undergoing conformational changes.
a change is usually induced upon binding of small molecules that provide chemical moieties to interact with nucleic acids, often products of metabolic pathways they thereby regulate  <cit> .
the occurrence of riboswitches in the 5′-utr region of metabolic genes that directly bind diverse metabolites and influence both at transcriptional and translational level have been described for gram-positive and gram-negative bacteria  <cit> .
for c. glutamicum, the existence and function of these riboswitches is indeed likely as some genes containing putative riboswitches within their 5′-utr are annotated as enzymes with a clearly associated metabolic function.
in other cases, the existence of a riboswitch of a certain class will be helpful in identification of the functional context of this gene.
beside the metabolite-sensing riboswitches, one rna thermometer  could also be validated by the annotation of its gene as putative cold-shock protein .
in e. coli the cspa thermosensor is described to modulate the translation of the cspa mrna  more efficiently at low temperatures than at higher temperatures, resulting in the adaption of cellular mechanisms at different temperatures as a cold-shock response  <cit> .
the structural prediction of the rna thermometer within the 5′-utr of cg <dig> and the cold-shock protein encoded by cg <dig> mrna indicate a similar function of the putative rna thermometer in c. glutamicum, although this remains to be proven experimentally.
moreover, a wealth of candidates that might harbor novel cis-regulatory rna structures was also identified.
for example, genes involved in the metabolism of amino acids  contain 5′-utrs longer than  <dig> nt that might contain riboswitch-like structures .
previously, several rna elements have been described to regulate amino acid metabolism genes in bacteria  <cit> .
such cis-regulatory elements allow a fine-tuning of gene expression  <cit> , and this might be advantageous for fast-growing, metabolically versatile bacteria like c. glutamicum.
beside the occurrence of riboswitches and rna thermometers within the 5′-utrs of mrnas, stem-loop structures of 5′-utr which have a protective effect against mrna degradation play a role in mrna decay  <cit> .
this might also be an explanation for the relatively high number of 5′-utrs longer than  <dig> nt in c. glutamicum.
abundance of leaderless mrnas in c. glutamicum
approximately 33% of mrnas were identified to be leaderless in c. glutamicum.
previous transcriptional studies have revealed ~ <dig> %  leaderless mrnas in helicobacter pylori <cit> , ~28%  in streptomyces coelicolor <cit> , and ~ <dig> %  in pseudomonas putida kt <dig>  <cit> , so that the fraction of leaderless transcripts  in c. glutamicum is the highest described in any bacterium.
it is already known that leaderless transcripts, although occurring in many taxonomic branches of bacteria, are especially abundant in gram-positives  <cit> .
interestingly, it was previously found that archaeal genes are commonly expressed as leaderless transcripts  <cit> , although there are also archaea which carry mainly long 5′-utrs  <cit> .
however, it is obscure whether there is a similarity between archaeal transcription and the transcriptional machinery of c. glutamicum since the transcription in archaea resembles more closely that in eukaryotes  <cit> .
the translational mechanism of leaderless transcripts and the recognition of such mrnas without a 5′-utr and a canonical rbs by the ribosomes were unknown for a long time.
however, in e. coli it was shown that the aug start codon and the initiator trna can promote stable binding between the mrna and the ribosome  <cit> , and that leaderless transcripts have a preference to 70s ribosomal monomers over 30s subunits, proposing an alternative translation initiation pathway for such mrnas  <cit> .
the work presented here revealed aug  and gug  as initiation codon for leaderless transcripts in c. glutamicum.
although the mechanism, by which gug can replace aug in leaderless translation is not clear, it is described that gug might function as start codon in leaderless transcripts also in other organisms, potentially providing a lower translational level  <cit> .
it can be assumed that leaderless transcripts might be remnants of ancestral mrnas, because heterologous leaderless mrnas derived from bacteria can be reliably translated in archaea and eukaryotes  <cit> .
the commonality and simplicity in the translation initiation on leaderless mrnas in archaea, bacteria, and eukaryotes is also indicative for an early evolutionary origin of such a translation mechanism  <cit> .
rnaseq data reveal aggag as ribosome binding site for c. glutamicum
based on our data, it was possible to obtain the most accurate and comprehensive analysis of the rbs in c. glutamicum so far.
our data show an increase in the fraction of purines  in the region between the 6th and 18th nt upstream of the initiation codon, indicating a recognition region for the ribosome  <cit> .
the search for a motif within this region identified the conserved motif aggag in about 89% of 5′-utr sequences in c. glutamicum that represents the reverse complement of the 3′-terminus of the 16s rrna.
these discoveries fit very well to previously published data for e. coli <cit> .
beside the initiation codon, the translation efficiency is also dependent on the hybridization of the rbs of the mrna to the 3′-end of the 16s rrna, and thus, on its sequence conservation  <cit> .
it was shown that translation can be increased using optimal rbs that are perfectly complementary to the 3′-terminus of the 16s rrna  <cit> .
therefore, it is likely that the most conserved bases from the 1st to the 3rd position  within the rbs motif aggag in c. glutamicum are essential for the translation initiation mechanism, whereas the remaining bases modulate translation efficiency.
the spacing between rbs and start codon was also shown to have a strong effect on translation, suggesting that the physical distance between the 3′-terminus of the 16s rrna and the anti-codon of the initiator fmet-trna  <cit>  is of importance.
in c. glutamicum, a spacing between  <dig> -  <dig> nt was found to be the most common .
the calculated mean spacing of  <dig>  ±  <dig>  nt fits well to the number derived for e. coli   <cit> .
therefore, approx.
<dig> -  <dig> nt is likely to be the “optimal” spacing.
revealing operon structures for c. glutamicum
in this rnaseq approach, about 97% of the annotated genes were found to be transcribed under at least one of the nine different growth conditions used.
besides validating the annotated coding sequence, the genes could furthermore be assigned to  <dig> primary operons or to  <dig>  monocistronic transcripts.
genes that are located next to each other and are transcribed coordinately from a single promoter form a genetic unit, an operon  <cit> .
due to the resolving power of rnaseq, executed on 5′-ends of native transcripts, it was furthermore found that  <dig> sub-operons are present.
the existence of additional sub-operons was previously described for different bacteria: for example, the mapping of tsss in helicobacter pylori identified  <dig> primary operons,  <dig> sub-operons, and  <dig> monocistrons within the primary operons  <cit> , whereas in mycoplasma pneumoniae  <dig> identified operons could be divided into  <dig> smaller alternative transcription units by analyzing transcription at  <dig> different conditions  <cit> .
the use of internal tsss greatly increases the transcriptomic complexity and regulatory capacity  <cit> .
furthermore, other transcriptomic analyses revealed a condition-dependent modulation of operon structures.
in such cases, a gene assigned to a polycistron in one condition can be transcribed as a monocistron in another condition  <cit>  suggesting switchable operon structures  <cit> .
in addition to the primary operons, we found that a third of the protein-coding regions in c. glutamicum are located on transcripts that are transcribed monocistronic.
this large number indicates that in general, the advantage to regulate each gene individually, e.g.
by transcription factors, outweighs the advantage of coordinated expression as part of a polycistron as well as the drawback of an increasing “regulatory burden” associated with an increasing number of monocistrons  <cit> .
on the other hand, we found that genes involved in the synthesis of enzyme cofactors or in protein metabolism  are organized in long operons.
this finding might be interpreted that especially in these cases coordinated expression is the major organizational force.
detection of novel transcripts unveil a huge amount of antisense transcription
our data revealed  <dig> transcriptionally active, but yet unknown regions for c. glutamicum.
out of them,  <dig> were identified as intergenic,  <dig> as intragenic, and  <dig> as antisense transcripts .
the intergenic transcripts most probably represent genes by themselves, either encoding small proteins or being a non-coding rna.
small, non-coding rnas are widely distributed in bacteria and also present in high numbers in c. glutamicum.
since the small rna fraction of the bacterial transcriptome requires special preparation protocols for analysis by rnaseq, it is not properly represented in the data sets obtained here and subject of another study that addresses the small rna fraction of the c. glutamicum transcriptome  <cit> .
quite a high number of intragenic tsss  that mapped within an annotated gene were determined for c. glutamicum.
yet, it is not clear whether this tsss result in alternative, shorter proteins, novel protein-coding or non-coding genes.
such internal tsss were also described for h. pylori <cit> .
furthermore, such internal tsss were also found in viruses and in human, producing a second, shorter gene product  <cit> .
it is speculated that such internal transcripts that might harbor multi-functional, possibly regulatory regions increase the genomic information content  <cit> .
furthermore,  <dig> new antisense transcripts were identified for c. glutamicum.
yet, only for  <dig> of those a tss was mapped.
it is likely that the remaining transcripts arise from sigma factor σa-independent transcription present only under stress conditions since they appeared in the library that included rna from stress conditions but not in the other that was built from normal growth conditions.
these antisense transcripts are expected to be non-coding rnas and may have regulatory roles in gene expression.
strand-specific transcriptome sequencing has led to the description of massive amounts of antisense rnas in various bacteria, eukaryotes, and archaea, and might represent a common form of regulation within all domains of life  <cit> .
antisense rnas act on gene expression by a variety of different mechanisms.
on one hand, they can hybridize to a part or the whole sense transcript, causing structural changes in the target affecting transcription termination , introduce or block cleavage sites for ribonucleases , or have an effect on translation of the target gene by blocking or releasing the rbs   <cit> .
on the other hand, their transcription can alter sense transcription by interference, suppressing the production of the transcript due to polymerase collisions   <cit> .
the use of antisense rnas for controlling gene expression allows an additional and tight regulation of target genes  <cit> , and permits adaptation of gene expression to more different conditions.
for c. glutamicum, the function and the regulation of expression of these antisense rnas is hitherto unknown, but represent an interesting research target for future studies.
transcript ends and predicted rho-independent terminators
rho-dependent and -independent termination are principal mechanisms by which bacterial transcription units are defined  <cit> .
intrinsic, rho-independent terminators are composed of a hairpin structure with g + c-rich stem followed by a t-stretch  <cit> .
it is proposed that the rna polymerase complex is destabilized after transcription and formation of the hairpin structure and that the transcribed rna molecule is released within the t-stretch region  <cit> .
the rnaseq data obtained here shows that transcription termination in c. glutamicum seems to take place within the t-tail region in only a minor number of transcripts.
the most 3′-ends of transcripts were found in the first g + c-rich region of the intrinsic terminator.
this observation is contradictory to the literature  <cit> .
a possible explanation might be an rna processing within the stem structure of the terminator by rnases that cleave double-stranded rna molecules.
for e. coli it was shown that a read-through of the int gene  leads to a formation of an rnase iii cleavage site within the terminator to regulate the expression of this gene  <cit> .
since the chosen criteria were a sharp drop-off in the number of reads within the terminator, we might have selected for transcripts in which processing of double stranded rna occurred.
another explanation for the transcript ends within the terminator stem might be that a cruciform structure formed on the dna within the terminator region displaces the rna polymerase thereby terminating transcription in the region of the terminator stem.
although it was shown in vitro that a + t-rich sequences can more easily form cruciform structures than g + c-rich sequences, it was never shown in vivo <cit> .
however, we cannot exclude that the use of the stem-loop dna adapter with seven wobble bases did not hybridize well in such stem-loop regions and therefore did not reach a perfect resolution of 3′-transcript ends.
to prove or disprove this and to get a better resolution of the 3′-transcript ends, other library preparation methods might be used.
suitable methods are already described for eukaryotic polya-rna: rna-pet .
in rna-pet the 5′- and 3′-end tags of full-length cdna fragments are fused and subsequently sequenced  <cit> .
it should be mentioned that for about 80% of the predicted rho-independent terminators for c. glutamicum no clear transcript end could be determined, either because of insufficient sequence coverage or because of a gradual and slow transcription drop-off.
this indicates that the connection between rnaseq analysis and 3′-ends or the transcripts or the processes involved in rho-independent termination in c. glutamicum or more general in actinobacteria are not yet fully understood.
CONCLUSIONS
in this study we have created the c. glutamicum transcriptome profiles using an improved rnaseq method.
the generated transcriptome data reported in this manuscript is strand-specific and allows novel insights into the transcriptomic organization and a comprehensive discovery of novel transcripts from intergenic and antisense regions throughout the genome.
we were able to map and describe operon structures for this bacterium and classified more than 70% of the new transcripts as antisense transcripts.
we identified more than  <dig>  tsss supported by promoter search of the housekeeping sigma factor σa and re-annotated more than  <dig> gene starts.
surprisingly, we observed that about 33% of the mrnas in c. glutamicum are leaderless transcripts without any preference for functional categories of the proteins encoded by these transcripts.
in this sense, it appears to be the ultimate tool for an information-rich genome annotation.
the transcriptomic data established in this study deliver an enormous amount of information on various subjects and opens many new fields to be further investigated.
among these are hitherto unknown transcriptional mechanisms in c. glutamicum and transcription-based regulation on the transcriptional as well as on the translational level.

 corynebacterium glutamicum is a non-pathogenic, non-sporulating, gram-positive soil bacterium that belongs to the order actinomycetales. this microorganism is widely used for the production of various amino acids and other industrially relevant compounds. furthermore, the availability of genetic engineering methods, an easy cultivation and a generally-regarded-as-safe status has helped to make it a model organism for systems biology investigations in the corynebacterineae, comprising important pathogens such as corynebacterium diphtheriae and mycobacterium tuberculosis. the genome sequence of the  mb circular chromosome was established a decade ago and contains 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. in addition, c. glutamicum possesses seven sigma factors regulating transcription on a global scale, recognizing specific promoter signals. although the promoters of about genes have been identified in the last two decades, this leaves the majority of genes without known transcription signals. recently, small rnas were studied in c. glutamicum on a global scale, demonstrating the usefulness of the recently developed method of high-throughput sequencing of cdna. the understanding and deciphering of transcriptome complexity of an organism and the underlying functionalities have become a major focus for post-genome research in recent years. beside the classical approaches for profiling transcripts like northern blots, reverse-transcriptase pcr, race, and microarrays, the recent development of rnaseq has revolutionized transcriptomics. this allows to analyze transcriptomes not only in a completely comprehensive way but also with single-nucleotide resolution. the features of rnaseq that are unmatched by the classical approaches, i.e. no background or saturation effects as in fluorescence-based detection, no cross-hybridization, and therefore an almost unrestricted dynamic range of detection, make rnaseq an attractive approach to analyze the entire transcriptome also quantitatively. this novel sequencing approach has been successfully applied for studying whole-genome transcription for various prokaryotes and eukaryotes and revealed an unexpected complexity of these transcriptomes, e.g. widespread antisense transcription and an enormous amount of small and novel rnas in bacterial genomes. additionally, this method offers the opportunity to improve genome annotation for prokaryotes and eukaryotes, in the latter especially regarding exon identification and alternative splicing effects. beside the use of rnaseq results mapping of complete transcripts, detection of transcription start sites and the analysis of the respective promoters, the transcriptome data can be further analyzed to characterize rbss, providing important information also on translational processes. quite often 5′-utrs have sizes up to several hundred bases, indicating more complex transcriptional and translational functions such as riboswitches, rna thermometers or binding sites for regulatory rnas. in contrast, rnaseq analyses have demonstrated that some transcripts are leaderless, imposing a different translation mechanism. further important information obtainable from rnaseq data is the arrangement of genes in operons. the classical operon has multiple genes which are transcribed from a single promoter. operons typically contain genes that are functionally connected, e.g. in a metabolic pathway. hence, this feature might be helpful in prediction of gene function. moreover, recent rnaseq data showed that various operons have to be divided into sub-operons due to internal transcription start sites which often respond to different conditions. thus, operon structures are not always simple, but can have rather complex architectures that can be fully resolved by rnaseq analysis. the rnaseq workflow is complex and a number of technical obstacles have to be overcome. first, the majority of total rna in a bacterial cell consists of ribosomal rna. this rrna has to be removed efficiently either by hybridization-based rrna depletion or enzyme-based degradation of processed transcripts. second, it is important to maintain the strand information to be able to discriminate between sense and antisense transcripts. methods allowing to obtain this information have been developed for eukaryotes, and were also adapted for prokaryotes. for bacterial rnaseq studies illumina, and solid sequencing platforms have been used. for rnaseq analyses of organisms with known genome sequence, a high number of short reads is preferable to a small number of long reads, at least for microbial genomes. in this study we describe an improved rnaseq method that provides a strand-specific characterization of entire transcriptomes at a whole genome level using high-throughput sequencing. furthermore, we developed two rnaseq library preparation protocols that allow for analyzing the primary transcriptome and the total transcriptome of an organism separately. additionally, we applied these two rnaseq protocols successfully to the transcriptome of c. glutamicum. by sequencing the primary transcriptome, we utilized rna samples from exponential growth phase to analyze tsss recognized by the housekeeping sigma factor σa. the investigation of the obtained rnaseq data delivered more than tsss which helped to correct more than gene starts and the detection of a quite high number of leaderless transcripts. for sequencing the whole transcriptome we used rna samples from nine different conditions to obtain a broad range of transcripts. the analysis of this data leads to the identification of operon structures and the detection of novel transcripts in c. glutamicum.  development of native 5′-end and whole transcript rnaseq protocols to analyze the whole transcriptome as well as the native transcription start sites of c. glutamicum, i.e. those that originate from initiation of transcription by rna polymerase, a whole transcriptome rnaseq protocol and a native 5′-end rnaseq protocol were developed, adapting the differential rna-seq approach. key differences of our protocol compared to the differential rna-seq method include: i) depletion of stable rnas using the ribo-zero rrna removal kit, as preliminary tests indicated that rrna constitutes up to 99% of the total rna in c. glutamicum, necessitating an efficient removal in both libraries. ii) rna fragmentation by metal hydrolysis to allow for more efficient ligation of the 5′-end rna adapter and better accessibility for the rna loop adapter. this should result in an increase of completely reverse transcribed cdnas carrying both adapter sequences needed for high-throughput sequencing. in case of the native 5′-end protocol, this also causes only fragments containing either a 5′-triphosphate or an unphosphorylated 5′-end to be retained upon terminator 5′-phosphate-dependent exonuclease. iii) repair of the 5′-ends of the rna fragments to enable ligation of the 5′-end rna adapter. both protocols apply an rna 5′-polyphosphatase treatment converting the 5′-triphosphates of primary transcript ends to monophosphates to allow ligation. thus in case of the native 5′-end protocol, only fragments derived from a 5′-end of a primary transcript will retain a 5′-monophosphate. in case of the whole transcriptome protocol, an additional treatment with t polynucleotide kinase is performed, phosphorylating all 5′-ends lacking a phosphate group and thus enhancing adapter ligation efficiency. iv) for cdna synthesis, a loop adapter is used which hybridizes via a 3′-nnnnnnn-tail preferentially to the 3′-end of the rna fragments as the binding there is stabilized by the stacking energy of the formed dna-rna duplex. this approach saves a preparation step for the reverse transcription and allows the direct accessibility of the 3′-fragment ends as the sequence used in the stem of the loop adapter is identical to the illumina paired-read primer v) several size selection steps are performed to remove adapter dimers. vi) sequencing is done using illumina technology, allowing for a much deeper sequencing and the possibility to obtain paired-end information the whole transcriptome rnaseq protocol was developed to determine operon structures, so far unknown transcripts as well as transcript ends. during this procedure the primary transcripts are not enriched, enabling the sequencing of processed and native transcripts. moreover, the whole transcriptome protocol enables read coverage of entire transcripts, including their 3′-ends. as this approach should deliver expression of as many transcripts as possible, the rna samples for this data set originated from mixed c. glutamicum cell samples grown under different cultivation conditions. the objective of generating the native 5′-end data set was to identify as many tsss as possible and to localize promoters, rbss and 5′-utrs of c. glutamicum in a comprehensive way. we were mainly interested in tsss recognized by the housekeeping sigma factor σa. therefore the rna samples for this data set originated from mixed c. glutamicum cell samples grown in minimal and in complex media. the mixing of cells from both conditions should ensure transcription of most housekeeping genes, including those involved in anabolism and in catabolism. data generation by illumina sequencing and mapping of dna sequence reads to the corynebacterium glutamicum atcc genome in order to characterize the primary and the whole transcriptome of c. glutamicum using rnaseq, the two described rnaseq libraries were sequenced on a genome analyzer iix. a total of 2 ×  and million reads were generated from the whole and the primary transcriptome library, respectively. these reads of nt in length were mapped to the c. glutamicum genome. after mapping of reads and removal of duplicate mappings, and million reads were mapped uniquely to the genome from the whole and the primary transcriptome library, respectively. ain million. bin case of the whole transcriptome data: sum of combined read pairs, forward reads without reverse mates, and reverse reads without forward mates that map to the genome sequence of c. glutamicum; in case of primary 5′-transcript ends data: forward reads that map to the genome sequence of c. glutamicum. ccpr: combined pair of reads. comprehensive identification of transcription start sites from the native 5′-end data set the analysis of the native 5′-end data set resulted in the identification of tsss. this number also contains alternative tsss that originate from one promoter and such that belong to rrna and trna genes. the total number of tsss was reduced by merging alternative tsss and removing tsss that belong to rrna and trna genes or false-positive tsss. an alternative transcription start was assumed to be in a range of ≤  base. the + position with the highest number of read starts within this range was selected as transcription start. after the elimination of alternative, redundant rrna and trna, and false-positive signals, tsss remain. these tsss can be classified into two groups: tsss which belong to annotated genes and tsss that are assigned to novel transcripts. tsss belonging to annotated genes were further categorized into single tsss and multiple tsss, the latter occurring at genes. tss numbers per gene could be as high as six, e.g. as found for cmt which encodes a trehalose corynomycolyl transferase. tsss belonging to novel transcripts were classified into three categories: antisense tsss – the respective transcript is allocated in antisense orientation to an annotated gene; intragenic tsss – these tsss are located within annotated genes in sense orientation; intergenic tsss – these tsss are located between annotated genes. in total, tss were assigned to annotated genes, whereby of belong to genes encoding proteins and the remaining seven were assigned to non-coding rna. in summary, for of the actually annotated genes in c. glutamicum a tss was found. additionally, tsss could not be assigned to annotated genes, but to novel, so far unknown transcripts. overall, of these tsss belong to antisense transcripts, relate to intragenic transcripts and are assigned to new intergenic transcripts. identification of σa-dependent promoters promoter motifs are sites on the dna to which the rna polymerase attaches in order to start transcription. in bacteria, different species of sigma factors have been identified that are components of the rna polymerase holoenzyme, each recognizing a different promoter motif and thus contributing to transcription of a particular set of genes. the major sigma factor, σ has been found in all known bacterial species and is responsible for the transcription of housekeeping genes. σ binds to specific promoter elements, called the - and a - region. thus, using the identified tsss, it is possible to search for promoter motifs. for this search the web program improbizer was used to scan bases upstream of each of the identified tsss. the conserved - motif “tannnt” was found in about 97% of the upstream sequences of the identified tsss with a spacer of - nt. the motif as well as the distance of the motif to the tss are similar to the published σa consensus promoter of c. glutamicum. then, a - region was searched using the upstream sequences with identified - motifs. as a further requirement for the identification of the - motif the position of the found motif was taken into account. therefore, only a spacer length of - nt was allowed. regarding these requirements motifs could be determined showing a weakly conserved “ttgnca” motif. this motif fits the previously published data for c. glutamicum. re-annotation of coding sequences the rnaseq data containing the tsss turned out to be very useful for the correction of translational starts of coding sequence. especially leaderless mrnas were conducive for re-annotation. in total, the translational start codon positions of genes were corrected. as a result of this re-annotation, genes now encode leaderless mrnas and the remaining are genes containing 5′-utrs. the tsss of these leadered genes mapped within the particular gene but not on a start codon that is in-frame to the annotated stop codon, so that a leaderless mrna for the appropriate gene could be excluded and a new, corrected translational start codon was searched downstream of the tss. for this search, dna sequences from the tss position to the annotated stop codon of each of the genes were scanned using the web program orf finder with atg, gtg, ctg, and ttg as start codons and tga, taa, and tag as stop codons. the identified and most meaningful gene starts for the genes containing 5′-utrs are listed in additional file 1: table s for further rnaseq analyses, especially for the determination of 5′-utrs, analysis of rbss, identification of operons structures, and detection of novel transcripts, these re-annotated gene starts were used. characteristics of 5′-utrs and leaderless mrnas the tss that were assigned to annotated, protein coding sequences were used for an analysis of the 5′-utrs, the part of a transcript reaching from the tss to the start codon. quite surprisingly, ~33% of the mrnas in c. glutamicum were found to have no 5′-utr. these mrnas were classified as leaderless in this study. this high fraction of leaderless mrna do not show any significant preference to functional eggnog categories. the analysis of initiator codons of such mrnas in c. glutamicum revealed that all leaderless mrnas have an aug or gug start codon. beside the many leaderless genes, there is a further accumulation of 5′-utrs with lengths between - nt. these short leaders might only harbor a ribosome-binding site for translation. analysis of the distribution of the 5′-utr lengths revealed that in general the number of 5′-utrs within a certain bin decreased exponentially with increasing length. however, an interesting exception is represented by 5′-utrs with a length of - nt. these 5′-utrs do not provide enough sequence for a rbs and spacing to the initiation codon. hence, the mechanism of translation initiation for such mrnas and whether such transcripts result in functional proteins remains unclear. a relatively high number of mrnas contain 5′-utrs longer than nt. it can be assumed that these long 5′-utrs play a role in the translational regulation of their respective mrnas through folding into secondary structures. such cis-regulatory mechanisms of 5′-utrs were previously described for many bacteria and can harbor sequences encoding leader peptides, riboswitches, rna thermometers or binding sites for trans-encoded rnas. to detect putative cis-regulatory 5′-utr candidates, rfam database predicted regulatory regions within 5′-utrs in c. glutamicum were compared with the rnaseq data obtained. altogether, regulatory regions were predicted for c. glutamicum and of them matched the rnaseq data. supporting evidence for these riboswitches was provided by the annotation of the associated protein-coding regions. for example, the gene cg encodes a predicted mononucleotide transporter and shows a putative flavin mononucleotide riboswitch within the 5′-utr for sensing flavin mono- and/or dinucleotides. furthermore, the products of the genes cg, cg, and cg are involved in thiamine metabolism and these genes contain putative thiamine pyrophosphate -sensing riboswitches within their 5′-utrs. it is attractive to speculate that the two other genes with a predicted and validated tpp riboswitch, cg encoding a putative short-chain oxidoreductase and cg encoding part of an abc transporter, are also involved in thiamine-dependent processes. additionally, one rna thermometer, the cspa mrna 5′-utr, was observed within the 5′-utr of cg that encodes a predicted cold-shock protein. rfam predictions for regulatory regions in c. glutamicum compared to rnaseq data rfam aname, id, coordinates, and bit score of predicted regulatory 5′-utrs for c. glutamicum were taken from the rfam database. brefers to detected tsss and rnaseq ends relate to the last position of the 5′-utr. the secondary structures of the verified regulatory regions are shown in additional file 3: figure s to identify possible secondary structures within the regulatory 5′-utrs predicted by rfam and validated by rnaseq, the web server program rnafold was utilized. the sequences of the putative regulatory 5′-utrs including bases of the coding region were used for the structure predictions. in all cases, the 5′-utrs can fold into stable stem-loop structures, where the rbs is sequestered. it should also be mentioned here that small leader peptides within 5′-utrs of genes and operons involved in amino acid synthesis were detected in c. glutamicum by rnaseq analysis. these leader peptides are possibly involved in transcription attenuation and control the expression of the appropriated genes dependent on the amino acid level. analysis of ribosome binding sites based on the 5′-utrs of protein-coding genes, it is possible to analyze rbss. therefore, the frequency of purines compared to that of pyrimidines of each nucleotide within the nt upstream of the start codon was computed and showed an accumulation of purines in the region between to 18 bp upstream of the start codon with a peak around - relative to the first base of the start codon indicating a rbs within this region. a scan for a sequence motif within this region using the web program improbizer confirmed this indication by detecting the conserved motif aggag in about 92% of the 5′-utr sequences. next, the distance distribution between the rbs and the initiator codon was calculated by binning the length of the spacers. the distribution for all detected rbs revealed a spacing of - nt as being the most common, with  ±  nt as mean spacing. here, we analyzed only ribosome sites that are located in the 5′-utr of transcripts. rbss within intercistronic regions were excluded from this analysis and will be addressed in more detail in future studies. however, the identified rbs motif aggag can also be found within those intercistronic regions. identification of operon structures based on the whole transcriptome data set due to the usage of the developed whole transcriptome protocol and the paired-end sequencing, as well as the primary 5′-end data, we were able to identify operon structures in c. glutamicum and could assign genes to monocistronic transcripts, primary operons, and sub-operons. genes were assigned to a primary operon, if or more combined read pairs connect neighboring genes. thus, a primary operon builds a chain of co-transcribed genes. all remaining genes that could not be assigned to primary operons were categorizes as monocistronic transcripts. as rnaseq data from the whole transcriptome as well as data from the primary 5′-ends were available and can be combined, in several cases we found that polycistronic operons have internal transcription starts and posterior genes might also form alternative sub-operons within the larger primary operon. altogether, annotated genes could be assigned to primary operons, including sub-operon structures. furthermore, this analysis showed that two-thirds of the estimated ~ genes in c. glutamicum are transcribed as operons and one-third monocistronically. additionally, the non-coding rnas 6c rna, and s rna were identified as monocistronic transcripts, and the m rna was assigned to a primary operon as it was found co-transcribed with annotated protein-coding genes. furthermore, trnas were also found to be co-transcribed with protein-coding genes. the remaining genes that could not be allocated to primary operons or monocistronic transcripts had very low transcript levels. generally, the quantity of operons decreases with increasing number of genes. thus, only primary operons contain or more genes, whereas primary operons contain genes. the largest primary operon identified here contains genes coding for proteins with diverse cellular functions. upon manual inspection, the gene functions of this large transcription unit comprise riboflavin biosynthesis, protein modifying enzymes and the important s-adenosylmethionine synthase, responsible for the synthesis of the main methyl group donor of the cell, sam. it was apparent, that also the proteins of other cofactor biosyntheses are organized in long operons. this was the case for heme, cytochrome, folate and nicotinamide adenine dinucleotide. other long operons contain functions in protein turnover. eggnog aaccording to the first gene in operon; detected within the primary 5′-transcript ends data. bfunctional classes based on manual inspection are included in brackets. cgenes within one primary operon are classified to more than different eggnog functions. description and classification of novel transcripts besides the validation of already known genes, the obtained transcriptome data was mined to identify new transcripts that were so far unknown for c. glutamicum. for description of the start of a new transcript the primary 5′-end as well as the whole transcriptome data was used. for the determination of the 3′-end of a new transcript the whole transcriptome data was applied. due to the constraints used for the construction of the cdna library, the data mostly contain transcripts that are larger than nt. the identification and characterization of small rnas in c. glutamicum using rnaseq was studied in detail separately. altogether, the newly found transcripts were classified into three categories: intergenic transcripts that are located between annotated genes, antisense transcripts that are located on the opposite strand of a transcripts and transcripts that start within a cds. in total, our data revealed novel transcripts for c. glutamicum. of these, were identified as intergenic transcripts, as antisense transcripts and as intragenic transcripts. for of the intergenic transcripts, a tss was identified from the primary 5′-end data set. as intergenic transcripts might encode proteins, the sequences of these intergenic transcripts were scanned for open reading frames using the web program orf finder, resulting in the identification of at least one open reading frame for transcripts. in total, of identified open reading frames had at least one homologous protein in another species. furthermore, to obtain additional evidence for encoded proteins within the new intergenic transcripts, a rbs was searched upstream of the longest predicted open reading frame. while one transcript was predicted to be leaderless, a potential rbs was identified for of the remaining transcripts. in total, new transcripts were classified as antisense transcripts. of these, are located in antisense orientation to one or more annotated protein-coding sequences. the remaining four antisense transcripts are located on the opposite strand of other transcripts. yet, only for of those antisense transcripts a clear tss could be identified. the remaining transcripts probably arise from σa-independent transcription events occurring only under stress conditions since they were found in the total transcriptome library but not in the 5′-enriched data set. transcript ends and predicted rho-independent terminators in addition to the analysis of transcription start sites and operon structures, we also examined the 3′-transcript ends and compared those to rho-independent terminators predicted by transtermhp. therefore, we extracted the transtermhp predictions for all monocistronic transcripts and the last gene within the defined primary operons. overall, we obtained predicted intrinsic terminators and for only of these a clear transcript end could be determined, based on the following criteria: the mean coverage of the last bases before the first terminator stem must be at least and the ratio of this mean coverage and the coverage of a given base within the terminator must be ≥  of the remaining predicted terminators, transcript ends were insufficiently covered and displayed only a gradual decrease in coverage. for the predicted rho-independent terminators we found that the majority of transcript ends were located within stem of the predicted rho-independent terminator and the minority within the loop, stem or t-tail. it was surprising that only a low number of 3′-ends were located in the t-tail. discussion this study presents the first comprehensive transcriptome analysis of corynebacterium glutamicum. the complete genome sequence of c. glutamicum atcc and its annotation were published in, but knowledge on transcriptional organization, promoter elements, and non-coding transcripts was known only for a few selected examples. the use of rnaseq to analyze and characterize the transcriptomes in prokaryotic as well as in eukaryotic cells has been exploited in recent times. while commercial rnaseq kits were used in most of the above–mentioned studies, here, we introduce two improved rnaseq library preparation protocols which provide to the possibility to customize each step as required. the described approach can easily be adapted for other species. for the description of the transcriptome profile of c. glutamicum, we combined the results of two rnaseq protocols, one that addresses the primary and the other the whole transcriptome. combining both data sets, we were able to report defined 5′- and 3′-ends of annotated transcripts, novel transcripts, and operon structures. the sequencing of a library that contained enriched primary transcript ends enabled a mapping of tsss that represented the basis for the analysis of promoter motifs, 5′-utrs, rbss and operon structures. σa-dependent promoters in c. glutamicum the search for recurring motifs at the tsss revealed a - region and a less conserved - region of the sigma factor σa-dependent promoter in c. glutamicum. this primary sigma factor is present in all known eubacteria and in c. glutamicum its reported recognition sequence consists of two hexamers, ttgnca and tanant, located around the positions - and - relative to the tss both of which closely match our results. it was previously observed that c. glutamicum promoters do not have well conserved - regions. the weak conservation of the - motif in c. glutamicum can be explained by the fact that there is a high number of promoters with an extended - promoter element, tgntataat, able to specify the full functionality for sigma factor recognition. in addition to the house-keeping σa and the primary-like σb, c. glutamicum possesses five additional sigma factors of the ecf family, each responsible for recognizing promoters of genes involved in specific functions and stress responses. these sigma factors might be responsible for recognition of promoters under stress conditions that were analyzed only in the whole transcriptome sample. since the rna sample for 5′-enriched library was generated from unstressed conditions, promoter analyses were only performed for putative housekeeping promoters. additional analyses of these promoters and their motifs will be performed in future studies. the identification of tsss also revealed that genes contain multiple tsss that result in different lengths of the 5′-utr. the occurrence of multiple tsss can be explained by the presence of multiple promoters. the use of multiple promoters might be helpful for adaption to nutritional signals, with one promoter ensuring a constitutive gene expression and the others increased transcription in presence of specific stimuli. characterization of 5′-utrs indicate novel regulatory elements our analysis confirmed the existence of 5′-utrs that have been predicted by the rfam database, among them several riboswitches. riboswitches control gene expression at the mrna level by undergoing conformational changes. a change is usually induced upon binding of small molecules that provide chemical moieties to interact with nucleic acids, often products of metabolic pathways they thereby regulate. the occurrence of riboswitches in the 5′-utr region of metabolic genes that directly bind diverse metabolites and influence both at transcriptional and translational level have been described for gram-positive and gram-negative bacteria. for c. glutamicum, the existence and function of these riboswitches is indeed likely as some genes containing putative riboswitches within their 5′-utr are annotated as enzymes with a clearly associated metabolic function. in other cases, the existence of a riboswitch of a certain class will be helpful in identification of the functional context of this gene. beside the metabolite-sensing riboswitches, one rna thermometer could also be validated by the annotation of its gene as putative cold-shock protein. in e. coli the cspa thermosensor is described to modulate the translation of the cspa mrna more efficiently at low temperatures than at higher temperatures, resulting in the adaption of cellular mechanisms at different temperatures as a cold-shock response. the structural prediction of the rna thermometer within the 5′-utr of cg and the cold-shock protein encoded by cg mrna indicate a similar function of the putative rna thermometer in c. glutamicum, although this remains to be proven experimentally. moreover, a wealth of candidates that might harbor novel cis-regulatory rna structures was also identified. for example, genes involved in the metabolism of amino acids contain 5′-utrs longer than nt that might contain riboswitch-like structures. previously, several rna elements have been described to regulate amino acid metabolism genes in bacteria. such cis-regulatory elements allow a fine-tuning of gene expression, and this might be advantageous for fast-growing, metabolically versatile bacteria like c. glutamicum. beside the occurrence of riboswitches and rna thermometers within the 5′-utrs of mrnas, stem-loop structures of 5′-utr which have a protective effect against mrna degradation play a role in mrna decay. this might also be an explanation for the relatively high number of 5′-utrs longer than nt in c. glutamicum. abundance of leaderless mrnas in c. glutamicum approximately 33% of mrnas were identified to be leaderless in c. glutamicum. previous transcriptional studies have revealed ~ % leaderless mrnas in helicobacter pylori, ~28% in streptomyces coelicolor, and ~ % in pseudomonas putida kt, so that the fraction of leaderless transcripts in c. glutamicum is the highest described in any bacterium. it is already known that leaderless transcripts, although occurring in many taxonomic branches of bacteria, are especially abundant in gram-positives. interestingly, it was previously found that archaeal genes are commonly expressed as leaderless transcripts, although there are also archaea which carry mainly long 5′-utrs. however, it is obscure whether there is a similarity between archaeal transcription and the transcriptional machinery of c. glutamicum since the transcription in archaea resembles more closely that in eukaryotes. the translational mechanism of leaderless transcripts and the recognition of such mrnas without a 5′-utr and a canonical rbs by the ribosomes were unknown for a long time. however, in e. coli it was shown that the aug start codon and the initiator trna can promote stable binding between the mrna and the ribosome, and that leaderless transcripts have a preference to 70s ribosomal monomers over 30s subunits, proposing an alternative translation initiation pathway for such mrnas. the work presented here revealed aug and gug as initiation codon for leaderless transcripts in c. glutamicum. although the mechanism, by which gug can replace aug in leaderless translation is not clear, it is described that gug might function as start codon in leaderless transcripts also in other organisms, potentially providing a lower translational level. it can be assumed that leaderless transcripts might be remnants of ancestral mrnas, because heterologous leaderless mrnas derived from bacteria can be reliably translated in archaea and eukaryotes. the commonality and simplicity in the translation initiation on leaderless mrnas in archaea, bacteria, and eukaryotes is also indicative for an early evolutionary origin of such a translation mechanism. rnaseq data reveal aggag as ribosome binding site for c. glutamicum based on our data, it was possible to obtain the most accurate and comprehensive analysis of the rbs in c. glutamicum so far. our data show an increase in the fraction of purines in the region between the 6th and 18th nt upstream of the initiation codon, indicating a recognition region for the ribosome. the search for a motif within this region identified the conserved motif aggag in about 89% of 5′-utr sequences in c. glutamicum that represents the reverse complement of the 3′-terminus of the 16s rrna. these discoveries fit very well to previously published data for e. coli. beside the initiation codon, the translation efficiency is also dependent on the hybridization of the rbs of the mrna to the 3′-end of the 16s rrna, and thus, on its sequence conservation. it was shown that translation can be increased using optimal rbs that are perfectly complementary to the 3′-terminus of the 16s rrna. therefore, it is likely that the most conserved bases from the 1st to the 3rd position within the rbs motif aggag in c. glutamicum are essential for the translation initiation mechanism, whereas the remaining bases modulate translation efficiency. the spacing between rbs and start codon was also shown to have a strong effect on translation, suggesting that the physical distance between the 3′-terminus of the 16s rrna and the anti-codon of the initiator fmet-trna is of importance. in c. glutamicum, a spacing between - nt was found to be the most common. the calculated mean spacing of  ±  nt fits well to the number derived for e. coli. therefore, approx. - nt is likely to be the “optimal” spacing. revealing operon structures for c. glutamicum in this rnaseq approach, about 97% of the annotated genes were found to be transcribed under at least one of the nine different growth conditions used. besides validating the annotated coding sequence, the genes could furthermore be assigned to primary operons or to monocistronic transcripts. genes that are located next to each other and are transcribed coordinately from a single promoter form a genetic unit, an operon. due to the resolving power of rnaseq, executed on 5′-ends of native transcripts, it was furthermore found that sub-operons are present. the existence of additional sub-operons was previously described for different bacteria: for example, the mapping of tsss in helicobacter pylori identified primary operons, sub-operons, and monocistrons within the primary operons, whereas in mycoplasma pneumoniae identified operons could be divided into smaller alternative transcription units by analyzing transcription at different conditions. the use of internal tsss greatly increases the transcriptomic complexity and regulatory capacity. furthermore, other transcriptomic analyses revealed a condition-dependent modulation of operon structures. in such cases, a gene assigned to a polycistron in one condition can be transcribed as a monocistron in another condition suggesting switchable operon structures. in addition to the primary operons, we found that a third of the protein-coding regions in c. glutamicum are located on transcripts that are transcribed monocistronic. this large number indicates that in general, the advantage to regulate each gene individually, e.g. by transcription factors, outweighs the advantage of coordinated expression as part of a polycistron as well as the drawback of an increasing “regulatory burden” associated with an increasing number of monocistrons. on the other hand, we found that genes involved in the synthesis of enzyme cofactors or in protein metabolism are organized in long operons. this finding might be interpreted that especially in these cases coordinated expression is the major organizational force. detection of novel transcripts unveil a huge amount of antisense transcription our data revealed transcriptionally active, but yet unknown regions for c. glutamicum. out of them, were identified as intergenic, as intragenic, and as antisense transcripts. the intergenic transcripts most probably represent genes by themselves, either encoding small proteins or being a non-coding rna. small, non-coding rnas are widely distributed in bacteria and also present in high numbers in c. glutamicum. since the small rna fraction of the bacterial transcriptome requires special preparation protocols for analysis by rnaseq, it is not properly represented in the data sets obtained here and subject of another study that addresses the small rna fraction of the c. glutamicum transcriptome. quite a high number of intragenic tsss that mapped within an annotated gene were determined for c. glutamicum. yet, it is not clear whether this tsss result in alternative, shorter proteins, novel protein-coding or non-coding genes. such internal tsss were also described for h. pylori. furthermore, such internal tsss were also found in viruses and in human, producing a second, shorter gene product. it is speculated that such internal transcripts that might harbor multi-functional, possibly regulatory regions increase the genomic information content. furthermore, new antisense transcripts were identified for c. glutamicum. yet, only for of those a tss was mapped. it is likely that the remaining transcripts arise from sigma factor σa-independent transcription present only under stress conditions since they appeared in the library that included rna from stress conditions but not in the other that was built from normal growth conditions. these antisense transcripts are expected to be non-coding rnas and may have regulatory roles in gene expression. strand-specific transcriptome sequencing has led to the description of massive amounts of antisense rnas in various bacteria, eukaryotes, and archaea, and might represent a common form of regulation within all domains of life. antisense rnas act on gene expression by a variety of different mechanisms. on one hand, they can hybridize to a part or the whole sense transcript, causing structural changes in the target affecting transcription termination, introduce or block cleavage sites for ribonucleases, or have an effect on translation of the target gene by blocking or releasing the rbs. on the other hand, their transcription can alter sense transcription by interference, suppressing the production of the transcript due to polymerase collisions. the use of antisense rnas for controlling gene expression allows an additional and tight regulation of target genes, and permits adaptation of gene expression to more different conditions. for c. glutamicum, the function and the regulation of expression of these antisense rnas is hitherto unknown, but represent an interesting research target for future studies. transcript ends and predicted rho-independent terminators rho-dependent and -independent termination are principal mechanisms by which bacterial transcription units are defined. intrinsic, rho-independent terminators are composed of a hairpin structure with g + c-rich stem followed by a t-stretch. it is proposed that the rna polymerase complex is destabilized after transcription and formation of the hairpin structure and that the transcribed rna molecule is released within the t-stretch region. the rnaseq data obtained here shows that transcription termination in c. glutamicum seems to take place within the t-tail region in only a minor number of transcripts. the most 3′-ends of transcripts were found in the first g + c-rich region of the intrinsic terminator. this observation is contradictory to the literature. a possible explanation might be an rna processing within the stem structure of the terminator by rnases that cleave double-stranded rna molecules. for e. coli it was shown that a read-through of the int gene leads to a formation of an rnase iii cleavage site within the terminator to regulate the expression of this gene. since the chosen criteria were a sharp drop-off in the number of reads within the terminator, we might have selected for transcripts in which processing of double stranded rna occurred. another explanation for the transcript ends within the terminator stem might be that a cruciform structure formed on the dna within the terminator region displaces the rna polymerase thereby terminating transcription in the region of the terminator stem. although it was shown in vitro that a + t-rich sequences can more easily form cruciform structures than g + c-rich sequences, it was never shown in vivo. however, we cannot exclude that the use of the stem-loop dna adapter with seven wobble bases did not hybridize well in such stem-loop regions and therefore did not reach a perfect resolution of 3′-transcript ends. to prove or disprove this and to get a better resolution of the 3′-transcript ends, other library preparation methods might be used. suitable methods are already described for eukaryotic polya-rna: rna-pet. in rna-pet the 5′- and 3′-end tags of full-length cdna fragments are fused and subsequently sequenced. it should be mentioned that for about 80% of the predicted rho-independent terminators for c. glutamicum no clear transcript end could be determined, either because of insufficient sequence coverage or because of a gradual and slow transcription drop-off. this indicates that the connection between rnaseq analysis and 3′-ends or the transcripts or the processes involved in rho-independent termination in c. glutamicum or more general in actinobacteria are not yet fully understood.  in this study we have created the c. glutamicum transcriptome profiles using an improved rnaseq method. the generated transcriptome data reported in this manuscript is strand-specific and allows novel insights into the transcriptomic organization and a comprehensive discovery of novel transcripts from intergenic and antisense regions throughout the genome. we were able to map and describe operon structures for this bacterium and classified more than 70% of the new transcripts as antisense transcripts. we identified more than tsss supported by promoter search of the housekeeping sigma factor σa and re-annotated more than gene starts. surprisingly, we observed that about 33% of the mrnas in c. glutamicum are leaderless transcripts without any preference for functional categories of the proteins encoded by these transcripts. in this sense, it appears to be the ultimate tool for an information-rich genome annotation. the transcriptomic data established in this study deliver an enormous amount of information on various subjects and opens many new fields to be further investigated. among these are hitherto unknown transcriptional mechanisms in c. glutamicum and transcription-based regulation on the transcriptional as well as on the translational level. 
the use of rnaseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. subsequent data analysis led to the identification of more than transcription start sites, the definition of 5′-utrs for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. rnaseq data sets were obtained by two methods, one that focuses on 5′-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3′-ends of transcripts. interestingly, a high number of mrnas is transcribed as leaderless transcripts. from the data, consensus promoter and ribosome binding site motifs were identified and it was shown that the majority of genes in c. glutamicum are transcribed monocistronically, but operons containing up to genes are also present. the comprehensive transcriptome map of c. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. the aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism corynebacterium glutamicum by rnaseq in order to improve its genome annotation and to describe important features for transcription and translation.
 the use of rnaseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. the aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism corynebacterium glutamicum by rnaseq in order to improve its genome annotation and to describe important features for transcription and translation.  rnaseq data sets were obtained by two methods, one that focuses on 5′-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3′-ends of transcripts. subsequent data analysis led to the identification of more than transcription start sites, the definition of 5′-utrs for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. interestingly, a high number of mrnas is transcribed as leaderless transcripts. from the data, consensus promoter and ribosome binding site motifs were identified and it was shown that the majority of genes in c. glutamicum are transcribed monocistronically, but operons containing up to genes are also present.  the comprehensive transcriptome map of c. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. the methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future. corynebacterium glutamicumrnahigh-throughput sequencingtranscriptome 