BACKGROUND
bacillus licheniformis is a spore-forming soil bacterium closely related to the gram-positive model organism bacillus subtilis.
the species’ saprophytic life style, based on the secretion of biopolymer-degrading enzymes, predestinates strains of b. licheniformis as ideal candidates for the large-scale industrial production of exoenzymes, such as amylases and peptide antibiotics  <cit> .
especially its high capacity of secreting overexpressed alkaline serine proteases has made b. licheniformis one of the most important bacterial workhorses in industrial enzyme production  <cit> .
due to their high stability and relatively low substrate specificity, alkaline serine proteases like subtilisins are crucial additives to household detergents and the greatest share on the worldwide enzyme market  <cit> .
attempts to optimize the productivity have addressed the fermentation process  <cit> , protein-engineering  <cit> , and cellular influences on protein quality and quantity  <cit> .
since the  <dig>  mb circular genome of the type strain b. licheniformis dsm <dig> was published in  <dig>  <cit> , several genome-based studies targeting strain improvement have been performed successfully  <cit> .
however, genome-based studies are limited to information directly accessible from the dna sequence and cannot benefit from knowledge of the active transcriptome.
considering that the regulatory network represented by protein- and rna-based regulators determines the performance of an industrial-oriented fermentation process  <cit>  rna-seq data might contribute to further optimization approaches.
rna-based regulatory elements are involved in the regulation of metabolism, growth processes, the adaptation to stress and varying culture conditions  <cit>  and can be divided into two main categories.
the first category comprises non-coding rnas .
trans-encoded ncrnas, often referred to as small rnas , are encoded independently from protein genes and are able to modulate the mrna of genes located at distant chromosomal loci or to interact with target proteins  <cit> .
upon formation of secondary structures, trans-encoded ncrnas interact with their target rnas by imperfect base pairing, which is triggered by the binding of a seed region of at least six contiguous nucleotides  <cit> .
this mechanism allows the srna to address multiple targets, thus orchestrating different members of one regulon  <cit> .
it has been shown that srnas affect mrna degradation and translation and modulate protein activity  <cit> .
a second class of regulatory ncrnas is encoded in cis, which means that these ncrnas are transcribed from the antisense strand of protein-coding genes  <cit> .
hence, they are complementary in full-length and can therefore form rna duplexes with the mrna of the targeted genes  <cit> .
most described examples of these cis-encoded antisense rnas  range from  <dig> to  <dig> nt in size, but some asrnas are also shown to be substantially longer  <cit> .
antisense rnas have been proven to either positively or negatively affect transcription, translation and mrna stability  <cit> .
in addition, a cis-encoded asrna might work as a trans-encoded srna for another target  <cit> .
antisense transcription has been detected in multiple organisms  <cit>  and, with the growing number of explored species, it is assumed that antisense transcripts can be found for ~ <dig> to 20% of the bacterial genes  <cit> .
a second class of rna-based regulators encompasses cis-regulatory elements, mainly present at the 5′ untranslated region  of mrna transcripts, e.g.
riboswitches, t-boxes or thermosensors  <cit> .
whereas both 5′ as well as 3’untranslated regions can bear signals for the initiation and termination of translation  <cit> , respectively, 5′utrs additionally have the ability to fine-tune translation by cis-regulatory elements.
they can be prone to rna-binding proteins or antisense rnas, carry attenuation systems  <cit>  and play a role in mrna stability  <cit> .
in contrast, 3’utrs are not as well understood and have escaped the attention of most transcriptomic studies  <cit> .
it is known that long utrs can be localized antisense to adjacent genes on the opposite strand; in fact some of these overlapping utrs have been demonstrated to act as negative regulators for genes encoded on the opposite strand  <cit> .
the development of next-generation sequencing techniques including rna sequencing  enabled the genome-wide identification of rna-based regulatory elements in an unprecedented depth.
the high dynamic range of rna-seq allows the identification of transcripts which are expressed at vastly different levels.
also, this method does not exhibit background noise and is therefore appropriate for the identification of lowly abundant transcripts  <cit> .
rna-seq analyses targeting ncrna in particular, have been published for e.g.
mycobacterium tuberculosis <cit> , streptomyces coelicolor <cit>  and sinorhizobium meliloti <cit> .
the major goal of the project in which this study is embedded is the improvement of production strains and thus ultimately the enhancement of enzyme production.
this study is targeted on the identification of active regulatory rna elements within the different phases of a productive fermentation process.
therefore samples from crucial stages of an industrial-oriented b. licheniformis subtilisin fermentation process have been examined by strand-specific rna-seq and differential rna-seq   <cit> .
a comprehensive analysis of the data revealed a multitude of rna features which correlate to the physiology and the growth phases during the process.
the combination of genomic data and rna features provides an excellent basis to understand the regulatory events within an industrial fermentation process.
RESULTS
b. licheniformis mw3Δspo, a germination deficient mutant of b. licheniformis dsm <dig>  transformed with an expression plasmid encoding an alkaline serine protease, was grown in fed-batch mode in 6 l cultures.
the fermentations were carried out in complex amino acid broth under conditions resembling the parameters used in industrial fermentation processes .
to enhance the reliability of the analysis, the experiments were carried out in triplicate .
samples were taken at five selected time points of the fermentation process, which were chosen to follow the initial cell growth  and to determine the decisive changes within the early  and the late stage  of the protease-producing states .
total rna from each sample was prepared for strand-specific whole transcriptome sequencing  <cit> .
rna from samples l-i to l-v was additionally prepared for differential rna-seq for determination of transcription start sites , as described by sharma et al.
<cit> .
whole transcriptome sequencing
strand-specific deep sequencing of the whole transcriptome of  <dig> b. licheniformis samples yielded more than  <dig> million reads with a specific length of  <dig> nucleotides.
the number of reads for each library ranged from  <dig>  ×  <dig> to  <dig>  ×  <dig>
after the application of a strict quality processing ,  <dig>  to  <dig> % of these reads have been found to map to the chromosome and the expression plasmid used in this study .
due to repeat regions,  <dig> % of the b. licheniformis genome is not precisely mappable when considering the applied read length of  <dig> nucleotides.
thus, all reads mapping completely to such repeat regions have been excluded from further analysis.
this pertains mainly to those  <dig>  to  <dig> % of reads which map to trna and rrna genes.
the majority of these rrna matching reads can be assigned to 5s rrna genes, which is in accordance with the fact that the applied depletion targets especially 16s and 23s rrnas.
also, all reads mapping to the plasmid were removed from the dataset, as this analysis is focused on the transcriptional activity of the chromosome.
finally,  <dig>  to  <dig> % of the initial reads were taken for further analyses.
these reads enabled the identification of transcriptional units and the determination of their boundaries to assign the transcriptional activity of coding as well as non-coding regions of the chromosome .
to facilitate the comparison of different transcription levels between samples, we introduce the nucleotide activity per kilobase of exon model per million mapped reads  value as single nucleotide-resolution measure of transcriptional activity .
npkms for each rna feature and for every gene were calculated and are available at additional file 2: table s <dig> and table s <dig>
transcription start site determination and operon prediction
differential rna-seq  has been designed by sharma et al.
<cit>  to allow selective enrichment of native 5′ ends of transcripts for the determination of transcription start sites .
the method is based on the observation that 5′ triphosphorylated rna fragments are originating from native 5′ ends.
in contrast, 5′ monophosphorylated rnas are products of rna decay or processing and do not contain information of transcription initiation.
the drna-seq approach includes a treatment with 5′ phosphate-dependent exonuclease , which results in the depletion of all monophosphorylated transcripts.
it has been shown that tss identification based on drna-seq data is superior to an estimation of transcript boundaries based on whole transcriptome rna-seq reads  <cit> .
the differential sequencing of samples l-i to l-v resulted in  <dig> , <dig> reads .
a total of  <dig> putative tss was predicted ,  <dig> of which were detected in at least two samples .
a comparison of the latter with the transcript boundaries obtained by whole transcriptome sequencing  shows that  <dig> identified tss confirm the rna-seq data, whereas the other findings introduce tss not detectable by conventional rna-seq.
to allow the assignment of the identified tss to their putative origin, an allocation to four different classes was accomplished   <cit> .
naturally, the affiliation of tss according to this schema is ambiguous as some tss sort to multiple classes, e. g. some tss are located in a promoter region and within the upstream gene as well.
the distribution of the identified tss to each class is shown in figure 2b.
<dig> tss were detected in promoter regions,  <dig> genes are bearing more than one putative tss in this region.
the drna-seq data enabled conclusions for tss determination in cases in which read-through transcription of the upstream gene caused by leaky termination prohibits the identification of downstream tss by conventional rna-seq data.
of the identified  <dig> intragenic tss,  <dig> are not located in the 500 bp promoter region of the downstream gene, reflecting a high number of putative internal promoters.
orphan tss may indicate potential start sites of yet unknown genes or non-coding rnas, this is supported by the finding that  <dig> of the  <dig> detected orphan tss could be allocated to identified ncrnas.
operon prediction based on rna-seq and drna-seq data resulted in  <dig> putative operons structuring the genome of b. licheniformis .
while most operons are monocistronic  or bicistronic , seven operons seem to encompass more than ten genes .
this small number of long operons is not in accordance with the operon prediction made by kristoffersen et al.
<cit>  for b. cereus.
the difference is due to the varying operon concept employed here.
especially the consideration of internal tss in combination with distinct shifts of expression resulted in an increase of shortened operons in this study.
reannotation
the first genome annotation of b. licheniformis dsm <dig> has been published in  <dig>  <cit> .
it has been shown previously that mapping of rna-seq data to genomes allows the correction of open reading frames and supports the identification of not-annotated protein genes  <cit> .
therefore, we performed a complete reannotation of the genome in order to integrate the rna-seq data provided by this study as well as the progress in gene prediction and annotation of the recent years.
distinct transcription start sites determined by drna-seq and rna-seq-based whole transcriptome data have been used to identify putative mis-annotated genes .
these findings were validated by length comparisons to genes deposited in public databases and confirmation of ribosomal binding sites and - <dig> and - <dig> promoter regions.
this approach enabled the correction of reading frames of  <dig> protein genes,  <dig> pseudo genes,  <dig> rrna genes and two trna genes .
moreover,  <dig> previously not-annotated protein genes were identified based on transcriptional activity and protein conservation .
<dig> genes  were removed from the annotation as these previously predicted orfs could not be verified by detailed genome analysis and comparisons to public databases.
in total, the reannotation approach resulted in a dataset containing  <dig> orfs.
comparisons to the annotation of b. licheniformis dsm <dig> by rey et al.
<cit>  showed that  <dig> of the newly annotated genes have not been described for this organism before.
<dig> of the removed genes were annotated in both genomes.
more than  <dig> gene annotations have been improved.
these improvements mainly comprise former hypothetical proteins now assigned to a function and proteins with altered gene symbols.
in addition to gene-associated improvements, seven genomic regions were identified as prophage regions based on gc content deviations, significant similarities to known prophage genes and the presence of insertion repeats.
the transcriptional activity of the prophage regions was rather low, which is consistent with the observation that many prophages are induced during sos response, which should not occur within a fermentation process  <cit> .
5′ and 3′untranslated regions
in this study,  <dig> 5′untranslated regions  with a mean length of  <dig> nt  could be identified.
thirty of these 5′utrs are shorter than  <dig> nt, implicating that leaderless transcription, commonly found in many bacteria  <cit> , is not an abundant mechanism in b. licheniformis.
correspondingly, low occurrence of leaderless transcription has also been suggested for other members of the phylum firmicutes <cit> .
the most strongly transcribed 5’utrs ≥ <dig> nt and the 5’utrs discussed in the following passage are listed in table  <dig>
cis
*.
at sampling points i, ii, and iii, the gene of the sporulation inhibitor kapd  reveals a 5’utr of  <dig> nt , whereas an alternative, drna-seq-supported 5’utr  with a length of  <dig> nt is present at the later stages of the fermentation process.
the tss of both 5’utrs seem to be preceded by a σa recognition site.
at sampling point iv, the transcriptional activity of the gene is higher than the activity of the 5’utr region.
in b. subtilis, growth phase-dependent differentiation into subpopulations of distinct cell types with different gene expression patterns is well described  <cit> .
the divergent expression levels of kapd and the long 5’utr in b. licheniformis might therefore result from different usage of promoter sites dependent on the respective cell type.
however, the observed effect could also derive from slow decay rates of the short form of the kapd mrna transcribed earlier.
<dig> further 5’utrs exhibited antisense activity towards upstream genes , as shown for the untranslated region bli_r <dig> upstream of the glutamate synthase operon gltab .
the observed 5’utr is completely antisense to the gene of the corresponding transcriptional activator gltc .
the drna-seq data suggest the presence of only one tss.
this finding might be an example for a regulatory linkage between adjacent genes localized on different strands.
this concept has recently been termed the excludon by sesto et al.
<cit> , who demonstrated that long 5’utrs can act negatively on the transcription of the opposite gene.
following this idea in the case of the glutamate synthase operon, the preceding 5’utr might establish a negative feedback regulation of the transcriptional activator gltc.
a corresponding elongated utr of the gltab operon has not been found in b. subtilis <cit> , which indicates different regulations of glutamate homeostasis in the two species.
next to regulatory effects based on antisense orientation, 5’utrs can bear intrinsic, so-called cis-regulatory elements  <cit> .
at the time of this study,  <dig> cis-regulatory elements have been predicted for b. licheniformis dsm <dig> by covariance models  <cit> .
all elements have been shown to be transcriptionally active during the fermentation process , although some are not located in 5’utrs but in intergenic read-through regions.
three new t-boxes, located upstream of the serine acetyltransferase gene cyse and the trna ligase subunit genes glyq and phes, could be identified by comparison to the rfam database.
in b. subtilis,  <dig> cis-regulatory elements have been described  <cit> , comprising rna switches as well as protein-binding rnas.
for  <dig> of these instances, transcription could be shown at orthologous loci in b. licheniformis .
<dig> 3’utrs  with an average length of  <dig> nt have been identified according to figure  <dig>  the most strongly transcribed 3’utrs ≥ <dig> nt and the 3’utr discussed in this chapter are listed in table  <dig>  of the identified 3’utrs, 42% exceed  <dig> nt and 16% even exceed  <dig> nt in length .
in total,  <dig> 3’utrs are localized antisense to adjacent genes .
a detailed manual inspection revealed that all 3’untranslated regions longer than  <dig> nt seem to be protruding after incomplete termination  <cit> .
altogether,  <dig> 3’utrs with internal termination sites could be determined, whereas  <dig> 3’utrs end at predicted termination sites.
these findings suggest that the effect of fading-out at the end of operons due to imperfect termination might be a common effect in b. licheniformis.
an example is the  <dig> nt 3’utr  downstream of the cell envelope stress response operon liaihgfsr .
the mrna transcript of this operon protrudes beyond a termination signal, which is located directly behind the stop codon of liar.
this protruding mrna sequence is antisense to the next four genes which comprise the germination receptor operon geraaabac  and a hypothetical protein .
a second terminator structure can be found  <dig> nt upstream of the end of the transcript.
*.
non-coding rna features
non-coding rnas were identified in non-coding regions of the chromosome, for example in intergenic regions or localized in antisense direction to protein genes .
the boundaries of the identified transcripts were determined by upshifts or downshifts of transcriptional activity.
all identified rna features were checked for similarities to complete protein genes as well as protein domains to ensure that they indeed represent non-coding rnas.
to extract the basic types of ncrna expression profiles during the examined fermentation process, cluster analysis based on the k-means algorithm  <cit>  was applied to those  <dig> ncrnas with highly reliable replicates.
in total,  <dig> clusters of divergent expression profiles were generated .
cluster  <dig> contains 36% of the applied ncrnas and 50% of all ncrnas > <dig> nt.
it displays a strong up-shift of transcriptional activity at sampling point iv followed by a decrease at sampling point v. the high portion of transcripts in this cluster prompts the conclusion that rna-based regulation is especially important during the later stages of the fermentation process.
other ncrnas exhibiting up-shifts of transcriptional activity are displayed in clusters  <dig> to  <dig>  whereas clusters  <dig> to  <dig> include transcripts with activity down-shifts.
the further clusters comprise ncrnas with expression shifts during the early fermentation process, as well as an activity up-shift at sampling point v in clusters  <dig> to  <dig>
all assigned non-coding rnas were categorized according to the scheme displayed in figure  <dig> and subdivided into the classes a
5
, a
3
, a
i
, a
misc
and indep.
selected ncrnas are listed in table  <dig>  whereas an overview of all identified features is given in additional file 2: table s <dig>  several ncrnas have been selected for validation by northern blotting .
the analyzed ncrnas were chosen as they are exemplarily for their respective class.
the occurrence of eight ncrnas could be verified, especially ncrnas < <dig> nt are in good accordance with the results gained by rna-seq.
the results for transcripts > <dig> nt are indicative for rna degradation or processing and leaky transcription termination.
however, three ncrnas could not be validated, which is most probably due to their low expression levels.
misc
misc
misc
misc
i
misc
misc
i
misc
misc
misc
3'utr
misc
i
misc
*.
indep ncrnas
as depicted in figure  <dig>  indep transcripts are defined as non-coding rnas not localized antisense to any mrna.
instead they can be found in intergenic regions or any other position of the chromosome.
in total,  <dig> indep rnas with sizes between  <dig> and  <dig> nt have been identified, of which  <dig> have tss verified by drna-seq.
within this group five housekeeping srnas could be annotated: the tmrna ssra , the 6s rnas bsra  and bsrb , the rna component of rnase p rnpb  and the signal recognition particle scr   <cit> .
87% of the indep transcripts exhibited npkm values ≥ <dig> in at least three samples, reflecting a strong transcriptional activity of the encoding genomics regions.
for example the srna scr, an essential part of the protein secretion system  <cit> , reaches a maximal npkm value of almost  <dig> .
this is in perfect accordance with the fact that the cells are derived from a fermentation process optimized for protein secretion.
interestingly,  <dig> indep ncrnas seem to be transcribed constitutively under the examined conditions , whereas only thirteen indep rnas show differential expression   <cit> ), as illustrated exemplarily for bli_r <dig> .
antisense ncrnas
in contrast to the class of indep ncrnas, the antisense ncrnas  a
i
, a
3
, a
5
, and a
misc
comprise non-coding transcripts localized antisense to annotated protein-coding genes.
they either target the protein-coding region of a gene  or the 5’ and 3’ untranslated regions .
furthermore, ncrnas that target more than one gene or that are only partially antisense are classified .
in this study,  <dig> a
misc
rnas  could be identified.
approximately  <dig> of them  are located opposite to ribosome binding sites and could therefore function as inhibitors of translation, a very common mechanism of cis-encoded asrnas  <cit> .
the length distribution of the non-coding rna features is shown in figure 5c, and illustrates that 42% of the a
misc
rnas are less than 400 nt in length.
twenty-seven of these short a
misc
rnas reach maximal npkm values ≥ <dig>  suggesting putative srna mechanisms.
however, some a
misc
rnas are much longer, i.e.
bli_r <dig> is  <dig> nt in length and spans six genes.
the occurrence of antisense transcripts of such length is not unexpected, as asrnas with very diverse sizes, reaching more than  <dig> nt, have been described for several species  <cit> .
furthermore,  <dig> a
i
transcripts  could be assigned, ranging in size from  <dig> to  <dig> nt.
over 95% of the a
i
transcripts exhibit maximal npkm values ≤ <dig>  68% even ≤ <dig>  due to the low coverage only  <dig> tss could be verified by drna-seq for these asrnas.
in total,  <dig> non-coding asrnas were determined, comprising 89% of all identified non-coding rna transcripts and targeting 15% of all genes.
the number of identified antisense ncrnas is in accordance to previous studies which assume that antisense transcription concerns ~ <dig> to 20% of the bacterial genes  <cit> .
antisense transcripts with putative impact on productivity
the general aim of our group is the identification of productivity related features.
thus, a special focus has been set on the identification of antisense transcripts with a putative impact on protease production as targets for strain improvement.
the alkaline serine protease subtilisin carlsberg  represents the major secreted feeding protease of b. licheniformis.
thus, it competes for energetic and secretory resources with the production protease.
we identified a cis-encoded  <dig> nt a
i
asrna  which is located at the 3’end of the apr mrna .
a highly active tss determined from the drna-seq data and a terminator structure downstream of the adjacent gene yhfn confirm the characterization of the transcript as independently transcribed asrna.
bli_r <dig> is highly expressed at all fermentation stages, whereas the transcriptional activity of the subtilisin carlsberg gene increases at the productive stages of the process.
the presence of the cis-encoded asrna opposite to the 3’end of the target mrna resembles the b. subtilis rata/txpa toxin/antitoxin system or the escherichia coli gady/gadx system in which an antisense rna promotes either mrna degradation or stability  <cit> .
to elucidate the impact of the detected asrna, further analyses will be necessary, especially as a corresponding transcript is absent in the transcriptome of b. subtilis <cit> .
further antisense transcripts against genes involved in cell differentiation, cell stress response, and thiamine and folate biosynthesis could be observed and are presented in additional file 1: figure s <dig>
it is exciting to think about a regulatory impact of the mentioned ncrnas, but there are also some noteworthy limitations to putative effects.
the completed rna-seq experiments cannot discern if the sense and the antisense transcript are transcribed in the same type of differentiated cells, which especially challenges stoichiometric estimations of asrnas and their mrna targets.
whether they can influence each other or fulfill different purposes in different cell types has to be a topic of single cell targeted investigations.
it has been reported that functional srnas are produced in excess amounts over the targeted mrna  <cit> .
therefore, a regulatory mechanism of poorly transcribed antisense rna cannot be assumed bona fide, but has to be evaluated carefully.
nonetheless, our data implicate that there might be a biological function assignable to the rna features, especially when they are conserved within related species as b. subtilis.
at last, it has to be experimentally excluded, especially for low abundant instances, that the found ncrnas originate from spurious transcriptional events, for instance driven by alternative sigma factors  <cit> .
comparative transcriptomics
in total, we determined  <dig> candidate non-coding rna transcripts, including antisense, as well as indep ncrnas .
for synechocystis sp.
pcc <dig>  sinorhizobium meliloti and the archaea sulfolobus solfataricus p <dig> and methanosarcina mazei gö <dig> between  <dig> and  <dig> non-coding rnas per mb were identified  <cit> , matching our result of  <dig> ncrnas/mb.
for b. subtilis, the close relative of b. licheniformis, nicolas et al.
<cit>  have found  <dig> non-coding rna features in a tiling array-based, condition-dependent transcriptome study.
the majority  of these features are intergenic transcripts determined by promoter analysis, whereas only 32% are derived from independently transcribed  rnas.
in contrast, the majority of ncrnas identified in b. licheniformis are antisense rnas , transcribed independently from protein-coding genes.
the identification of more antisense transcripts in b. licheniformis might be accounted to the reduced background noise in rna-seq in comparison to tiling arrays, which allows a better detection of low abundant transcripts  <cit> .
<dig> of the b. licheniformis ncrnas are located in regions with high sequence similarity to b. subtilis <cit>  and  <dig> ncrnas are encoded at the frontiers of conserved and not conserved regions of the two genomes.
based on sequence similarity, only  <dig>  out of the, in total,  <dig> ncrnas located in these regions seem to occur in the b. subtilis transcriptome  <cit> , emphasizing the differences of the two closely related species.
comparisons to two earlier b. subtilis transcriptome studies show similar low levels of accordance  <cit> .
however, as mentioned above, it is also possible that the identified antisense ncrnas partly derive from spurious transcription events  <cit> , and hence do not introduce a species-specific effect.
for b. subtilis,  <dig> srnas have been validated experimentally  <cit> .
comparison to rfam and/or comparison of genomic locations facilitated the detection of eleven of these srnas in the transcriptome of b. licheniformis .
these include, in addition to the mentioned five housekeeping srnas  <cit> , two regulatory rnas with well-known function in b. subtilis: sr <dig> and rnaa  <cit> .
the other rnas found in b. licheniformis are bsri, csfg, surc and rsae  <cit> .
the b. subtilis srnas which could not be confirmed in b. licheniformis originate from loci with no conserved gene pattern in this organism and thus may contribute to the differences between the two species.
jahn et al.
<cit>  described the toxin-antitoxin system bsrg/sr <dig> located in the spβ prophage region of b. subtilis.
although b. licheniformis does not harbor a homolog of the spβ prophage, two distinct transcripts were found to encode peptides similar to the bsrg toxin .
additionally, the transcriptional activity of the corresponding loci revealed pairs of overlapping transcripts from both strands  as shown for the bsrg/sr <dig> type toxin-antitoxin system.
therefore both newly identified orfs were annotated as bsrg-like peptides .
furthermore, the antisense transcripts  resemble the sr <dig> antitoxin, especially in stem loops sl <dig>  sl <dig> and tsl  <cit>  directly antisense to the bsrg-encoding mrna.
CONCLUSIONS
the presented study generated substantial data on the transcriptional activity of b. licheniformis within five relevant growth stages of an industrial-oriented fermentation process.
a detailed analysis of the transcriptome data enabled us to accomplish a high quality functional genome reannotation of b. licheniformis dsm <dig> .
the integration of the reannotation and the transcriptionally active regions  resulted in the identification and quantification of hundreds of rna based regulatory elements as well as protein encoding genes.
in total,  <dig> rna features have been sorted into ten functional classes .
<dig> 5’utrs and  <dig> 3’utrs  as well as  <dig> ncrnas  and  <dig> antisense intergenic read-through  transcripts have been identified.
a striking observation was the identification of  <dig> rna features, which mapped antisense to annotated genomic features .
notably antisense rna features have been found in each of the functional classes and include transcripts of a size range from  <dig> to  <dig> base pairs in length.
we have identified both: constitutively as well as growth phase dependently expressed rna features.
our data represent a solid amount of knowledge on regulatory elements which orchestrate the cellular activities of b. licheniformis during the succession of growth phases within a productive fermentation.
to generate an overview of the functional diversity of the identified rna features, all instances have been screened against the rfam database.
this approach resulted in hits to experimentally well characterized rna features known from b. subtilis and other relatives, as well as in a multitude of so far unknown rna features without any rfam hit.
the knowledge on genes and regulatory rna features which are transcriptionally active during an industrial-oriented fermentation enables an excellent access to a rational strain design approach for the optimization of b. licheniformis as industrial workhorse.
especially the regulatory features which represent differences to the model organism b. subtilis give new insights to the still open question what makes strains of the species b. licheniformis superior to b. subtilis strains in terms of protease production capacity in industrial applications  <cit> .
in the future it may be promising to correlate the transcriptional activity of the rna features to the corresponding protein expression patterns.

 bacillus licheniformis is a spore-forming soil bacterium closely related to the gram-positive model organism bacillus subtilis. the species’ saprophytic life style, based on the secretion of biopolymer-degrading enzymes, predestinates strains of b. licheniformis as ideal candidates for the large-scale industrial production of exoenzymes, such as amylases and peptide antibiotics. especially its high capacity of secreting overexpressed alkaline serine proteases has made b. licheniformis one of the most important bacterial workhorses in industrial enzyme production. due to their high stability and relatively low substrate specificity, alkaline serine proteases like subtilisins are crucial additives to household detergents and the greatest share on the worldwide enzyme market. attempts to optimize the productivity have addressed the fermentation process, protein-engineering, and cellular influences on protein quality and quantity. since the  mb circular genome of the type strain b. licheniformis dsm was published in, several genome-based studies targeting strain improvement have been performed successfully. however, genome-based studies are limited to information directly accessible from the dna sequence and cannot benefit from knowledge of the active transcriptome. considering that the regulatory network represented by protein- and rna-based regulators determines the performance of an industrial-oriented fermentation process rna-seq data might contribute to further optimization approaches. rna-based regulatory elements are involved in the regulation of metabolism, growth processes, the adaptation to stress and varying culture conditions and can be divided into two main categories. the first category comprises non-coding rnas. trans-encoded ncrnas, often referred to as small rnas, are encoded independently from protein genes and are able to modulate the mrna of genes located at distant chromosomal loci or to interact with target proteins. upon formation of secondary structures, trans-encoded ncrnas interact with their target rnas by imperfect base pairing, which is triggered by the binding of a seed region of at least six contiguous nucleotides. this mechanism allows the srna to address multiple targets, thus orchestrating different members of one regulon. it has been shown that srnas affect mrna degradation and translation and modulate protein activity. a second class of regulatory ncrnas is encoded in cis, which means that these ncrnas are transcribed from the antisense strand of protein-coding genes. hence, they are complementary in full-length and can therefore form rna duplexes with the mrna of the targeted genes. most described examples of these cis-encoded antisense rnas range from to nt in size, but some asrnas are also shown to be substantially longer. antisense rnas have been proven to either positively or negatively affect transcription, translation and mrna stability. in addition, a cis-encoded asrna might work as a trans-encoded srna for another target. antisense transcription has been detected in multiple organisms and, with the growing number of explored species, it is assumed that antisense transcripts can be found for ~ to 20% of the bacterial genes. a second class of rna-based regulators encompasses cis-regulatory elements, mainly present at the 5′ untranslated region of mrna transcripts, e.g. riboswitches, t-boxes or thermosensors. whereas both 5′ as well as 3’untranslated regions can bear signals for the initiation and termination of translation, respectively, 5′utrs additionally have the ability to fine-tune translation by cis-regulatory elements. they can be prone to rna-binding proteins or antisense rnas, carry attenuation systems and play a role in mrna stability. in contrast, 3’utrs are not as well understood and have escaped the attention of most transcriptomic studies. it is known that long utrs can be localized antisense to adjacent genes on the opposite strand; in fact some of these overlapping utrs have been demonstrated to act as negative regulators for genes encoded on the opposite strand. the development of next-generation sequencing techniques including rna sequencing enabled the genome-wide identification of rna-based regulatory elements in an unprecedented depth. the high dynamic range of rna-seq allows the identification of transcripts which are expressed at vastly different levels. also, this method does not exhibit background noise and is therefore appropriate for the identification of lowly abundant transcripts. rna-seq analyses targeting ncrna in particular, have been published for e.g. mycobacterium tuberculosis, streptomyces coelicolor and sinorhizobium meliloti. the major goal of the project in which this study is embedded is the improvement of production strains and thus ultimately the enhancement of enzyme production. this study is targeted on the identification of active regulatory rna elements within the different phases of a productive fermentation process. therefore samples from crucial stages of an industrial-oriented b. licheniformis subtilisin fermentation process have been examined by strand-specific rna-seq and differential rna-seq. a comprehensive analysis of the data revealed a multitude of rna features which correlate to the physiology and the growth phases during the process. the combination of genomic data and rna features provides an excellent basis to understand the regulatory events within an industrial fermentation process.  b. licheniformis mw3Δspo, a germination deficient mutant of b. licheniformis dsm transformed with an expression plasmid encoding an alkaline serine protease, was grown in fed-batch mode in 6 l cultures. the fermentations were carried out in complex amino acid broth under conditions resembling the parameters used in industrial fermentation processes. to enhance the reliability of the analysis, the experiments were carried out in triplicate. samples were taken at five selected time points of the fermentation process, which were chosen to follow the initial cell growth and to determine the decisive changes within the early and the late stage of the protease-producing states. total rna from each sample was prepared for strand-specific whole transcriptome sequencing. rna from samples l-i to l-v was additionally prepared for differential rna-seq for determination of transcription start sites, as described by sharma et al.. whole transcriptome sequencing strand-specific deep sequencing of the whole transcriptome of b. licheniformis samples yielded more than million reads with a specific length of nucleotides. the number of reads for each library ranged from  ×  to  ×  after the application of a strict quality processing, to % of these reads have been found to map to the chromosome and the expression plasmid used in this study. due to repeat regions, % of the b. licheniformis genome is not precisely mappable when considering the applied read length of nucleotides. thus, all reads mapping completely to such repeat regions have been excluded from further analysis. this pertains mainly to those to % of reads which map to trna and rrna genes. the majority of these rrna matching reads can be assigned to 5s rrna genes, which is in accordance with the fact that the applied depletion targets especially 16s and 23s rrnas. also, all reads mapping to the plasmid were removed from the dataset, as this analysis is focused on the transcriptional activity of the chromosome. finally, to % of the initial reads were taken for further analyses. these reads enabled the identification of transcriptional units and the determination of their boundaries to assign the transcriptional activity of coding as well as non-coding regions of the chromosome. to facilitate the comparison of different transcription levels between samples, we introduce the nucleotide activity per kilobase of exon model per million mapped reads value as single nucleotide-resolution measure of transcriptional activity. npkms for each rna feature and for every gene were calculated and are available at additional file 2: table s and table s transcription start site determination and operon prediction differential rna-seq has been designed by sharma et al. to allow selective enrichment of native 5′ ends of transcripts for the determination of transcription start sites. the method is based on the observation that 5′ triphosphorylated rna fragments are originating from native 5′ ends. in contrast, 5′ monophosphorylated rnas are products of rna decay or processing and do not contain information of transcription initiation. the drna-seq approach includes a treatment with 5′ phosphate-dependent exonuclease, which results in the depletion of all monophosphorylated transcripts. it has been shown that tss identification based on drna-seq data is superior to an estimation of transcript boundaries based on whole transcriptome rna-seq reads. the differential sequencing of samples l-i to l-v resulted in, reads. a total of putative tss was predicted, of which were detected in at least two samples. a comparison of the latter with the transcript boundaries obtained by whole transcriptome sequencing shows that identified tss confirm the rna-seq data, whereas the other findings introduce tss not detectable by conventional rna-seq. to allow the assignment of the identified tss to their putative origin, an allocation to four different classes was accomplished. naturally, the affiliation of tss according to this schema is ambiguous as some tss sort to multiple classes, e. g. some tss are located in a promoter region and within the upstream gene as well. the distribution of the identified tss to each class is shown in figure 2b. tss were detected in promoter regions, genes are bearing more than one putative tss in this region. the drna-seq data enabled conclusions for tss determination in cases in which read-through transcription of the upstream gene caused by leaky termination prohibits the identification of downstream tss by conventional rna-seq data. of the identified intragenic tss, are not located in the 500 bp promoter region of the downstream gene, reflecting a high number of putative internal promoters. orphan tss may indicate potential start sites of yet unknown genes or non-coding rnas, this is supported by the finding that of the detected orphan tss could be allocated to identified ncrnas. operon prediction based on rna-seq and drna-seq data resulted in putative operons structuring the genome of b. licheniformis. while most operons are monocistronic or bicistronic, seven operons seem to encompass more than ten genes. this small number of long operons is not in accordance with the operon prediction made by kristoffersen et al. for b. cereus. the difference is due to the varying operon concept employed here. especially the consideration of internal tss in combination with distinct shifts of expression resulted in an increase of shortened operons in this study. reannotation the first genome annotation of b. licheniformis dsm has been published in. it has been shown previously that mapping of rna-seq data to genomes allows the correction of open reading frames and supports the identification of not-annotated protein genes. therefore, we performed a complete reannotation of the genome in order to integrate the rna-seq data provided by this study as well as the progress in gene prediction and annotation of the recent years. distinct transcription start sites determined by drna-seq and rna-seq-based whole transcriptome data have been used to identify putative mis-annotated genes. these findings were validated by length comparisons to genes deposited in public databases and confirmation of ribosomal binding sites and - and - promoter regions. this approach enabled the correction of reading frames of protein genes, pseudo genes, rrna genes and two trna genes. moreover, previously not-annotated protein genes were identified based on transcriptional activity and protein conservation. genes were removed from the annotation as these previously predicted orfs could not be verified by detailed genome analysis and comparisons to public databases. in total, the reannotation approach resulted in a dataset containing orfs. comparisons to the annotation of b. licheniformis dsm by rey et al. showed that of the newly annotated genes have not been described for this organism before. of the removed genes were annotated in both genomes. more than gene annotations have been improved. these improvements mainly comprise former hypothetical proteins now assigned to a function and proteins with altered gene symbols. in addition to gene-associated improvements, seven genomic regions were identified as prophage regions based on gc content deviations, significant similarities to known prophage genes and the presence of insertion repeats. the transcriptional activity of the prophage regions was rather low, which is consistent with the observation that many prophages are induced during sos response, which should not occur within a fermentation process. 5′ and 3′untranslated regions in this study, 5′untranslated regions with a mean length of nt could be identified. thirty of these 5′utrs are shorter than nt, implicating that leaderless transcription, commonly found in many bacteria, is not an abundant mechanism in b. licheniformis. correspondingly, low occurrence of leaderless transcription has also been suggested for other members of the phylum firmicutes. the most strongly transcribed 5’utrs ≥ nt and the 5’utrs discussed in the following passage are listed in table  cis *. at sampling points i, ii, and iii, the gene of the sporulation inhibitor kapd reveals a 5’utr of nt, whereas an alternative, drna-seq-supported 5’utr with a length of nt is present at the later stages of the fermentation process. the tss of both 5’utrs seem to be preceded by a σa recognition site. at sampling point iv, the transcriptional activity of the gene is higher than the activity of the 5’utr region. in b. subtilis, growth phase-dependent differentiation into subpopulations of distinct cell types with different gene expression patterns is well described. the divergent expression levels of kapd and the long 5’utr in b. licheniformis might therefore result from different usage of promoter sites dependent on the respective cell type. however, the observed effect could also derive from slow decay rates of the short form of the kapd mrna transcribed earlier. further 5’utrs exhibited antisense activity towards upstream genes, as shown for the untranslated region bli_r upstream of the glutamate synthase operon gltab. the observed 5’utr is completely antisense to the gene of the corresponding transcriptional activator gltc. the drna-seq data suggest the presence of only one tss. this finding might be an example for a regulatory linkage between adjacent genes localized on different strands. this concept has recently been termed the excludon by sesto et al., who demonstrated that long 5’utrs can act negatively on the transcription of the opposite gene. following this idea in the case of the glutamate synthase operon, the preceding 5’utr might establish a negative feedback regulation of the transcriptional activator gltc. a corresponding elongated utr of the gltab operon has not been found in b. subtilis, which indicates different regulations of glutamate homeostasis in the two species. next to regulatory effects based on antisense orientation, 5’utrs can bear intrinsic, so-called cis-regulatory elements. at the time of this study, cis-regulatory elements have been predicted for b. licheniformis dsm by covariance models. all elements have been shown to be transcriptionally active during the fermentation process, although some are not located in 5’utrs but in intergenic read-through regions. three new t-boxes, located upstream of the serine acetyltransferase gene cyse and the trna ligase subunit genes glyq and phes, could be identified by comparison to the rfam database. in b. subtilis, cis-regulatory elements have been described, comprising rna switches as well as protein-binding rnas. for of these instances, transcription could be shown at orthologous loci in b. licheniformis. 3’utrs with an average length of nt have been identified according to figure  the most strongly transcribed 3’utrs ≥ nt and the 3’utr discussed in this chapter are listed in table  of the identified 3’utrs, 42% exceed nt and 16% even exceed nt in length. in total, 3’utrs are localized antisense to adjacent genes. a detailed manual inspection revealed that all 3’untranslated regions longer than nt seem to be protruding after incomplete termination. altogether, 3’utrs with internal termination sites could be determined, whereas 3’utrs end at predicted termination sites. these findings suggest that the effect of fading-out at the end of operons due to imperfect termination might be a common effect in b. licheniformis. an example is the nt 3’utr downstream of the cell envelope stress response operon liaihgfsr. the mrna transcript of this operon protrudes beyond a termination signal, which is located directly behind the stop codon of liar. this protruding mrna sequence is antisense to the next four genes which comprise the germination receptor operon geraaabac and a hypothetical protein. a second terminator structure can be found nt upstream of the end of the transcript. *. non-coding rna features non-coding rnas were identified in non-coding regions of the chromosome, for example in intergenic regions or localized in antisense direction to protein genes. the boundaries of the identified transcripts were determined by upshifts or downshifts of transcriptional activity. all identified rna features were checked for similarities to complete protein genes as well as protein domains to ensure that they indeed represent non-coding rnas. to extract the basic types of ncrna expression profiles during the examined fermentation process, cluster analysis based on the k-means algorithm was applied to those ncrnas with highly reliable replicates. in total, clusters of divergent expression profiles were generated. cluster contains 36% of the applied ncrnas and 50% of all ncrnas > nt. it displays a strong up-shift of transcriptional activity at sampling point iv followed by a decrease at sampling point v. the high portion of transcripts in this cluster prompts the conclusion that rna-based regulation is especially important during the later stages of the fermentation process. other ncrnas exhibiting up-shifts of transcriptional activity are displayed in clusters to whereas clusters to include transcripts with activity down-shifts. the further clusters comprise ncrnas with expression shifts during the early fermentation process, as well as an activity up-shift at sampling point v in clusters to all assigned non-coding rnas were categorized according to the scheme displayed in figure  and subdivided into the classes a 5, a 3, a i, a misc and indep. selected ncrnas are listed in table  whereas an overview of all identified features is given in additional file 2: table s several ncrnas have been selected for validation by northern blotting. the analyzed ncrnas were chosen as they are exemplarily for their respective class. the occurrence of eight ncrnas could be verified, especially ncrnas < nt are in good accordance with the results gained by rna-seq. the results for transcripts > nt are indicative for rna degradation or processing and leaky transcription termination. however, three ncrnas could not be validated, which is most probably due to their low expression levels. misc misc misc misc i misc misc i misc misc misc 3'utr misc i misc *. indep ncrnas as depicted in figure  indep transcripts are defined as non-coding rnas not localized antisense to any mrna. instead they can be found in intergenic regions or any other position of the chromosome. in total, indep rnas with sizes between and nt have been identified, of which have tss verified by drna-seq. within this group five housekeeping srnas could be annotated: the tmrna ssra, the 6s rnas bsra and bsrb, the rna component of rnase p rnpb and the signal recognition particle scr. 87% of the indep transcripts exhibited npkm values ≥ in at least three samples, reflecting a strong transcriptional activity of the encoding genomics regions. for example the srna scr, an essential part of the protein secretion system, reaches a maximal npkm value of almost. this is in perfect accordance with the fact that the cells are derived from a fermentation process optimized for protein secretion. interestingly, indep ncrnas seem to be transcribed constitutively under the examined conditions, whereas only thirteen indep rnas show differential expression ), as illustrated exemplarily for bli_r. antisense ncrnas in contrast to the class of indep ncrnas, the antisense ncrnas a i, a 3, a 5, and a misc comprise non-coding transcripts localized antisense to annotated protein-coding genes. they either target the protein-coding region of a gene or the 5’ and 3’ untranslated regions. furthermore, ncrnas that target more than one gene or that are only partially antisense are classified. in this study, a misc rnas could be identified. approximately of them are located opposite to ribosome binding sites and could therefore function as inhibitors of translation, a very common mechanism of cis-encoded asrnas. the length distribution of the non-coding rna features is shown in figure 5c, and illustrates that 42% of the a misc rnas are less than 400 nt in length. twenty-seven of these short a misc rnas reach maximal npkm values ≥ suggesting putative srna mechanisms. however, some a misc rnas are much longer, i.e. bli_r is nt in length and spans six genes. the occurrence of antisense transcripts of such length is not unexpected, as asrnas with very diverse sizes, reaching more than nt, have been described for several species. furthermore, a i transcripts could be assigned, ranging in size from to nt. over 95% of the a i transcripts exhibit maximal npkm values ≤ 68% even ≤ due to the low coverage only tss could be verified by drna-seq for these asrnas. in total, non-coding asrnas were determined, comprising 89% of all identified non-coding rna transcripts and targeting 15% of all genes. the number of identified antisense ncrnas is in accordance to previous studies which assume that antisense transcription concerns ~ to 20% of the bacterial genes. antisense transcripts with putative impact on productivity the general aim of our group is the identification of productivity related features. thus, a special focus has been set on the identification of antisense transcripts with a putative impact on protease production as targets for strain improvement. the alkaline serine protease subtilisin carlsberg represents the major secreted feeding protease of b. licheniformis. thus, it competes for energetic and secretory resources with the production protease. we identified a cis-encoded nt a i asrna which is located at the 3’end of the apr mrna. a highly active tss determined from the drna-seq data and a terminator structure downstream of the adjacent gene yhfn confirm the characterization of the transcript as independently transcribed asrna. bli_r is highly expressed at all fermentation stages, whereas the transcriptional activity of the subtilisin carlsberg gene increases at the productive stages of the process. the presence of the cis-encoded asrna opposite to the 3’end of the target mrna resembles the b. subtilis rata/txpa toxin/antitoxin system or the escherichia coli gady/gadx system in which an antisense rna promotes either mrna degradation or stability. to elucidate the impact of the detected asrna, further analyses will be necessary, especially as a corresponding transcript is absent in the transcriptome of b. subtilis. further antisense transcripts against genes involved in cell differentiation, cell stress response, and thiamine and folate biosynthesis could be observed and are presented in additional file 1: figure s it is exciting to think about a regulatory impact of the mentioned ncrnas, but there are also some noteworthy limitations to putative effects. the completed rna-seq experiments cannot discern if the sense and the antisense transcript are transcribed in the same type of differentiated cells, which especially challenges stoichiometric estimations of asrnas and their mrna targets. whether they can influence each other or fulfill different purposes in different cell types has to be a topic of single cell targeted investigations. it has been reported that functional srnas are produced in excess amounts over the targeted mrna. therefore, a regulatory mechanism of poorly transcribed antisense rna cannot be assumed bona fide, but has to be evaluated carefully. nonetheless, our data implicate that there might be a biological function assignable to the rna features, especially when they are conserved within related species as b. subtilis. at last, it has to be experimentally excluded, especially for low abundant instances, that the found ncrnas originate from spurious transcriptional events, for instance driven by alternative sigma factors. comparative transcriptomics in total, we determined candidate non-coding rna transcripts, including antisense, as well as indep ncrnas. for synechocystis sp. pcc sinorhizobium meliloti and the archaea sulfolobus solfataricus p and methanosarcina mazei gö between and non-coding rnas per mb were identified, matching our result of ncrnas/mb. for b. subtilis, the close relative of b. licheniformis, nicolas et al. have found non-coding rna features in a tiling array-based, condition-dependent transcriptome study. the majority of these features are intergenic transcripts determined by promoter analysis, whereas only 32% are derived from independently transcribed rnas. in contrast, the majority of ncrnas identified in b. licheniformis are antisense rnas, transcribed independently from protein-coding genes. the identification of more antisense transcripts in b. licheniformis might be accounted to the reduced background noise in rna-seq in comparison to tiling arrays, which allows a better detection of low abundant transcripts. of the b. licheniformis ncrnas are located in regions with high sequence similarity to b. subtilis and ncrnas are encoded at the frontiers of conserved and not conserved regions of the two genomes. based on sequence similarity, only out of the, in total, ncrnas located in these regions seem to occur in the b. subtilis transcriptome, emphasizing the differences of the two closely related species. comparisons to two earlier b. subtilis transcriptome studies show similar low levels of accordance. however, as mentioned above, it is also possible that the identified antisense ncrnas partly derive from spurious transcription events, and hence do not introduce a species-specific effect. for b. subtilis, srnas have been validated experimentally. comparison to rfam and/or comparison of genomic locations facilitated the detection of eleven of these srnas in the transcriptome of b. licheniformis. these include, in addition to the mentioned five housekeeping srnas, two regulatory rnas with well-known function in b. subtilis: sr and rnaa. the other rnas found in b. licheniformis are bsri, csfg, surc and rsae. the b. subtilis srnas which could not be confirmed in b. licheniformis originate from loci with no conserved gene pattern in this organism and thus may contribute to the differences between the two species. jahn et al. described the toxin-antitoxin system bsrg/sr located in the spβ prophage region of b. subtilis. although b. licheniformis does not harbor a homolog of the spβ prophage, two distinct transcripts were found to encode peptides similar to the bsrg toxin. additionally, the transcriptional activity of the corresponding loci revealed pairs of overlapping transcripts from both strands as shown for the bsrg/sr type toxin-antitoxin system. therefore both newly identified orfs were annotated as bsrg-like peptides. furthermore, the antisense transcripts resemble the sr antitoxin, especially in stem loops sl sl and tsl directly antisense to the bsrg-encoding mrna.  the presented study generated substantial data on the transcriptional activity of b. licheniformis within five relevant growth stages of an industrial-oriented fermentation process. a detailed analysis of the transcriptome data enabled us to accomplish a high quality functional genome reannotation of b. licheniformis dsm. the integration of the reannotation and the transcriptionally active regions resulted in the identification and quantification of hundreds of rna based regulatory elements as well as protein encoding genes. in total, rna features have been sorted into ten functional classes. 5’utrs and 3’utrs as well as ncrnas and antisense intergenic read-through transcripts have been identified. a striking observation was the identification of rna features, which mapped antisense to annotated genomic features. notably antisense rna features have been found in each of the functional classes and include transcripts of a size range from to base pairs in length. we have identified both: constitutively as well as growth phase dependently expressed rna features. our data represent a solid amount of knowledge on regulatory elements which orchestrate the cellular activities of b. licheniformis during the succession of growth phases within a productive fermentation. to generate an overview of the functional diversity of the identified rna features, all instances have been screened against the rfam database. this approach resulted in hits to experimentally well characterized rna features known from b. subtilis and other relatives, as well as in a multitude of so far unknown rna features without any rfam hit. the knowledge on genes and regulatory rna features which are transcriptionally active during an industrial-oriented fermentation enables an excellent access to a rational strain design approach for the optimization of b. licheniformis as industrial workhorse. especially the regulatory features which represent differences to the model organism b. subtilis give new insights to the still open question what makes strains of the species b. licheniformis superior to b. subtilis strains in terms of protease production capacity in industrial applications. in the future it may be promising to correlate the transcriptional activity of the rna features to the corresponding protein expression patterns. 
regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. the identified sequences include the expected rna features like housekeeping srnas, metabolic riboswitches and rna switches well known from studies on bacillus subtilis as well as a multitude of completely new candidates for regulatory rnas. an unexpectedly high number of rna features are encoded antisense to annotated protein and rna genes, in addition to independently transcribed small rnas. the hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. the identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of bacillus licheniformis strains. rna features encoded by such active loci have been identified and sorted into ten functional classes. here we present an rna-seq based study considering selected phases of an industry-oriented fermentation of bacillus licheniformis. the genome of the type strain b. licheniformis dsm was completely reannotated using data obtained from rna-seq analyses and from public databases. these antisense transcripts contain molecules with a remarkable size range variation from to base pairs in length.
 the production of enzymes by an industrial strain requires a complex adaption of the bacterial metabolism to the conditions within the fermenter. regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. this complex network of genes is orchestrated by proteins as well as regulatory rna elements. here we present an rna-seq based study considering selected phases of an industry-oriented fermentation of bacillus licheniformis.  a detailed analysis of strand-specific rna-seq datasets revealed a multitude of transcriptionally active genomic regions. rna features encoded by such active loci have been identified and sorted into ten functional classes. the identified sequences include the expected rna features like housekeeping srnas, metabolic riboswitches and rna switches well known from studies on bacillus subtilis as well as a multitude of completely new candidates for regulatory rnas. an unexpectedly high number of rna features are encoded antisense to annotated protein and rna genes, in addition to independently transcribed small rnas. these antisense transcripts contain molecules with a remarkable size range variation from to base pairs in length. the genome of the type strain b. licheniformis dsm was completely reannotated using data obtained from rna-seq analyses and from public databases.  the hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. the identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of bacillus licheniformis strains. drna-seqrna-based regulationutrncrnasrnaantisense rnasubtilisintranscription start siteoperon predictionreannotation 