BACKGROUND
actinobacteria represent one of the most dominant and distinct bacterial phylum.
the evolutionary divergence of actinobacteria is an ancient event, reflected in their unique biology.
the majority of actinobacteria are found in soil, where they play a vital role in carbon cycling.
in human medicine, they comprise a large number of pathogens and provide a major source of antibiotics  <cit> .
they have a large, high gc content genome that equips them for growth in hostile, nutrient poor environments.
they are also able to synthesise secondary metabolites that have important commercial and medical significance  <cit> .
saccharopolyspora erythraea  is a model gram-positive filamentous actinomycete that has served for the study of antibiotic production for several decades.
the bacteria produces erythromycin a, the first macrolide antibiotic to be discovered, and the backbone for numerous modern antibiotics  <cit> .
s.erythraea exhibits a distinct and complex lifecycle, in which an initial growth phase is followed by a transition period, known as the metabolic switch, followed by a secondary growth phase.
like in streptomyces coelicolor , the switch in s.erythraea is followed by morphological changes that coincide with potential cell death and the transcription of secondary metabolite gene clusters  <cit> .
a detailed study of transcriptional activity during this transitional period is of major importance to understand the complex, yet little understood, life cycle of this sophisticated bacterial phylum.
studies have profiled actinomycete transcriptomes using microarrays  <cit> .
in fact all annotated coding sequences have been profiled using affymetrix arrays in s.coelicolor <cit> .
however, using long probes which are less likely to produce false negatives has proved challenging due to the high gc content of actinobacterial genomes.
the recent advent of rna sequencing provides a new opportunity to profile the global transcriptome of high gc content microorganisms.
here, we present a detailed transcriptional description of s.erythraea life cycle from in-depth rna sequencing.
our study shows that the major morphological changes observed at the metabolic switch are accompanied by an extreme transcriptional phenomenon, with the repression of large regions of the genome.
in addition, we characterise a restrictive bottleneck event mediated by targeted rnase activity that results in a wholesale and rapid global transcriptional rearrangement event.
RESULTS
rna sequencing shows pervasive transcription of the genome
like most actinobacteria, s.erythraea has a  <dig> % gc content genome which prevented us designing and performing microarray experiments that contained all orfs .
after performing microarray experiments in a control sample, we employed rna sequencing to profile the whole transcriptome.
we first assessed the ability of two alternative rna sequencing approaches, duplex-specific thermostable normalisation   <cit>  and microbexpress depletion  <cit> , to deplete rrna in gc-rich transcriptomes.
enriched mrna samples were compared to untreated matched reference controls .
we did not observe any systematic divergence in nucleotide composition of sequenced reads between treated and untreated libraries .
we observed a high correlation between gene abundance estimates by independent validation by qrt-pcr and the expression of low gc content genes represented in the microarray , thereby confirming the fidelity of both strategies to profile gc rich transcriptomes.
prior to harvesting rna, several fermentations were performed to carefully characterize the metabolic switch in bioreactors.
we determined that rna harvested at seven time-points,  would accurately describe the entire genetic program .
we prepared unstranded rna libraries  <cit>  and sequenced  <dig> million reads for a total of  <dig>  gb and a mean ~ <dig>  fold coverage of the genome .
a replicate fermentation was sampled across the metabolic switch .
the replicate samples were used to prepare strand-specific libraries that were sequenced to produce  <dig>  million alignable reads and an additional  <dig>  gb of sequencing .
our comprehensive sequencing showed the s.erythraea genome to be prevalently transcribed, with at least  <dig> % of genome sequence from either strand being represented .
this is similar to recent studies in yeast and other eukaryotes that show pervasive transcription of complex genomes that express intergenic and noncoding rna  <cit> .
furthermore, given less than  <dig> % of expressed nucleotides were represented singly suggests that close to saturating sequencing coverage of the transcriptome was attained, thereby permitting us to detect rare and transient transcriptional events.
the transcription of genomic macro-regions alternates at the switch
we next applied rna sequencing to resolve large-scale topological features of the s.erythraea transcriptome.
our sequencing clearly illustrates the strand-specific enrichment for transcription from the leading strand, consistent with co-directional transcription of essential and highly expressed genes with replication  <cit>  .
like other actinomycetes, the genome is divided into two large regions, with a core region thought to contain the majority of genes essential for survival, and a non-core region enriched for genes for conditionally adaptive functions  <cit> .
alignment of sequenced reads across these regions allowed the resolution of genome-scale, contiguous transcriptional regions associated with these core/non-core regions .
these macro-regions undergo alternative enrichment across the switch, with marked repression of the non-core transcriptional loci following the switch .
the borders of the core/non-core macro regions are delineated by transposable sequences   <cit> .
given the capacity for rna sequencing to resolve the expression of repetitive sequences, we were able to detect a notably strong and specific induction  of paralogous transposon clusters at these region borders, coincident with the shifting of non-core to core transcriptional regions.
these borders orientate the transcriptional regions perpendicular to the strand-specific enrichment for leading/lagging strands .
when superimposed, these regions dissect the genome into four distinct quarters; with each region containing distinctly co-expressed and functionally related gene families .
collectively this demonstrates a broad regulatory context provided by contiguous transcriptional macro-regions for the functional organisation and alternate expression of genes across the metabolic switch.
the metabolic switch defines a distinct gene expression program
the high temporal resolution and sequencing depth with which we profiled the growth cycle and metabolic switch using rna-seq , revealed considerable complexity in gene expression profiles.
although we detected transcription from all genes, for clarity we restricted our analysis to prominently expressed genes .
shifts to gene expression invoked at the switch were so broad as to affect  <dig> % of the genes that were profiled .
to delineate cogent trends within this complexity, we identified major gene groups that exhibited a normalised enrichment at exponential, transitional or stationary phases across the s.erythraea life cycle .
a comparative analysis delineated two distinct gene expression programs with diverging functional trends before and following the switch .
for example, the greater number of genes expressed prior to the switch were enriched for programs associated with central carbon metabolism, such as those involved in carbohydrate synthesis , cell wall biogenesis , energy production  and lipid metabolism .
in contrast, genes associated with secretion and transport , posttranslational modification  and ribosomal structure and biogenesis  were enriched after the switch.
similarly, the erythromycin gene cluster was enriched after the metabolic switch .
during the metabolic switch the greatest enrichment was for genes associated with signal transduction  and transcription regulation .
these alternate programs were reciprocally enriched for core/non-core and leading/lagging strand organisation , reflecting the overarching context these macro-regions provide for gene expression.
interestingly we found similar patterns of up or down regulation to previously reported homologous genes in closely related microorganisms  <cit> , suggesting that the transcriptional phenomenon described here comprise a characteristic event that punctuates and defines gene expression in related microorganisms .
rna sequencing facilitates operon annotation
by the contiguous transcription across shared and co-expressed genes, we looked at the predicted organization of the  <dig>  previously predicted operons that encompass  <dig>  genes  <cit> .
our de novo assembly using oases <cit> , from the strand specific rna sequencing after trimming all reads to  <dig> bases and assembling them using a  <dig> bases k-mer, resulted in  <dig>  contigs.
we found several of these contigs aligned with previously predicted operons .
moreover, using the strand specific sequencing, we found  <dig> % of total transcription to be antisense to these annotations, with an enrichment of antisense transcription at the 3’ end of the genes.
lastly, we employed a range of metrics, including presence, size and structure to annotate novel independent ncrnas within intergenic regions.
by combining small rna sequencing and long rna-seq data, we identified  <dig> putative ncrna from transcribed sections in intergenic genomic regions .
the novel ncrnas display distinct cpc scores  <cit> , dynamic transcriptional pattern and  <dig> of them displayed a distinct ncrna secondary structure   <cit> .
rna degradation mediates a transcriptional bottleneck event
the two alternative genetic programs are demarcated at the switch by a bottleneck event that is readily apparent in the transcriptome, with up to a  <dig> -fold suppression in gene counts for genes associated with translation .
this bottleneck event, happening at the metabolic switch, is aptly illustrated in the suppression of essential components to the transcriptional, translation and energy production machinery .
this group of genes persisting at the switch was enriched for transcriptional regulators , consistent with the major phenotypic changes that occur during this phase.
furthermore, the wide imbalance between the highest and lowest expressed genes  suggests the majority of mrnas to be present at less than one copy per cell, consistent with recent reports of heterogeneity between individual cell transcriptomes  <cit> .
this imbalance narrows during the bottleneck event, suggesting decreased diversity between individual cell transcriptomes.
this observation is in accordance with the previously described programmed cell death that occurs at the switch and involves expression of enzymes for cellular dismantling  <cit> .
notably, we observed that bioanalyzer traces from two independent fermentations showed degraded profiles at the switch .
furthermore, we observe the pronounced expression of ribonucleases and proteases at the inception of the switch ), including rnase e and rnase iii.
given the critical roles ascribed to these ribonucleases in regulating the half-life of developmental mrnas in related species  <cit> , in combination with specific rna depletion at the switch, we next considered the role of targeted rna degradation in the global rearrangement of the transcriptome.
degradation targets specific mrna classes
our analysis of rna integrity across the s.erythraea life cycle revealed extensive rna degradation coincident with the metabolic switch .
given that samples were treated with rna-later and that we obtained high rin numbers , it was unlikely that degradation could occurred post-extraction.
although the qiagen columns have less binding affinity for rnas smaller than  <dig> bp, it does not completely remove srnas  <cit> .
thus, to provide an insight into the degradation pattern, we isolated and sequenced these degraded rna fractions.
we firstly isolated fragments corresponding to 20-50nt in size, thereby depleting the preparation of small regulatory rnas that were greater than 50nt in length  <cit> .
we also reasoned that, unlike the 5’ monophosphate termini of a fragmented transcript, primary rna transcripts with a 5’ triphosphate would be refractory to library preparation and sequencing   <cit> .
for comparison we also sequenced matching fractions from before  and after  the switch, returning approximately  <dig> million sequenced small rnas .
we next omitted any reads that did not align in sense to transcribed regions from matched rna sequencing libraries, reasoning they may reflect bona-fide small rnas.
alignment of the remaining small rna fragments exhibit no apparent size or nucleotide enrichment to annotated coding genes previously resolved.
we next identified those mrnas subject to targeted degradation and turnover across the switch by comparing the ratio of full-length and fragmented reads that align to each transcript.
although we observed broadly accelerated mrna degradation at the switch , we noted this degradation to be relatively slower or faster for specific genes and functional classes and to often occur in concert with our previously described gene expression profiles.
for example, consistent with their enrichment at the switch, we found that while transcripts encoding transcription factors generally exhibit higher stability , some specific classes, such as xre transcription factors, known to regulate developmental life cycle and secondary metabolism  <cit> , undergo a cohesive and accelerated degradation .
this observation establishes that the degradation can be targeted to specific mrna classes, thereby contributing to the stage-specific enrichments observed across the s.erythraea growth cycle.
gene ontology analysis of s.erythraea genes most intensively targeted for destruction at the metabolic switch returned a large enrichment for transcripts associated with the translational process, including ribosomal genesis , ribosomal proteins  and other associated translational factors.
we detected the targeted degradation of additional functional gene networks, such as energy production and regulation, which undergo suppression at the bottleneck.
collectively, these trends demonstrate the contribution of targeted mrna degradation to the transcriptional bottleneck and the modulation of genetic programs subsequently deployed .
they also anticipate a pivotal and global role for ribonucleases, such as rnase e and iii, in regulating the metabolic switch  <cit> .
discussion
our analysis of the s.erythraea transcriptome describes a major transcriptional phenomenon occurring during fermentation in bioreactors.
a genome-wide transcriptional non-core region, delineated by genes of markedly lower gc content, sparse gene density and inverted repeats, is broadly repressed following the switch.
this region organization is present in related microorganisms, including some with linear genomes, such as the terminal regions of the s.coelicolor linear chromosome where analogous insertion elements mark the inner boundary of terminal inverted repeats  <cit> .
similar low gc regions have been reported in streptomyces ambofaciens, where these regions have been associated to genome plasticity and may be used to elucidate mechanisms of evolution  <cit> .
the presence of analogous transposable elements delineating the core/non-core regions is apparent in other related microorganisms anticipating a role in the maintenance and possible regulation of these macro-regions.
the alternating expression of these regions, previously observed in s.erythraea using microarrays  <cit> , is able to mediate a broad coordinated suite of genetic changes, representing a broad architectural mechanism by which a cell can rapidly modulate large changes in the transcriptome in response to environmental cues  <cit> .
this transcriptional reorganization is complemented by a targeted bottleneck event where mrnas are degraded.
the bottleneck event establishes that the degradation may be targeted to specific mrna classes, thereby contributing to the stage-specific enrichments following the s.erythraea growth cycle.
the importance of these degradation processes in global transcriptomic regulation has been recently dissected in yeast  <cit>  and individual cases described in s.coelicolor, whereby endoribonuclease activity of rnase iii has been shown to bind to specific transcript and to regulate antibiotic production  <cit> .
studies have also described the role of ppgpp in regulating mrna half-life in s.coelicolor by inhibiting pnpase activity which preserves mrna for crucial cellular processes  <cit> .
such widespread degradation may also indicate a role for programmed cell death of a distinct cellular subpopulation, in mediating the changes at the metabolic switch.
the new transcriptome observed after the switch may be reflective of the transcriptome of the surviving subpopulation.
collectively, these observations of cellular degradation and targeted mrna degradation supports the emergence of key, yet little studied, mechanism by which gene expression are regulated.
our results, which attained close to saturating sequencing coverage of the transcriptome, reveal unanticipated additional transcriptional complexity with almost one third of transcriptional content originating from unannotated sequences.
combining rna sequencing with sequencing of small rnas showed the importance of non-coding rnas in bacterial transcription.
CONCLUSIONS
like other related microorganisms, s.erythraea undergoes a distinct transitional switch that is characterized by growth arrest, morphological changes and the production of secondary metabolites.
we show this to be accompanied by a massive transcriptional modulation whose speed and scale, revealed here for the first time by rna sequencing, represents a significant and novel transcriptional phenomenon.
given similarities shared between the life cycle and genome organization with other microorganisms, we suggest that this transcriptional phenomenon may be a widespread feature across the large critically important bacterial group of industrial microorganisms.

 actinobacteria represent one of the most dominant and distinct bacterial phylum. the evolutionary divergence of actinobacteria is an ancient event, reflected in their unique biology. the majority of actinobacteria are found in soil, where they play a vital role in carbon cycling. in human medicine, they comprise a large number of pathogens and provide a major source of antibiotics. they have a large, high gc content genome that equips them for growth in hostile, nutrient poor environments. they are also able to synthesise secondary metabolites that have important commercial and medical significance. saccharopolyspora erythraea is a model gram-positive filamentous actinomycete that has served for the study of antibiotic production for several decades. the bacteria produces erythromycin a, the first macrolide antibiotic to be discovered, and the backbone for numerous modern antibiotics. s.erythraea exhibits a distinct and complex lifecycle, in which an initial growth phase is followed by a transition period, known as the metabolic switch, followed by a secondary growth phase. like in streptomyces coelicolor, the switch in s.erythraea is followed by morphological changes that coincide with potential cell death and the transcription of secondary metabolite gene clusters. a detailed study of transcriptional activity during this transitional period is of major importance to understand the complex, yet little understood, life cycle of this sophisticated bacterial phylum. studies have profiled actinomycete transcriptomes using microarrays. in fact all annotated coding sequences have been profiled using affymetrix arrays in s.coelicolor. however, using long probes which are less likely to produce false negatives has proved challenging due to the high gc content of actinobacterial genomes. the recent advent of rna sequencing provides a new opportunity to profile the global transcriptome of high gc content microorganisms. here, we present a detailed transcriptional description of s.erythraea life cycle from in-depth rna sequencing. our study shows that the major morphological changes observed at the metabolic switch are accompanied by an extreme transcriptional phenomenon, with the repression of large regions of the genome. in addition, we characterise a restrictive bottleneck event mediated by targeted rnase activity that results in a wholesale and rapid global transcriptional rearrangement event.  rna sequencing shows pervasive transcription of the genome like most actinobacteria, s.erythraea has a % gc content genome which prevented us designing and performing microarray experiments that contained all orfs. after performing microarray experiments in a control sample, we employed rna sequencing to profile the whole transcriptome. we first assessed the ability of two alternative rna sequencing approaches, duplex-specific thermostable normalisation and microbexpress depletion, to deplete rrna in gc-rich transcriptomes. enriched mrna samples were compared to untreated matched reference controls. we did not observe any systematic divergence in nucleotide composition of sequenced reads between treated and untreated libraries. we observed a high correlation between gene abundance estimates by independent validation by qrt-pcr and the expression of low gc content genes represented in the microarray, thereby confirming the fidelity of both strategies to profile gc rich transcriptomes. prior to harvesting rna, several fermentations were performed to carefully characterize the metabolic switch in bioreactors. we determined that rna harvested at seven time-points, would accurately describe the entire genetic program. we prepared unstranded rna libraries and sequenced million reads for a total of gb and a mean ~ fold coverage of the genome. a replicate fermentation was sampled across the metabolic switch. the replicate samples were used to prepare strand-specific libraries that were sequenced to produce million alignable reads and an additional gb of sequencing. our comprehensive sequencing showed the s.erythraea genome to be prevalently transcribed, with at least % of genome sequence from either strand being represented. this is similar to recent studies in yeast and other eukaryotes that show pervasive transcription of complex genomes that express intergenic and noncoding rna. furthermore, given less than % of expressed nucleotides were represented singly suggests that close to saturating sequencing coverage of the transcriptome was attained, thereby permitting us to detect rare and transient transcriptional events. the transcription of genomic macro-regions alternates at the switch we next applied rna sequencing to resolve large-scale topological features of the s.erythraea transcriptome. our sequencing clearly illustrates the strand-specific enrichment for transcription from the leading strand, consistent with co-directional transcription of essential and highly expressed genes with replication. like other actinomycetes, the genome is divided into two large regions, with a core region thought to contain the majority of genes essential for survival, and a non-core region enriched for genes for conditionally adaptive functions. alignment of sequenced reads across these regions allowed the resolution of genome-scale, contiguous transcriptional regions associated with these core/non-core regions. these macro-regions undergo alternative enrichment across the switch, with marked repression of the non-core transcriptional loci following the switch. the borders of the core/non-core macro regions are delineated by transposable sequences. given the capacity for rna sequencing to resolve the expression of repetitive sequences, we were able to detect a notably strong and specific induction of paralogous transposon clusters at these region borders, coincident with the shifting of non-core to core transcriptional regions. these borders orientate the transcriptional regions perpendicular to the strand-specific enrichment for leading/lagging strands. when superimposed, these regions dissect the genome into four distinct quarters; with each region containing distinctly co-expressed and functionally related gene families. collectively this demonstrates a broad regulatory context provided by contiguous transcriptional macro-regions for the functional organisation and alternate expression of genes across the metabolic switch. the metabolic switch defines a distinct gene expression program the high temporal resolution and sequencing depth with which we profiled the growth cycle and metabolic switch using rna-seq, revealed considerable complexity in gene expression profiles. although we detected transcription from all genes, for clarity we restricted our analysis to prominently expressed genes. shifts to gene expression invoked at the switch were so broad as to affect % of the genes that were profiled. to delineate cogent trends within this complexity, we identified major gene groups that exhibited a normalised enrichment at exponential, transitional or stationary phases across the s.erythraea life cycle. a comparative analysis delineated two distinct gene expression programs with diverging functional trends before and following the switch. for example, the greater number of genes expressed prior to the switch were enriched for programs associated with central carbon metabolism, such as those involved in carbohydrate synthesis, cell wall biogenesis, energy production and lipid metabolism. in contrast, genes associated with secretion and transport, posttranslational modification and ribosomal structure and biogenesis were enriched after the switch. similarly, the erythromycin gene cluster was enriched after the metabolic switch. during the metabolic switch the greatest enrichment was for genes associated with signal transduction and transcription regulation. these alternate programs were reciprocally enriched for core/non-core and leading/lagging strand organisation, reflecting the overarching context these macro-regions provide for gene expression. interestingly we found similar patterns of up or down regulation to previously reported homologous genes in closely related microorganisms, suggesting that the transcriptional phenomenon described here comprise a characteristic event that punctuates and defines gene expression in related microorganisms. rna sequencing facilitates operon annotation by the contiguous transcription across shared and co-expressed genes, we looked at the predicted organization of the previously predicted operons that encompass genes. our de novo assembly using oases, from the strand specific rna sequencing after trimming all reads to bases and assembling them using a bases k-mer, resulted in contigs. we found several of these contigs aligned with previously predicted operons. moreover, using the strand specific sequencing, we found % of total transcription to be antisense to these annotations, with an enrichment of antisense transcription at the 3’ end of the genes. lastly, we employed a range of metrics, including presence, size and structure to annotate novel independent ncrnas within intergenic regions. by combining small rna sequencing and long rna-seq data, we identified putative ncrna from transcribed sections in intergenic genomic regions. the novel ncrnas display distinct cpc scores, dynamic transcriptional pattern and of them displayed a distinct ncrna secondary structure. rna degradation mediates a transcriptional bottleneck event the two alternative genetic programs are demarcated at the switch by a bottleneck event that is readily apparent in the transcriptome, with up to a -fold suppression in gene counts for genes associated with translation. this bottleneck event, happening at the metabolic switch, is aptly illustrated in the suppression of essential components to the transcriptional, translation and energy production machinery. this group of genes persisting at the switch was enriched for transcriptional regulators, consistent with the major phenotypic changes that occur during this phase. furthermore, the wide imbalance between the highest and lowest expressed genes suggests the majority of mrnas to be present at less than one copy per cell, consistent with recent reports of heterogeneity between individual cell transcriptomes. this imbalance narrows during the bottleneck event, suggesting decreased diversity between individual cell transcriptomes. this observation is in accordance with the previously described programmed cell death that occurs at the switch and involves expression of enzymes for cellular dismantling. notably, we observed that bioanalyzer traces from two independent fermentations showed degraded profiles at the switch. furthermore, we observe the pronounced expression of ribonucleases and proteases at the inception of the switch ), including rnase e and rnase iii. given the critical roles ascribed to these ribonucleases in regulating the half-life of developmental mrnas in related species, in combination with specific rna depletion at the switch, we next considered the role of targeted rna degradation in the global rearrangement of the transcriptome. degradation targets specific mrna classes our analysis of rna integrity across the s.erythraea life cycle revealed extensive rna degradation coincident with the metabolic switch. given that samples were treated with rna-later and that we obtained high rin numbers, it was unlikely that degradation could occurred post-extraction. although the qiagen columns have less binding affinity for rnas smaller than bp, it does not completely remove srnas. thus, to provide an insight into the degradation pattern, we isolated and sequenced these degraded rna fractions. we firstly isolated fragments corresponding to 20-50nt in size, thereby depleting the preparation of small regulatory rnas that were greater than 50nt in length. we also reasoned that, unlike the 5’ monophosphate termini of a fragmented transcript, primary rna transcripts with a 5’ triphosphate would be refractory to library preparation and sequencing. for comparison we also sequenced matching fractions from before and after the switch, returning approximately million sequenced small rnas. we next omitted any reads that did not align in sense to transcribed regions from matched rna sequencing libraries, reasoning they may reflect bona-fide small rnas. alignment of the remaining small rna fragments exhibit no apparent size or nucleotide enrichment to annotated coding genes previously resolved. we next identified those mrnas subject to targeted degradation and turnover across the switch by comparing the ratio of full-length and fragmented reads that align to each transcript. although we observed broadly accelerated mrna degradation at the switch, we noted this degradation to be relatively slower or faster for specific genes and functional classes and to often occur in concert with our previously described gene expression profiles. for example, consistent with their enrichment at the switch, we found that while transcripts encoding transcription factors generally exhibit higher stability, some specific classes, such as xre transcription factors, known to regulate developmental life cycle and secondary metabolism, undergo a cohesive and accelerated degradation. this observation establishes that the degradation can be targeted to specific mrna classes, thereby contributing to the stage-specific enrichments observed across the s.erythraea growth cycle. gene ontology analysis of s.erythraea genes most intensively targeted for destruction at the metabolic switch returned a large enrichment for transcripts associated with the translational process, including ribosomal genesis, ribosomal proteins and other associated translational factors. we detected the targeted degradation of additional functional gene networks, such as energy production and regulation, which undergo suppression at the bottleneck. collectively, these trends demonstrate the contribution of targeted mrna degradation to the transcriptional bottleneck and the modulation of genetic programs subsequently deployed. they also anticipate a pivotal and global role for ribonucleases, such as rnase e and iii, in regulating the metabolic switch. discussion our analysis of the s.erythraea transcriptome describes a major transcriptional phenomenon occurring during fermentation in bioreactors. a genome-wide transcriptional non-core region, delineated by genes of markedly lower gc content, sparse gene density and inverted repeats, is broadly repressed following the switch. this region organization is present in related microorganisms, including some with linear genomes, such as the terminal regions of the s.coelicolor linear chromosome where analogous insertion elements mark the inner boundary of terminal inverted repeats. similar low gc regions have been reported in streptomyces ambofaciens, where these regions have been associated to genome plasticity and may be used to elucidate mechanisms of evolution. the presence of analogous transposable elements delineating the core/non-core regions is apparent in other related microorganisms anticipating a role in the maintenance and possible regulation of these macro-regions. the alternating expression of these regions, previously observed in s.erythraea using microarrays, is able to mediate a broad coordinated suite of genetic changes, representing a broad architectural mechanism by which a cell can rapidly modulate large changes in the transcriptome in response to environmental cues. this transcriptional reorganization is complemented by a targeted bottleneck event where mrnas are degraded. the bottleneck event establishes that the degradation may be targeted to specific mrna classes, thereby contributing to the stage-specific enrichments following the s.erythraea growth cycle. the importance of these degradation processes in global transcriptomic regulation has been recently dissected in yeast and individual cases described in s.coelicolor, whereby endoribonuclease activity of rnase iii has been shown to bind to specific transcript and to regulate antibiotic production. studies have also described the role of ppgpp in regulating mrna half-life in s.coelicolor by inhibiting pnpase activity which preserves mrna for crucial cellular processes. such widespread degradation may also indicate a role for programmed cell death of a distinct cellular subpopulation, in mediating the changes at the metabolic switch. the new transcriptome observed after the switch may be reflective of the transcriptome of the surviving subpopulation. collectively, these observations of cellular degradation and targeted mrna degradation supports the emergence of key, yet little studied, mechanism by which gene expression are regulated. our results, which attained close to saturating sequencing coverage of the transcriptome, reveal unanticipated additional transcriptional complexity with almost one third of transcriptional content originating from unannotated sequences. combining rna sequencing with sequencing of small rnas showed the importance of non-coding rnas in bacterial transcription.  like other related microorganisms, s.erythraea undergoes a distinct transitional switch that is characterized by growth arrest, morphological changes and the production of secondary metabolites. we show this to be accompanied by a massive transcriptional modulation whose speed and scale, revealed here for the first time by rna sequencing, represents a significant and novel transcriptional phenomenon. given similarities shared between the life cycle and genome organization with other microorganisms, we suggest that this transcriptional phenomenon may be a widespread feature across the large critically important bacterial group of industrial microorganisms. 
during the metabolic switch, global changes in gene expression influence the metabolic machinery of saccharopolyspora erythraea, resetting an entirely novel gene expression program. we showed that the metabolic switch is a sophisticated mechanism of transcriptional regulation capable of resetting and re-synchronizing gene expression programs at extraordinary speed and scale. our results, which attained close to saturating sequencing coverage of the transcriptome, revealed unanticipated transcriptional complexity with almost one third of transcriptional content originating from un-annotated sequences. the new transcriptional program is orchestrated by a bottleneck event during which mrna levels are severely restricted by targeted mrna degradation. consistent with other members of the actinobacterial phylum, saccharopolyspora erythraea undergo a transitional switch. we performed rna sequencing to analyze the transcriptional changes that occur during growth of saccharopolyspora erythraea in batch culture. this switch is characterized by numerous metabolic and morphological changes. paralogous transposon clusters, delineate these transcriptional programs.
 actinobacteria form a major bacterial phylum that includes numerous human pathogens. actinobacteria are primary contributors to carbon cycling and also represent a primary source of industrial high value products such as antibiotics and biopesticides. consistent with other members of the actinobacterial phylum, saccharopolyspora erythraea undergo a transitional switch. this switch is characterized by numerous metabolic and morphological changes.  we performed rna sequencing to analyze the transcriptional changes that occur during growth of saccharopolyspora erythraea in batch culture. by sequencing rna across the fermentation time course, at a mean coverage of 4000x, we found the vast majority of genes to be prominently expressed, showing that we attained close to saturating sequencing coverage of the transcriptome. during the metabolic switch, global changes in gene expression influence the metabolic machinery of saccharopolyspora erythraea, resetting an entirely novel gene expression program. after the switch, global changes include the broad repression of half the genes regulated by complex transcriptional mechanisms. paralogous transposon clusters, delineate these transcriptional programs. the new transcriptional program is orchestrated by a bottleneck event during which mrna levels are severely restricted by targeted mrna degradation.  our results, which attained close to saturating sequencing coverage of the transcriptome, revealed unanticipated transcriptional complexity with almost one third of transcriptional content originating from un-annotated sequences. we showed that the metabolic switch is a sophisticated mechanism of transcriptional regulation capable of resetting and re-synchronizing gene expression programs at extraordinary speed and scale. rna-sequencingactinobacteriasaccharopolyspora erythraeaerythromycinmetabolic switch 