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Background: Accurate bacterial genome annotations provide a framework to understanding cellular functions, behavior and pathogenicity and are essential for metabolic engineering. Annotations based only on in silico predictions are inaccurate, particularly for large, high G + C content genomes due to the lack of similarities in gene length and gene organization to model organisms., Results: Here we describe a 2D systems biology driven re-annotation of the Saccharopolyspora erythraea genome using proteogenomics, a genome-scale metabolic reconstruction, RNA-sequencing and small-RNA-sequencing. We observed transcription of more than 300 intergenic regions, detected 59 peptides in intergenic regions, confirmed 164 open reading frames previously annotated as hypothetical proteins and reassigned function to open reading frames using the genome-scale metabolic reconstruction. Finally, we present a novel way of mapping ribosomal binding sites across the genome by sequencing small RNAs., Conclusions: The work presented here describes a novel framework for annotation of the Saccharopolyspora erythraea genome. Based on experimental observations, the 2D annotation framework greatly reduces errors that are commonly made when annotating large-high G + C content genomes using computational prediction algorithms.

Background: 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., Results: 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., Conclusions: 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.