Abstract
BackgroundThe use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. The aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism Corynebacterium glutamicum by RNAseq in order to improve its genome annotation and to describe important features for transcription and translation.ResultsRNAseq data sets were obtained by two methods, one that focuses on 5′-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3′-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5′-UTRs (untranslated regions) for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. Interestingly, a high number of mRNAs (33%) is transcribed as leaderless transcripts. From the data, consensus promoter and ribosome binding site (RBS) motifs were identified and it was shown that the majority of genes in C. glutamicum are transcribed monocistronically, but operons containing up to 16 genes are also present.ConclusionsThe comprehensive transcriptome map of C. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements (e.g. promoter regions, gene starts and stops, 5′-UTRs, RBSs, transcript starts and ends) and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. The methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future.
Highlights
The use of High-throughput RNA sequencing (RNAseq) to resolve the transcriptional organization of an organism was established in recent years and showed the complexity and dynamics of bacterial transcriptomes
In this study we describe an improved RNAseq method that provides a strand-specific characterization of entire transcriptomes at a whole genome level using high-throughput sequencing
Development of native 5′-end and whole transcript RNAseq protocols To analyze the whole transcriptome as well as the native transcription start sites of C. glutamicum, i.e. those that originate from initiation of transcription by RNA polymerase, a whole transcriptome RNAseq protocol (Figure 1a) and a native 5′-end RNAseq protocol were developed (Figure 1b), adapting the differential RNA-seq approach [15]
Summary
The use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and showed the complexity and dynamics of bacterial transcriptomes. Corynebacterium glutamicum is a non-pathogenic, nonsporulating, gram-positive soil bacterium that belongs to the order Actinomycetales This microorganism is widely used for the production of various amino acids and other industrially relevant compounds [1,2]. Beside the classical approaches for profiling transcripts like Northern blots, reverse-transcriptase (q)PCR, RACE (rapid amplification of cDNA ends), and microarrays, the recent development of RNAseq has revolutionized transcriptomics. This allows to analyze transcriptomes in a completely comprehensive way and with singlenucleotide resolution [12,13]. The features of RNAseq that are unmatched by the classical approaches, i.e. no background or saturation effects as in fluorescence-based detection, no cross-hybridization, and an almost unrestricted dynamic range of detection, make RNAseq an attractive approach to analyze the entire transcriptome quantitatively [12]
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