Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique

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.

Transcriptional Regulation of the Vanillate Utilization Genes ( vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

“…The whole C. glutamicum transcriptome was analyzed recently, describing promoters, transcripts, and operons in a mixed RNA sample harvested under different growth conditions (42). In this way, it was intended to be as comprehensive as possible, since many, if not all, transcripts should be represented by the data sets.…”

“…The promoter of the vanABK operon (P vanABK ) is negatively regulated by its specific repressor, VanR. The exact locations of the promoters of vanR and the vanABK operon were deduced from transcriptome sequencing experiments (42). It was interesting that vanR is transcribed leaderless.…”

“…Briefly, RNA samples isolated from cells grown under different cultivation conditions were mixed and treated to retain only transcripts with native 5= ends or a whole transcriptome consisting of native and processed transcripts. Generation of sequencing libraries and sequence analysis were described in detail in Pfeifer-Sancar et al (42). Due to sequencing-mixed RNA samples, the data sets generated identified nearly all transcription start points and operons without assigning certain transcription start points or transcripts to a specific culture condition.…”

“…Transcriptome sequencing (RNA-seq) data were taken from the study of Pfeifer-Sancar et al (42). Briefly, RNA samples isolated from cells grown under different cultivation conditions were mixed and treated to retain only transcripts with native 5= ends or a whole transcriptome consisting of native and processed transcripts.…”

ReadXplorer—visualization and analysis of mapped sequences

“…When using a suitable RNA-Seq protocol, such as the selective analysis of primary transcripts (Borries et al , 2012) or a 5′ RNA adapter ligation (Pfeifer-Sancar et al , 2013), the mapped reads can be analyzed for TSSs.…”