Entwicklung der Transkriptomsequenzierung und Anwendung zur Analyse des Transkriptoms von Corynebacterium glutamicum

Pfeifer-Sancar K. Entwicklung der Transkriptomsequenzierung und Anwendung zur Analyse des Transkriptoms von Corynebacterium glutamicum. Bielefeld: Universität Bielefeld; 2014

Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032

Mentz A, Neshat A, Pfeifer-Sancar K, Pühler A, Rückert C, Kalinowski J. Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032. BMC Genomics. 2013;14(1): 714.BACKGROUND: Recent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis. RESULTS: In our RNA-Seq approach, total RNA from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical (heat shock, cold shock) or by chemical stresses (diamide, H2O2, NaCl) at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5[prime]- and 3[prime]-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found. CONCLUSIONS: The study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs

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

Pfeifer-Sancar K, Mentz A, Rückert C, Kalinowski J. Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique. BMC Genomics. 2013;14(1): 888.BACKGROUND: The 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. RESULTS: RNAseq data sets were obtained by two methods, one that focuses on 5[prime]-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3[prime]-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5[prime]-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. CONCLUSIONS: The 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[prime]-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

Genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete Corynebacterium glutamicum.

Albersmeier A, Pfeifer-Sancar K, Rückert C, Kalinowski J. Genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete Corynebacterium glutamicum. Journal of Biotechnology. 2017;257:99-109.The genome-wide identification of transcription start sites, enabled by high-throughput sequencing of a cDNA library enriched for native 5' transcript ends, is ideally suited for the analysis of promoters. Here, the transcriptome of Corynebacterium glutamicum, a non-pathogenic soil bacterium from the actinomycetes branch that is used in industry for the production of amino acids, was analysed by transcriptome sequencing of the 5'-ends of native transcripts. Total RNA samples were harvested from the exponential phase of growth, therefore the study mainly addressed promoters recognized by the main house-keeping sigma factor σA. The identification of 2454 transcription start sites (TSS) allowed the detailed analysis of most promoters recognized by σA and furthermore enabled us to form different promoter groups according to their location relative to protein-coding regions. These groups included leaderless transcripts (546 promoters), short-leadered (500 bases) transcripts (173) as well as intragenic (557) and antisense transcripts (261). All promoters and the individual groups were searched for information, e.g. conserved residues and promoter motifs, and general design features as well as group-specific preferences were identified. A purine was found highly favored as TSS, whereas the -1 position was dominated by pyrimidines. The spacer between TSS and -10 region were consistently 6-7 bases and the -10 promoter motif was generally visible, whereas a recognizable -35 region was only occurring in a smaller fraction of promoters (7.5%) and enriched for leadered and antisense transcripts but depleted for leaderless transcripts. Promoters showing an extended -10 region were especially frequent in case of non-canonical -10 motifs (45.5%). Two bases downstream of the -10 core region, a G was conserved, exceeding 40% abundance in most groups. This fraction reached 74.6% for a group of putative σB-dependent promoters, thus giving a hint to a specific property of these promoters. In addition, the high number of promoters analysed allowed finding of subtle signals only showing up significantly with this large set. This included the observation of a periodically changing A+T-content with maxima spaced by a full turn of the DNA helix. This periodic structure includes the A+T-rich UP-element of bacterial promoters known before but was found to extend up to -100, indicating hitherto unknown constraints influencing promoter architecture and possibly also promoter function

Identifizierung von Promotoren in Corynebacterium glutamicum

Busche T, Pfeifer-Sancar K, Rückert C, Kalinowski J. Identifizierung von Promotoren in Corynebacterium glutamicum. BIOspektrum. 2014;20(3):284-287