Identifizierung und Funktionsanalysen von kleinen RNAs in Corynebakterium glutamicum

Mentz A. Identifizierung und Funktionsanalysen von kleinen RNAs in Corynebakterium 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

RNA-protein correlation of liver toxicity markers in HepaRG cells

The liver is a main target organ for the toxicity of many different compounds. While in general, in vivo testing is still routinely used for assessing the hepatotoxic potential of test chemicals, the use of in vitro models offers advantages with regard to throughput, consumption of resources, and animal welfare aspects. Using the human hepatoma cell line HepaRG, we performed a comparative evaluation of a panel of hepatotoxicity marker mRNAs and proteins after exposure of the cells to 30 different pesticidal active compounds comprising herbizides, fungicides, insecticides, and others. The panel of hepatotoxicity markers included nuclear receptor target genes, key players of fatty acid and bile acid metabolism-related pathways, as well as recently identified biomarkers of drug-induced liver injury. Moreover, marker genes and proteins were identified, for example, S100P, ANXA10, CYP1A1, and CYP7A1. These markers respond with high sensitivity to stimulation with chemically diverse test compounds already at non-cytotoxic concentrations. The potency of the test compounds, determined as an overall parameter of their ability to deregulate marker expression in vitro, was very similar between the mRNA and protein levels. Thus, this study does not only characterize the response of human liver cells to 30 different pesticides but also demonstrates that hepatotoxicity testing in human HepaRG cells yields well comparable results at the mRNA and protein levels. Furthermore, robust hepatotoxicity marker genes and proteins were identified in HepaRG cells

RNA-protein correlation of liver toxicity markers in HepaRG cells

The liver is a main target organ for the toxicity of many different compounds. While in general, in vivo testing is still routinely used for assessing the hepatotoxic potential of test chemicals, the use of in vitro models offers advantages with regard to throughput, consumption of resources, and animal welfare aspects. Using the human hepatoma cell line HepaRG, we performed a comparative evaluation of a panel of hepatotoxicity marker mRNAs and proteins after exposure of the cells to 30 different pesticidal active compounds comprising herbizides, fungicides, insecticides, and others. The panel of hepatotoxicity markers included nuclear receptor target genes, key players of fatty acid and bile acid metabolism-related pathways, as well as recently identified biomarkers of drug-induced liver injury. Moreover, marker genes and proteins were identified, for example, S100P, ANXA10, CYP1A1, and CYP7A1. These markers respond with high sensitivity to stimulation with chemically diverse test compounds already at non-cytotoxic concentrations. The potency of the test compounds, determined as an overall parameter of their ability to deregulate marker expression in vitro, was very similar between the mRNA and protein levels. Thus, this study does not only characterize the response of human liver cells to 30 different pesticides but also demonstrates that hepatotoxicity testing in human HepaRG cells yields well comparable results at the mRNA and protein levels. Furthermore, robust hepatotoxicity marker genes and proteins were identified in HepaRG cells

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

Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis

Neshat A, Mentz A, Rückert C, Kalinowski J. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis. Journal of biotechnology. 2014;190:55-63.: The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts

Molecular Diagnostics in cutaneous T-Cell Lymphomas - Identification of new potentially therapy-relevant Biomarkers

Hain C, Cieslak C, Mentz A, Stranzenbach R, Kalinowski J, Stadler R. Molekulare Diagnostik bei kutanen T-Zell Lymphomen – Identifizierung neuer potentiell therapierelevanter Biomarke. JOURNAL DER DEUTSCHEN DERMATOLOGISCHEN GESELLSCHAFT. 2019;17(Suppl. 1):1

A targeted transcriptomic and proteomic approach to assess hepatotoxic mixture effects in vitro

Braeuning A, Lichtenstein D, Mentz A, et al. A targeted transcriptomic and proteomic approach to assess hepatotoxic mixture effects in vitro. In: NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY. Vol 392. Springer; 2019: S76-S77

A targeted transcriptomic and proteomic approach to assess hepatotoxic mixture effects in vitro

Braeuning A, Lichtenstein D, Mentz A, et al. A targeted transcriptomic and proteomic approach to assess hepatotoxic mixture effects in vitro. In: NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY. Vol 392. Springer; 2019: S76-S77

Mixture effects on toxicologically relevant liver proteins after pesticide treatment in hepaRG cells

Schmidt F, Steinhilber A, Mentz A, et al. Mixture effects on toxicologically relevant liver proteins after pesticide treatment in hepaRG cells. In: NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY. Vol 393. New york: Springer; 2020: 15