RNAseq analysis of global gene expression patterns in Corynebacterium glutamicum Adaptive Laboratory Evolution (ALE) strains GluA T5 and GluA T7 with accelerated growth and glutarate production.
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ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 1500, NextSeq 500
ORGANISM(S): Corynebacterium glutamicum ATCC 13032
SUBMITTER: Tobias Busche
PROVIDER: E-MTAB-10025 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The growth rate (µ) of microbes is a fundamental property influencing its production capacity. To identify the transcriptomic changes of Corynebacterium glutamicum ATCC13032 with increasing growth rate, three transitions, induced by different pre-culture conditions, were sampled in triplicate at growth rates ranging from 0.02 to 0.4 h-1. The pre-culture conditions differed in limiting substrate (phosphate, nitrogen, carbon) and the length of the stationary phase. Samples of 2 mL were withdrawn from the bioreactor in biological triplicates and immediately centrifuged at 20000 g for 30 seconds at 4 °C. The supernatant was discarded and the remaining cell pellet was immediately flash frozen in liquid nitrogen. Total RNA was isolated from three biological replicates using RNeasy Mini Kit along with a DNase Kit (both from Qiagen). Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries. The resulting cDNAs were sequenced paired end on an Illumina MiSeq system using 75 bp read length and on Illumina HiSeq 1500 system using 70 bp read length and 50 bp read length for one single sample. Through the comparison of these three datasets, each containing three biological replicates, the pre-condition independent gene expression changes could be deduced and the growth rate modulon was identified.
2019-04-01 | E-MTAB-7436 | biostudies-arrayexpress
Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in ï??hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 ± 0.03 gg-1 and a volumetric productivity of 0.04 ± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA. Two conditions tested, 200 mM NaCl Vs 200 mM pipecolic supplemented in the culture medium, control experiments done with the addition of 200mM of NaCl. Four technical replicates.
2016-03-30 | E-GEOD-79690 | biostudies-arrayexpress
Project description:Methanol is considered as an interesting carbon source in biobased microbial production processes. As Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies on the response of this organism to methanol. C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be up-regulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The deletion mutants M-oM-^AM-^DaldM-oM-^AM-^DadhE and M-oM-^AM-^DaldM-oM-^AM-^DmshC were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF recently identified by us. Similar to ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogeneous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway. Whole-genome DNA microarray analyses were performed to monitor changes in the global gene expression of C. glutamicum wild type in response to the presence of methanol.
2013-09-27 | E-GEOD-49936 | biostudies-arrayexpress
Project description:We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study ChrSA was shown to be crucial for heme resistance of C. glutamicum by activation of the putative heme-detoxifying ABC-transporter HrtBA in the presence of heme. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards heme. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA provided evidence for ChrA being a repressor of the heme biosynthesis operon hemAC and hemH and an activator of the ctaE-qcrCAB operon, encoding subunits of the cytochrome bc1-aa3 supercomplex of the respiratory chain. The heme oxygenase gene, hmuO, showed a strongly decreased mRNA level in the M-NM-^TchrSA mutant, but no significant binding of ChrA was observed in vitro. Promoter fusion studies of PchrSA with eyfp indicated positive autoregulation of the chrSA operon in the presence of heme. Interestingly, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralog response regulator, suggesting a close link between HrrSA and ChrSA. Mutational analysis and in silico prediction resulted in the deduction of a 16-bp weakly conserved inverted repeat as consensus DNA-binding motif of ChrA. Altogether, the present study emphasizes ChrSA as a second TCS, besides HrrSA, involved in heme-dependent gene regulation in C. glutamicum. To identify the influence of ChrSA on global gene expression , DNA microarray analyses were performed with the M-NM-^TchrSA mutant compared to C. glutamicum wild type. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII minimal medium with 4% glucose and either 2.5 M-BM-5M FeSO4 or 2.5 M-BM-5M hemin as iron source. Three biological replicates were performed.
2012-10-31 | E-GEOD-37327 | biostudies-arrayexpress
Project description:Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu2+ was studied using DNA microarrays and revealed 26 genes that showed a ≥3-fold increased mRNA level, including cg3280-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase and a copper-transport ATPase. In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3280 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress. Four or five biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown with 1.25 µM or with 21.25 µM CuSO4; Exp. 2: WT vs. copRS deletion mutant grown with 21.25 µM CuSO4; For analysis via DNA microarrays, the RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 1.25 uM CuSO4 or 21.25 uM CuSO4.
2011-09-04 | E-GEOD-27510 | biostudies-arrayexpress
Project description:Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing all annotated sigma factor genes on C. glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. HPLC analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA Microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes of the pentose phosphate pathway and for enzymes dependent on FMN, FAD or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. Endogenous sigma factor gene, sigH, was overexpressed in C. glutamicum ATCC13032 from IPTG inducible vector, pEKEx3. Two different concentration of IPTG (10 μM and 15 μM) was used for induction of SigH expression.
2015-06-20 | E-GEOD-70017 | biostudies-arrayexpress
Project description:In this study, disruption and overexpression of sigD were performed in Corynebacterium glutamicum and analyzed by transcriptome sequencing (RNA-seq) to understand the SigD regulon in C. glutamicum. For the effect of sigD overexpression, the relative abundance of mRNA was compared in WT(pVWEx1-sigD) without IPTG or with 50 M of IPTG. For the effect of sigD disruption, the abundance was compared between the sigD disrupted mutant and the wild type strain.
2017-01-27 | E-MTAB-5411 | biostudies-arrayexpress
Project description:Comparative transcriptome analyses revealed 69 genes exhibiting ?2-fold mRNA level changes in C. glutamicum DfkpA.Overall, 34 genes exhibited ?2-fold increased and 35 genes at least 0.5-fold decreased mRNA levels in JVO1 DfkpA. About half of these genes (32) encode for hypothetical/putative proteins. LdhA encoding L-lactate dehydrogenase exhibited by far the strongest increased mRNA level (8.5-fold) in the absence of FkpA. Determination of the respective specific LdhA activities revealed about 2-fold increased activity in JVO1 DfkpA (0.73 ± 0.05 U/mg) compared to the reference JVO1 (0.41 ± 0.02 U/mg). Notably, the two short gene lists include four genes encoding transcriptional regulators exhibiting increased mRNA levels (farR, lexA, divS, znr) and two exhibiting decreased mRNA levels (ramB, mmpLR) in the absence of FkpA under the conditions tested. The mRNA level of cysK was increased (2.05-fold) which is repressed by RamB whose mRNA level was decreased (0.27-fold). In contrast, the mRNA level of RamB-repressed ald was decreased (0.31-fold) and not increased. To identify the influence of PPIase FkpA on global gene expression, DNA microarray analyses were performed with a ?fkpA mutant compared to the reference C. glutamicum JVO1. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII minimal medium with 4% glucose at increased temperature (35°C) above growth optimum of C. glutamicum. Two biological replicates were performed with each representing an intra-array duplicate.
2015-09-08 | E-GEOD-65294 | biostudies-arrayexpress
Project description:DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase (PEPCk), led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2 and IolR. Determination of the PEPCk activity of the deletion mutants M-NM-^TramA, M-NM-^TgntR1M-NM-^TgntR2, and M-NM-^TiolR indicated that RamA represses pck during growth on glucose about twofold, whereas GntR1, GntR2, and IolR activate pck expression about twofold, irrespective whether glucose or acetate served as carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in divergent orientation to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the M-NM-^TiolR mutant and the parental wild-type revealed strongly elevated (>100-fold) mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT and iolR itself, which presumably is subject to negative autoregulation. A consensus DNA-binding motif was identified (5M-bM-^@M-^Y-KGWCHTRACA-3M-bM-^@M-^Y) which corresponds well to those of other GntR-type regulators of the HutC family. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism. To identify genes that are potentially regulated by IolR, the transcriptome profile of the iolR deletion strain was compared to that of the WT using DNA microarray analysis. The strains were grown in CGXII minimal medium with 4% (wt/vol) glucose as sole carbon source and RNA was isolated from cells harvested in the early exponential growth phase (OD600 of about 5). The transcriptome comparison was performed in triplicate starting from independent cultures. The second replicate included a dye-swap.
2013-07-29 | E-GEOD-44812 | biostudies-arrayexpress
Project description:Corynebacterium glutamicum, a gram-positive soil bacterium used for the industrial production of amino acids such as L-glutamate and L-lysine, is able to use a number of different nitrogen sources, such as ammonium, urea, or creatinine. In this communication, we show that L-glutamine serves as an excellent nitrogen source for C. glutamicum and allows similar growth rates in glucose minimal medium as ammonium. A transcriptome comparison revealed a strong induction of the nitrogen starvation response when glutamine was used as nitrogen source. Subsequent growth experiments with a variety of mutants defective in nitrogen metabolism showed that glutamate synthase is crucial for glutamine utilization, while a putative glutaminase is dispensable under the experimental conditions used. The fact that the glutamate synthase encoding gltBD operon is under strict nitrogen control explains the necessity for induction of the nitrogen starvation response. The paradox situation that the nitrogen starvation response is induced although intracellular L-glutamine levels are high has implications on nitrogen sensing. In contrast to other gram-positive and gram-negative bacteria such as Bacillus subtilis, Salmonella typhimurium, and Klebsiella pneumoniae, a drop in glutamine concentration obviously does not serve as a nitrogen starvation signal in C. glutamicum. Three biological replicates were performed. To analyse how L-glutamine influences global gene expression when used as sole nitrogen source instead of ammonium, DNA microarray analyses were performed. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either L-glutamine or ammonium sulphate as nitrogen source.
2010-10-05 | E-GEOD-19779 | biostudies-arrayexpress