Transcriptome analyses of the Corynebacterium glutamicum strains adapted to supraoptimal growth temperatures
ABSTRACT: C. glutamicum strains adapted to higher growth temperatures were obtained through an adaptive laboratory evolution experiment. To elucidate molecular basis for thermotolerance acquired by the evolved strains, we examined transcriptional responses of the evolved and parental strains to thermal stress using microarray technology. Each strain was grown at the optimal growth temperature (33 ℃). When OD610 reached around 7, the growth temperature increased to 41 ℃, and transcriptional changes of the evolved and parental strains were examined with microarray technology.
Project description:The transcriptional regulator GntR1 downregulates the genes for gluconate catabolism and pentose phosphate pathway in Corynebacterium glutamicum. Gluconate lowers the DNA binding affinity of GntR1, which is probably the mechanism of gluconate-dependent induction of these genes. In addition, GntR1 positively regulates the ptsG, a gene encoding for a major glucose transporter, and the pck, a gene encoding phosphoenolpyruvate carboxykinase. Here, we searched for the new target of GntR1 at genome-wide scale by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analysis. This analysis identified 56 in vivo GntR1 binding sites, of which 7 sites were previously reported. The newly identified GntR1 sites include the upstream regions of carbon metabolism genes such as pyk, maeB, gapB, and icd. Binding of GntR1 to the promoter region of these genes was confirmed by electrophoretic mobility shift assay. The activity of the icd, gapB, and maeB promoters were reduced by the mutation at GntR1 binding site in contrast to the pyk promoter activity increased, indicating that GntR1 is a transcriptional activator of icd, gapB, and maeB and is a repressor of pyk. Thus, it is likely that GntR1 stimulates glucose uptake by inducing the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) gene while repressing pyk to increase PEP availability in the absence of gluconate. Repression of zwf and gnd may reduce NADPH supply which may be compensated by the induction of maeB, and icd. Upregulation of icd, gapB, and maeB and downregulation of pyk by GntR1 probably supports gluconeogenesis. Gene expression profile of the wild type at the exponential phase was compared with that of the rshA deletion mutant. Three independent experiments were performed.
Project description:Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. To this end, we used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 ºC. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. Thus we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms. The first aim of this study was to assess the most competitive strains that grow under wine fermentation conditions at low temperature. To this end, we performed a growth competition assay with 27 commercial wine strains inoculated at equal population size in synthetic grape must. In spite of the economical and industrial importance of these strains, their phenotypic variation in the main enological traits, particularly those related to optimum growth temperature, and their ability to adapt to low temperature fermentation have been poorly investigated. The second goal was to obtain an improved strain to grow and ferment at low temperature by evolutionary engineering. For this purpose, we maintained growth competition in synthetic grape must during 200 generations to select for the mutations that produce phenotypes with improved growth in this medium. One of these evolved cultures was previously treated with ethyl methanesulfonate (EMS) to increase the mutation rate. Finally, we aimed to decipher the molecular basis underlying this improvement by analyzing the genomic and transcriptional differences between the parental strain and the strain evolved at low temperature.
Project description:A population of Saccharomyces cerevisiae was cultured for approximately 450 generations in the presence of high glucose to select for genetic variants. This experiment allows for a controlled model of adaptive evolution under natural selection. Using the parental strain BY4741 as the starting population, an evolved culture was obtained after continuous aerobic growth in a glucose-high medium for approximately 450 generations. After the evolution period three single colony isolates were selected for analysis. Next-generation Ion Torrent sequencing was used to evaluate genetic changes. Greater than 100 deletion/insertion changes were found with approximately half of these effecting genes. Additionally, over 180 single-nucleotide polymorphisms (SNPs) were identified with more than one quarter of these resulting in a non-synonymous mutation. Affymetrix DNA microarrays and RNseq analysis were used to determine differences in gene expression in the evolved strains compared to the parental strain. It was established that approximately 900 genes demonstrated significantly altered expression in the evolved strains relative to the parental strain. Many of these genes showed similar alterations in their expression in all three evolved strains. Interestingly, genes with altered expression in the three evolved strains included genes with a role in the TCA cycle. Overall these results are consistent with the physiological observations of decreased ethanol production and suggest that the underlying metabolism switched from fermentation to respiration during aerobic growth. Overall design: Yeast strains were grown in the presence of high glucose for approximately 450 generations. Individual isolates were then grown and total RNA was collected to compare difference between the evolved strains and the parental strain.
Project description:A population of Saccharomyces cerevisiae was cultured for approximately 450 generations in the presence of high glucose to select for genetic variants. This experiment allows for a controlled model of adaptive evolution under natural selection. Using the parental strain BY4741 as the starting population, an evolved culture was obtained after continuous aerobic growth in a glucose-high medium for approximately 450 generations. After the evolution period three single colony isolates were selected for analysis. Next-generation Ion Torrent sequencing was used to evaluate genetic changes. Greater than 100 deletion/insertion changes were found with approximately half of these effecting genes. Additionally, over 180 single-nucleotide polymorphisms (SNPs) were identified with more than one quarter of these resulting in a non-synonymous mutation. Affymetrix DNA microarrays and RNseq analysis were used to determine differences in gene expression in the evolved strains compared to the parental strain. It was established that approximately 900 genes demonstrated significantly altered expression in the evolved strains relative to the parental strain. Many of these genes showed similar alterations in their expression in all three evolved strains. Interestingly, genes with altered expression in the three evolved strains included genes with a role in the TCA cycle. Overall these results are consistent with the physiological observations of decreased ethanol production and suggest that the underlying metabolism switched from fermentation to respiration during aerobic growth. Yeast strains were grown in the presence of high glucose for approximately 450 generations. Individual isolates were then grown and total RNA was collected to compare difference between the evolved strains and the parental strain.
Project description:Bacteria modify expression of different types of terminal oxidase in response to oxygen availability. Corynebacterium glutamicum, a facultative anaerobic bacterium in Actinobacteria, possesses aa3-type cytochrome c oxidase and cytochrome bd-type quinol oxidase, the latter of which is induced upon oxygen limitation. We report here that an extracytoplasmic function sigma factor, SigC, is unprecedentedly responsible for the regulation. Chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analysis detected eight SigC-binding regions in the genome, leading to identification of a consensus promoter sequence for SigC recognition. The promoter sequences were found upstream of genes for cytochrome bd, heme a synthesis enzymes, and uncharacterized membrane proteins, all of which were upregulated by sigC overexpression. In contrast, that found on the antisense strand upstream of an operon encoding the cytochrome bc1 complex conferred a SigC-dependent negative effect on the operon expression. The SigC regulon was induced by cytochrome aa3 deficiency without modification of expression of sigC itself, but not by deficiency of the bc1 complex. These findings suggest that SigC is activated in response to impairment of electron transfer via cytochrome aa3, not directly to shift in oxygen levels. Our results provide a novel paradigm for transcriptional regulation of the aerobic respiratory system in bacteria. Gene expression profile of the wild type at the exponential phase was compared with that of the sigC deletion mutant. Two indepent experiments using two independent mutants were performed.
Project description:To determine potential roles of three transcript factors (Rbf1p, Hfl1p and Dpb4p) in cell metabolic activities and other cellular bioprocesses, we have performed genomic microarray in each gene knockout strain and transcription profiles were compared to its parental strain SN250 The total RNAs were extracted from exponential growth of null mutants and SN250, then used to synthesize cDNA for microarray assays in Aglient array that contains 6101 genes in duplicate
Project description:Analysis of evolved changes in transcriptional plasticity and parental effects on plasticity induced by mild heat stress in the nematode Caenorhabditis remanei. Results of this study highlight the importance of the broad environmental context of an organism and its influence on phenotypic plasticity, parental effects, and evolutionary responses. mRNA profiles of ancestral and two experimentally evolved populations of C. remanei. Parents of the sampled worms were raised at either 20°C or 30°C, then the resulting embryos were divided and reared at either 20°C or 30°C prior to collection (as L1 larvae). 6 replicates/larval temperature for each population if the parents were raised at 20°C, and 2 replicates/larval temperature for each population if the parents were raised at 30°C.
Project description:Salicylic acid (SA) has long been implicated in plant responses to oxidative stress. A direct assessment of SA effects in planta has been difficult, as SA-overproducing Arabidopsis mutants are compromised in growth or other developmental processes. We now report that transgenic Populus expressing a bacterial bifunctional SA synthase accumulated two to three orders of magnitude more total SA than wild type without affecting growth. Microarray experiments were performed to gauge the transcriptional responses of young source leaves to SA manipulation and/or heat stress. Differentially expressed genes due to SA perturbation or temperature treatment were identified. Co-expression network analysis was performed to identify key driver genes in SA-modulated response. Wild-type and transgenic Populus expressing cytosolic Irp9 (Irp9 line I6), plastidic Irp9 (FD-Irp9 lines F10 and F52) or NahG (NahG line N31) genes were maintained in two identical growth chambers at normal (NT, 27°C/17°C, day/night) or high (HT, 35°C/25°C) temperatures. Young source leaves at leaf plastochron index (LPI) 5 were sampled one week after the start of heat stress. Two biological replicates were used.
Project description:The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (Δrnc mutant) showed an elongated cell shape, and presence of several lines on the cell surface, indicating a required of RNase III for maintaining normal cell morphology in C. glutamicum. The level of mraZ mRNA was increased, whereas cgR_1596 mRNA encoding a putative cell wall hydrolase and ftsEX mRNA were decreased in the Δrnc mutant. The half-life of mraZ mRNA was significantly prolonged in the Δrnc and the Δpnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3′-to-5′ exoribonuclease, PNPase. Northern hybridization and primer extension analysis revealed that the cleavage site for mraZ mRNA by RNase III is in the coding region. Overproduction of MraZ resulted in an elongated cell shape. The expression of ftsEX decreased while that of cgR_1596 unchanged in an MraZ-overexpressing strain. An electrophoretic mobility shift assay and a transcriptional reporter assay indicate that MraZ is a transcriptional repressor of ftsEX in C. glutamicum. These results indicate that RNase III is required for efficient expression of MraZ-dependent ftsEX and MraZ-independent cgR_1596. Gene expression profile of the wild type at the exponential phase was compared with that of the rnc mutant. Three indepent experiments were performed.
Project description:Abiotic stress causes disturbances in the cellular homeostasis. Re-adjustment of balance in carbon, nitrogen and phosphorus metabolism therefore plays a central role in stress adaptation. However, it is currently unknown which parts of the primary cell metabolism follow common patterns under different stress conditions and which represent specific responses. To address these questions, changes in transcriptome, metabolome and ionome were analyzed in maize source leaves from plants suffering low temperature, low nitrogen (N) and low phosphorus (P) stress. The selection of maize as study object provided data directly from an important crop species and the so far underexplored C4 metabolism. Growth retardation was comparable under all tested stress conditions. The only primary metabolic pathway responding similar to all stresses was nitrate assimilation, which was down-regulated. The largest group of commonly regulated transcripts followed the expression pattern: down under low temperature and low N, but up under low P. Several members of this transcript cluster could be connected to P metabolism and correlated negatively to different phosphate concentration in the leaf tissue. Accumulation of starch under low temperature and low N stress, but decrease in starch levels under low under low P conditions indicated that only low P treated leaves suffered carbon starvation. In conclusion, maize employs very different strategies for management of nitrogen and phosphorus metabolism under stress. While nitrate assimilation was regulated depending on demand by growth processes, phosphate concentrations changed depending on availability, thus building up reserves under excess conditions. Carbon and energy metabolism of the C4 maize leaves were particularly sensitive to P starvation. Responses of maize source leaves to low temperature, low nitrogen and low phosphorus conditions were tested in independent single-stress experiments. Seedlings were cultivated in pots containing nutrient-poor peat soil under the controlled conditions of a growth chamber. The plants were fertilized with modified Hoagland solutions, containing 15mM KNO3 and 0.5mM KH2PO4 for control conditions; for low N and low P treatment, the nutrient concentrations were reduced to 0.15mM KNO3 and 0.1mM KH2PO4, respectively. Low temperature treated plants were always supplied with control nutrient solution. Plants from the nitrogen and phosphorus experiment as well as the control temperature plants were exposed to 28°C during the day and 20°C during the night. Low temperature treatment was limited to the night period and was reduced to 4°C for the 10h dark period. Source leaf lamina were harvested at day 20 (low temperature experiment) or day 30 after start of germination (low nitrogen and low phosphorus experiment) for parallel analysis of transcriptome, metabolome and ion profiles. The molecular data is further supplemented by phenotypic characterization of the maize seedlings under investigation.