RESUMO
c-raf is a serine-threonine kinase and a downstream effector of ras signaling. This kinase plays an essential role in cell proliferation, differentiation, and apoptosis. In the past, we reported induction of c-raf gene expression in rat liver cancer on treatment with a mixture of aryl hydrocarbon receptor (AhR) agonists. This prompted our interest in investigating the role of AhR in the transcriptional regulation of c-raf. Initially, we cloned the rat c-raf promoter and sequenced the genomic DNA and cDNA by Southern blotting and capillary electrophoresis. Then, a genetic algorithm was applied to search for putative AhR-binding sites. DNA-binding activity of AhR was confirmed by electromobility shift assay. We also studied c-raf gene expression in rat hepatoma cell lines with functional and/or devoid AhR and in primary human and rat hepatocyte cultures. Overall, we identified five and three AhR-binding sites in the human and rat c-raf gene, respectively. Treatment of hepatocyte cultures with the AhR antagonist resveratrol reduced DNA binding of AhR. Only rat hepatoma cells with functional AhR responded to 1 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment with >10-fold c-raf mRNA induction. Treatment of human and rat hepatocyte cultures with various AhR-activating chemicals resulted in induction of c-raf gene expression, albeit at different levels. Taken collectively, we show AhR to be a master regulator of c-raf and propose cross-talk between AhR and the mitogen-activated protein kinase signaling pathway in chemically induced hepatocarcinogenesis.
Assuntos
Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-raf/genética , Receptor Cross-Talk , Receptores de Hidrocarboneto Arílico/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Dioxinas/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Dados de Sequência Molecular , Bifenilos Policlorados/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/agonistas , Alinhamento de Sequência , Transcrição Gênica/efeitos dos fármacosRESUMO
The influence of acetohydroxy acid synthase (AHAS) on L-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about twofold-lower K(m) for the substrate pyruvate and an about fourfold-lower V(max); (ii) a slightly increased K(m) for the substrate alpha-ketobutyrate with an about twofold-lower V(max); and (iii) insensitivity against the inhibitors L-valine, L-isoleucine, and L-leucine (10 mM each). Introduction of the modified AHAS into the L-lysine producers C. glutamicum DM1729 and DM1933 increased L-lysine formation by 43% (30 mM versus 21 mM) and 36% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity is linked to increased L-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to L-valine, L-isoleucine, and L-leucine auxotrophy and to further-improved L-lysine production. In batch fermentations, C. glutamicum DM1729 Delta ilvB produced about 85% more L-lysine (70 mM versus 38 mM) and showed an 85%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 Delta ilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the L-lysine biosynthetic pathway.
Assuntos
Acetolactato Sintase/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/metabolismo , Lisina/biossíntese , Acetolactato Sintase/genética , Proteínas de Bactérias/genética , Butiratos/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Isoleucina/metabolismo , Isoleucina/farmacologia , Cinética , Leucina/metabolismo , Leucina/farmacologia , Ácido Pirúvico/metabolismo , Deleção de Sequência , Valina/metabolismo , Valina/farmacologiaRESUMO
Targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. Recently, we created a library of 26 genome-reduced strains of Corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. Here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders, and the resulting library of 28 strains was characterized under various environmental conditions. The final chassis strain C1* carries a genome reduction of 13.4% (412 deleted genes) and shows wild-type-like growth behavior in defined medium with d-glucose as carbon and energy source. Moreover, C1* proves to be robust against several stresses (including oxygen limitation) and shows long-term growth stability under defined and complex medium conditions. In addition to providing a novel prokaryotic chassis strain, our results comprise a large strain library and a revised genome annotation list, which will be valuable sources for future systemic studies of C. glutamicum.
Assuntos
Biotecnologia/métodos , Corynebacterium glutamicum/genética , Genoma Bacteriano , Biologia Sintética/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Corynebacterium glutamicum/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Frequência do Gene , Família Multigênica/genética , Fenótipo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The first physics operation phase on the stellarator experiment Wendelstein 7-X was successfully completed in March 2016 after about 10 weeks of operation. Experiments in this phase were conducted with five graphite limiters as the primary plasma-facing components. Overall, the results were beyond the expectations published shortly before the start of operation [Sunn Pedersen et al., Nucl. Fusion 55, 126001 (2015)] both with respect to parameters reached and with respect to physics themes addressed. We report here on some of the most important plasma experiments that were conducted. The importance of electric fields on global confinement will be discussed, and the obtained results will be compared and contrasted with results from other devices, quantified in terms of the fusion triple product. Expected values for the triple product in future operation phases will also be described and put into a broader fusion perspective.
RESUMO
For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top-down approach starting from any existing organism. In this study, we initiated the top-down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage-free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application.
Assuntos
Corynebacterium glutamicum/crescimento & desenvolvimento , Genes Essenciais , Genoma Bacteriano , Corynebacterium glutamicum/genética , Engenharia Genética , Aptidão Genética , Família Multigênica , Filogenia , Deleção de SequênciaRESUMO
BACKGROUND AND PURPOSE: This work reports a detailed study carried out in two UK radiotherapy centres of the dosimetric accuracy of the collapsed cone algorithm of a commercial treatment planning system (Helax-TMS) in simulated clinical situations. MATERIALS AND METHODS: Initially the accuracy of the collapsed cone algorithm in homogeneous media is evaluated for an extensive set of simple and complex fields. Water, lung and bone substitute epoxy resin material were then used to assess the algorithm in inhomogeneous media and compare its accuracy with the pencil beam algorithm currently in clinical use. Finally a semi-anatomic phantom and an anthropomorphic phantom were employed to assess the dosimetric accuracy using simulated clinical set ups. Thermoluminescence dosimeter (TLD) measurements were made with the anthropomorphic phantom and ionisation chambers otherwise. Nominal 4, 6 and 15 MV photon beams were studied. RESULTS: For most homogeneous cases agreement between measured and calculated dose is within +/-2% or +/-2 mm. In cases with heterogeneities and simulated clinical situations it is observed that the accuracy is also generally within +/-2% or +/-2 mm. Specific instances where the difference between measured and calculated values exceed this are highlighted. CONCLUSIONS: It can be concluded that in clinical treatment planning situations where lung is present the collapsed cone algorithm should be considered in preference to pencil beam algorithms normally used but that there may still be some discrepancy between calculations and measurement.
Assuntos
Algoritmos , Planejamento da Radioterapia Assistida por Computador/métodos , Humanos , Imagens de FantasmasRESUMO
A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.
Assuntos
Corynebacterium/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Melhoramento Genético/métodos , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Lisina/biossíntese , Fenótipo , Engenharia de Proteínas/métodos , Divisão Celular/fisiologia , Corynebacterium/citologia , Lisina/genética , Mutagênese Sítio-Dirigida , Mutação , NADP/metabolismo , Consumo de Oxigênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Adenylyltransferases regulate glutamine synthetase activity in enterobacteria and actinomycetes such as Streptomyces coelicolor, Mycobacterium tuberculosis and Corynebacterium glutamicum. In this study the effects of a mutation of the glnE gene, coding for adenylyltransferase, on transcriptome and metabolome profiles of C. glutamicum was investigated. As expected, the glnE deletion led to a loss of activity regulation of glutamine synthetase. Astonishingly, additionally the glnE mutation caused a nitrogen limitation response on the transcript level as well. Interestingly, induction of the nitrogen starvation response in the mutant strain was unusually weak and GlnK was present in adenylylated form even without nitrogen starvation. The results obtained might hint to a moonlighting function of adenylyltransferase and might be explained by protein interaction of adenylyltransferase and an unknown interaction partner of the nitrogen regulatory network.
Assuntos
Corynebacterium glutamicum/enzimologia , Nitrogênio/deficiência , Nucleotidiltransferases/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Teste de Complementação Genética , Metaboloma/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
The effects of a deletion of the amtR gene, encoding the master regulator of nitrogen control in Corynebacterium glutamicum, were investigated by metabolome and transcriptome analyses. Compared to the wild type, different metabolite patterns were observed in respect to glycolysis, pentose phosphate pathway, citric acid cycle, and most amino acid pools. Not all of these alterations could be attributed to changes at the level of mRNA and must be caused by posttranscriptional regulatory processes. However, subsequently carried out transcriptome analyses, which were confirmed by gel retardation experiments, revealed two new targets of AmtR, the dapD gene, encoding succinylase involved in m-diaminopimelate synthesis, and the mez gene, coding for malic enzyme. The regulation of dapD connects the AmtR-dependent nitrogen control with l-lysine biosynthesis, the regulation of mez with carbon metabolism. An increased l-glutamine pool in the amtR mutant compared to the wild type was correlated with deregulated expression of the AmtR-regulated glnA gene and an increased glutamine synthetase activity. The glutamate pool was decreased in the mutant and also glutamate excretion was impaired.
Assuntos
Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Perfilação da Expressão Gênica , Metabolômica , Proteínas Repressoras/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Corynebacterium glutamicum/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Cromatografia Gasosa-Espectrometria de Massas , Deleção de Genes , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA , Proteínas Repressoras/metabolismoRESUMO
Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.