scholarly journals Regulation of l-Lactate Utilization by the FadR-Type Regulator LldR of Corynebacterium glutamicum

2007 ◽  
Vol 190 (3) ◽  
pp. 963-971 ◽  
Author(s):  
Tobias Georgi ◽  
Verena Engels ◽  
Volker F. Wendisch
Keyword(s):  
Energy Source ◽  
Corynebacterium Glutamicum ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Carbon Sources ◽  
Upstream Region ◽  
Growth Defect ◽  
Mrna Levels ◽  
Lactate Utilization ◽  
In Cells

ABSTRACT Corynebacterium glutamicum can grow on l-lactate as a sole carbon and energy source. The NCgl2816-lldD operon encoding a putative transporter (NCgl2816) and a quinone-dependent l-lactate dehydrogenase (LldD) is required for l-lactate utilization. DNA affinity chromatography revealed that the FadR-type regulator LldR (encoded by NCgl2814) binds to the upstream region of NCgl2816-lldD. Overexpression of lldR resulted in strongly reduced NCgl2816-lldD mRNA levels and strongly reduced LldD activity, and as a consequence, a severe growth defect was observed in cells grown on l-lactate as the sole carbon and energy source, but not in cells grown on glucose, ribose, or acetate. Deletion of lldR had no effect on growth on these carbon sources but resulted in high NCgl2816-lldD mRNA levels and high LldD activity in the presence and absence of l-lactate. Purified His-tagged LldR bound to a 54-bp fragment of the NCgl2816-lldD promoter, which overlaps with the transcriptional start site determined by random amplification of cDNA ends-PCR and contains a putative operator motif typical of FadR-type regulators, which is −1TNGTNNNACNA10. Mutational analysis revealed that this motif with hyphenated dyad symmetry is essential for binding of LldD to the NCgl2816-lldD promoter. l-Lactate, but not d-lactate, interfered with binding of LldRHis to the NCgl2816-lldD promoter. Thus, during growth on media lacking l-lactate, LldR represses expression of NCgl2816-lldD. In the presence of l-lactate in the growth medium or under conditions leading to intracellular l-lactate accumulation, the l-lactate utilization operon is induced.

scholarly journals Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

2005 ◽  
Vol 71 (10) ◽  
pp. 5920-5928 ◽  
Author(s):  
Corinna Stansen ◽  
Davin Uy ◽  
Stephane Delaunay ◽  
Lothar Eggeling ◽  
Jean-Louis Goergen ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Specific Activity ◽  
Temperature Shift ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Reverse Transcription Pcr ◽  
Glutamate Production ◽  
Lactate Utilization

ABSTRACT Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

scholarly journals Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

2008 ◽  
Vol 190 (19) ◽  
pp. 6458-6466 ◽  
Author(s):  
Jung-Won Youn ◽  
Elena Jolkver ◽  
Reinhard Krämer ◽  
Kay Marin ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Uptake System ◽  
Dependent Manner ◽  
Wild Type ◽  
Prolonged Incubation ◽  
In The Wild ◽  
Biochemical Analyses

ABSTRACT Many bacteria can utilize C4-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l-malate, and likely also oxaloacetate in a sodium-dependent manner.

scholarly journals c-Jun Controls Histone Modifications, NF-κB Recruitment, and RNA Polymerase II Function To Activate the ccl2 Gene

2008 ◽  
Vol 28 (13) ◽  
pp. 4407-4423 ◽  
Author(s):  
Sabine Wolter ◽  
Anneke Doerrie ◽  
Axel Weber ◽  
Heike Schneider ◽  
Elke Hoffmann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Interleukin 1 ◽  
Histone Deacetylase 3 ◽  
H3 Acetylation ◽  
Ccl2 Gene ◽  
Dependent Interaction ◽  
In Cells

ABSTRACT Interleukin-1 (IL-1)-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or Jun N-terminal protein kinase 1 (JNK1)/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant protein. We used chromatin immunoprecipitation to identify three c-Jun-binding sites located in the far 5′ region close to the transcriptional start site and in the far 3′ region of murine and human ccl2 genes. Mutational analysis revealed that the latter two sites contribute to ccl2 transcription in response to the presence of IL-1 or of ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild-type and c-Jun-deficient cells revealed that c-Jun regulates Ser10 phosphorylation of histone H3, acetylation of histones H3 and H4, and recruitment of histone deacetylase 3 (HDAC3), NF-κB subunits, and RNA polymerase II across the ccl2 locus. c-Jun also coimmunoprecipitated with p65 NF-κB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support the idea of an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-κB and HDAC3 at the level of chromatin.

scholarly journals The DeoR-Type Regulator SugR Represses Expression of ptsG in Corynebacterium glutamicum

2007 ◽  
Vol 189 (8) ◽  
pp. 2955-2966 ◽  
Author(s):  
Verena Engels ◽  
Volker F. Wendisch
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Dna Microarray ◽  
Sole Carbon Source ◽  
Glucose Utilization ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Phosphotransferase System ◽  
High Fructose ◽  
Promoter Dna

ABSTRACT Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids. Uptake of the preferred carbon source glucose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is reduced during coutilization of glucose with acetate, sucrose, or fructose compared to growth on glucose as the sole carbon source. Here we show that the DeoR-type regulator SugR (NCgl1856) represses expression of ptsG, which encodes the glucose-specific PTS enzyme II. Overexpression of sugR resulted in reduced ptsG mRNA levels, decreased glucose utilization, and perturbed growth on media containing glucose. In mutants lacking sugR, expression of the ptsG′-′cat fusion was increased two- to sevenfold during growth on gluconeogenic carbon sources but remained similar during growth on glucose or other sugars. As shown by DNA microarray analysis, SugR also regulates expression of other genes, including ptsS and the putative NCgl1859-fruK-ptsF operon. Purified SugR bound to DNA regions upstream of ptsG, ptsS, and NCgl1859, and a 75-bp ptsG promoter fragment was sufficient for SugR binding. Fructose-6-phosphate interfered with binding of SugR to the ptsG promoter DNA. Thus, while during growth on gluconeogenic carbon sources SugR represses ptsG, ptsG expression is derepressed during growth on glucose or under other conditions characterized by high fructose-6-phosphate concentrations, representing one mechanism which allows C. glutamicum to adapt glucose uptake to carbon source availability.

Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen

1989 ◽  
Vol 9 (2) ◽  
pp. 484-491
Author(s):  
D L Marykwas ◽  
T D Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Transcriptional Repression ◽  
Regulatory Gene ◽  
Carbon Sources ◽  
Nuclear Gene ◽  
Mrna Levels ◽  
Diploid Strain ◽  
In Cells

The product of the Saccharomyces cerevisiae nuclear gene PET494 is required to promote the translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII). The level of cytochrome c oxidase activity is affected by several different environmental conditions, which also influence coxIII expression. We have studied the regulation of PET494 to test whether the level of its expression might modulate coxIII translation in response to these conditions. A pet494::lacZ fusion was constructed and used to monitor PET494 expression. PET494 was regulated by oxygen availability: expression in a respiration-competent diploid strain grown anaerobically was one-fifth the level of expression in aerobically grown cells. However, since PET494 mRNA levels did not vary in response to oxygen deprivation, regulation by oxygen appears to occur at the translational level. This oxygen regulation was not mediated by heme, and PET494 expression was independent of the heme activator protein HAP2. The regulation of PET494 expression by carbon source was also examined. In cells grown on glucose-containing medium, PET494 was expressed at levels four- to sixfold lower than in cells grown on ethanol and glycerol. However, addition of ethanol to glucose-containing medium induced PET494 expression approximately twofold. PET494 transcript levels varied over a fourfold range in response to different carbon sources. The effects on PET494 expression of mutations in the SNF1, SNF2, SSN6, and HXK2 genes were also determined and found to be minimal.

scholarly journals Thr435 phosphorylation regulates RelA (p65) NF-κB subunit transactivation

2010 ◽  
Vol 426 (3) ◽  
pp. 345-354 ◽  
Author(s):  
John M. O'Shea ◽  
Neil D. Perkins
Keyword(s):  
Target Genes ◽  
Mutational Analysis ◽  
Wild Type Mouse ◽  
Mrna Levels ◽  
Transactivation Domain ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Histone Deacetylase 1 ◽  
In Cells

Phosphorylation of the RelA (p65) NF-κB (nuclear factor κB) subunit has been previously shown to modulate its ability to induce or repress transcription. In the present study we have investigated the consequences of Thr435 phosphorylation within the C-terminal transactivation domain of RelA. We confirm that Thr435 is phosphorylated in cells and is induced by TNFα (tumour necrosis factor α) treatment. Mutational analysis of this site revealed gene-specific effects on transcription, with a T435D phosphomimetic mutant significantly enhancing Cxcl2 (CXC chemokine ligand 2) mRNA levels in reconstituted Rela−/− mouse embryonic fibroblasts. Chromatin immunoprecipitation analysis revealed that this mutation results in enhanced levels of histone acetylation associated with decreased recruitment of HDAC1 (histone deacetylase 1). Moreover, mutation of this site disrupted RelA interaction with HDAC1 in vitro. Thr435 phosphorylation of promoter-bound RelA was also detected at NF-κB target genes following TNFα treatment in wild-type mouse embryonic fibroblasts. Phosphorylation at this site therefore provides an additional mechanism through which the specificity of NF-κB transcriptional activity can be modulated in cells.

scholarly journals Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen.

1989 ◽  
Vol 9 (2) ◽  
pp. 484-491 ◽  
Author(s):  
D L Marykwas ◽  
T D Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Transcriptional Repression ◽  
Regulatory Gene ◽  
Carbon Sources ◽  
Nuclear Gene ◽  
Mrna Levels ◽  
Diploid Strain ◽  
In Cells

The product of the Saccharomyces cerevisiae nuclear gene PET494 is required to promote the translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII). The level of cytochrome c oxidase activity is affected by several different environmental conditions, which also influence coxIII expression. We have studied the regulation of PET494 to test whether the level of its expression might modulate coxIII translation in response to these conditions. A pet494::lacZ fusion was constructed and used to monitor PET494 expression. PET494 was regulated by oxygen availability: expression in a respiration-competent diploid strain grown anaerobically was one-fifth the level of expression in aerobically grown cells. However, since PET494 mRNA levels did not vary in response to oxygen deprivation, regulation by oxygen appears to occur at the translational level. This oxygen regulation was not mediated by heme, and PET494 expression was independent of the heme activator protein HAP2. The regulation of PET494 expression by carbon source was also examined. In cells grown on glucose-containing medium, PET494 was expressed at levels four- to sixfold lower than in cells grown on ethanol and glycerol. However, addition of ethanol to glucose-containing medium induced PET494 expression approximately twofold. PET494 transcript levels varied over a fourfold range in response to different carbon sources. The effects on PET494 expression of mutations in the SNF1, SNF2, SSN6, and HXK2 genes were also determined and found to be minimal.

scholarly journals Regulation of the Acetoin Catabolic Pathway Is Controlled by Sigma L in Bacillus subtilis

2001 ◽  
Vol 183 (8) ◽  
pp. 2497-2504 ◽  
Author(s):  
Naima Ould Ali ◽  
Joelle Bignon ◽  
Georges Rapoport ◽  
Michel Debarbouille
Keyword(s):  
Bacillus Subtilis ◽  
Growth Medium ◽  
Dna Sequences ◽  
Transcriptional Start Site ◽  
Specific Interaction ◽  
Carbon Sources ◽  
Upstream Region ◽  
Global Regulator ◽  
Sigma 54 ◽  
Dnase I Footprinting

ABSTRACT Bacillus subtilis grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. Acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. TheacoABCL operon encodes the E1α, E1β, E2, and E3 subunits of the acetoin dehydrogenase complex in B. subtilis. Expression of this operon is induced by acetoin and repressed by glucose in the growth medium. The acoR gene is located downstream from the acoABCL operon and encodes a positive regulator which stimulates the transcription of the operon. The product of acoR has similarities to transcriptional activators of sigma 54-dependent promoters. The four genes of the operon are transcribed from a −12, −24 promoter, and transcription is abolished in acoR and sigL mutants. Deletion analysis showed that DNA sequences more than 85 bp upstream from the transcriptional start site are necessary for full induction of the operon. These upstream activating sequences are probably the targets of AcoR. Analysis of an acoR′-′lacZ strain ofB. subtilis showed that the expression of acoRis not induced by acetoin and is repressed by the presence of glucose in the growth medium. Transcription of acoR is also negatively controlled by CcpA, a global regulator of carbon catabolite repression. A specific interaction of CcpA in the upstream region ofacoR was demonstrated by DNase I footprinting experiments, suggesting that repression of transcription of acoR is mediated by the binding of CcpA to the promoter region ofacoR.

scholarly journals Coordinated Regulation ofgnd, Which Encodes 6-Phosphogluconate Dehydrogenase, by the Two Transcriptional Regulators GntR1 and RamA in Corynebacterium glutamicum

2012 ◽  
Vol 194 (23) ◽  
pp. 6527-6536 ◽  
Author(s):  
Yuya Tanaka ◽  
Shigeki Ehira ◽  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Dna Binding ◽  
Corynebacterium Glutamicum ◽  
Binding Sites ◽  
Affinity Purification ◽  
Transcriptional Start Site ◽  
Transcriptional Regulators ◽  
Binding Activity ◽  
Upstream Region ◽  
Content Type ◽  
Phosphogluconate Dehydrogenase

ABSTRACTThe transcriptional regulation ofCorynebacterium glutamicum gnd, encoding 6-phosphogluconate dehydrogenase, was investigated. Two transcriptional regulators, GntR1 and RamA, were isolated by affinity purification usinggndpromoter DNA. GntR1 was previously identified as a repressor of gluconate utilization genes, includinggnd. Involvement of RamA ingndexpression had not been investigated to date. The level ofgndmRNA was barely affected by the single deletion oframA. However,gndexpression was downregulated in theramA gntR1double mutant compared to that of thegntR1single mutant, suggesting that RamA activatesgndexpression. Two RamA binding sites are found in the 5′ upstream region ofgnd. Mutation proximal to the transcriptional start site diminished the gluconate-dependent induction ofgnd-lacZ. DNase I footprinting assay revealed two GntR1 binding sites, with one corresponding to a previously proposed site that overlaps with the −10 region. The other site overlaps the RamA binding site. GntR1 binding to this newly identified site inhibits DNA binding of RamA. Therefore, it is likely that GntR1 repressesgndexpression by preventing both RNA polymerase and RamA binding to the promoter. In addition, DNA binding activity of RamA was reduced by high concentrations of NAD(P)H but not by NAD(P), implying that RamA senses the redox perturbation of the cell.

scholarly journals Impressic Acid Ameliorates Atopic Dermatitis-Like Skin Lesions by Inhibiting ERK1/2-Mediated Phosphorylation of NF-κB and STAT1

2021 ◽  
Vol 22 (5) ◽  
pp. 2334
Author(s):  
Jae Ho Choi ◽  
Gi Ho Lee ◽  
Sun Woo Jin ◽  
Ji Yeon Kim ◽  
Yong Pil Hwang ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Skin Lesions ◽  
Histopathological Analysis ◽  
Mrna Levels ◽  
Chemokine Production ◽  
Dermal Thickness ◽  
Skin Symptoms ◽  
Tnf Α ◽  
Ifn Γ ◽  
In Cells

Impressic acid (IPA), a lupane-type triterpenoid from Acanthopanax koreanum, has many pharmacological activities, including the attenuation of vascular endothelium dysfunction, cartilage destruction, and inflammatory diseases, but its influence on atopic dermatitis (AD)-like skin lesions is unknown. Therefore, we investigated the suppressive effect of IPA on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin symptoms in mice and the underlying mechanisms in cells. IPA attenuated the DNCB-induced increase in the serum concentrations of IgE and thymic stromal lymphopoietin (TSLP), and in the mRNA levels of thymus and activation regulated chemokine(TARC), macrophage derived chemokine (MDC), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) in mice. Histopathological analysis showed that IPA reduced the epidermal/dermal thickness and inflammatory and mast cell infiltration of ear tissue. In addition, IPA attenuated the phosphorylation of NF-κB and IκBα, and the degradation of IκBα in ear lesions. Furthermore, IPA treatment suppressed TNF-α/IFN-γ-induced TARC expression by inhibiting the NF-κB activation in cells. Phosphorylation of extracellular signalregulated protein kinase (ERK1/2) and the signal transducer and activator of transcription 1 (STAT1), the upstream signaling proteins, was reduced by IPA treatment in HaCaT cells. In conclusion, IPA ameliorated AD-like skin symptoms by regulating cytokine and chemokine production and so has therapeutic potential for AD-like skin lesions.

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