scholarly journals Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1360-1375 ◽  
Author(s):  
Christiane Dietrich ◽  
Aimé Nato ◽  
Bruno Bost ◽  
Pierre Le Maréchal ◽  
Armel Guyonvarch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Time Course ◽  
Site Directed Mutagenesis ◽  
Transcriptional Level ◽  
Phosphotransferase System ◽  
Limited Growth ◽  
Mobility Shift ◽  
Batch Cultures ◽  
Fructose 1,6 Bisphosphate ◽  
Electrophoretic Mobility Shift Assays

Corynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding.

scholarly journals Involvement of the LuxR-Type Transcriptional Regulator RamA in Regulation of Expression of the gapA Gene, Encoding Glyceraldehyde-3-Phosphate Dehydrogenase of Corynebacterium glutamicum

2008 ◽  
Vol 191 (3) ◽  
pp. 968-977 ◽  
Author(s):  
Koichi Toyoda ◽  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sugar Transport ◽  
Transcriptional Regulator ◽  
Phosphotransferase System ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
Gene Encoding ◽  
Global Regulators ◽  
Gapdh Activity ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT SugR, RamA, GlxR, GntR1, and a MarR-type transcriptional regulator bind to the promoter region of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), essential for glycolysis in Corynebacterium glutamicum. We previously showed that SugR, a transcriptional repressor of phosphotransferase system genes for the sugar transport system, is involved in the downregulation of gapA expression in the absence of sugar. In this study, the role of RamA in the expression of the gapA gene was examined. Comparing the gapA expression and GAPDH activity of a ramA mutant with those of the wild type revealed that RamA is involved in upregulation of gapA expression in glucose-grown cells. DNase I footprint analyses and electrophoretic mobility shift assays revealed that RamA binds with different affinities to three sites in the gapA promoter. lacZ reporter assays with mutated RamA binding sites in the gapA promoter showed that the middle binding site is the most important for RamA to activate gapA expression and that binding of RamA to the gapA promoter activates the gene expression not only in glucose-grown cells but also in acetate-grown cells. Furthermore, RamA also directly activates sugR expression, indicating that two global regulators, RamA and SugR, are coordinately involved in the complex regulation of gapA expression in C. glutamicum.

scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
Author(s):  
José R. Blesa ◽  
José Hernández-Yago
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Outer Mitochondrial Membrane ◽  
Mobility Shift ◽  
A Subunit ◽  
Expression Evidence ◽  
Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

2008 ◽  
Vol 410 (3) ◽  
pp. 605-611 ◽  
Author(s):  
Kwang Soo Kim ◽  
Hee Young Kim ◽  
Eun-hye Joe ◽  
Ilo Jou
Keyword(s):  
Rat Brain ◽  
Interferon Γ ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Activator Protein 1 ◽  
Mobility Shift ◽  
Matrix Metalloproteinase 3 ◽  
Mitogen Activated Protein ◽  
Electrophoretic Mobility Shift Assays

Many brain cells secrete MMPs (matrix metalloproteinases), and increased or misregulated MMP levels are found in neurodegenerative disorders. Here we report that MMP-3 transcription and protein secretion were increased in rat brain astrocytes stimulated with lipopolysaccharide, gangliosides or interferon-γ. Sequential deletion of the MMP-3 promoter revealed that sequences between −0.5 kb and the start codon were crucial for the transcriptional induction of MMP-3. In addition, experiments using pharmacological inhibitors of individual mitogen-activated protein kinases revealed that MMP-3 induction and promoter activity involved Jun N-terminal kinase, a representative upstream signal of AP-1 (activator protein-1). Sequence analyses of the region of the MMP-3 promoter 500 bp from the start codon indicated the presence of three AP-1 binding sequences. Among them, electrophoretic-mobility-shift assays as well as site-directed mutagenesis of individual AP-1 sequences revealed that distal and middle, but not proximal, sequences largely mediated its induction. Together, these results indicate that AP-1 could control MMP-3 induction in brain astrocytes and that its regulation through specific AP-1 elements could be exploited in the treatment of brain pathologies in which increased expression of MMP-3 plays crucial roles.

Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Kambiz Morabbi Heravi ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Phosphotransferase System ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Proposed Model ◽  
Rna Polymerase Holoenzyme ◽  
Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

scholarly journals Regulation ofperRExpression by Iron and PerR in Campylobacter jejuni

2011 ◽  
Vol 193 (22) ◽  
pp. 6171-6178 ◽  
Author(s):  
Minkyeong Kim ◽  
Sunyoung Hwang ◽  
Sangryeol Ryu ◽  
Byeonghwa Jeon
Keyword(s):  
Oxidative Stress ◽  
Campylobacter Jejuni ◽  
Regulatory Region ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Transcriptional Changes ◽  
Electrophoretic Mobility Shift Assays ◽  
Binding Sequence

Campylobacter jejuniis a leading food-borne pathogen causing gastroenteritis in humans. Although OxyR is a widespread oxidative stress regulator in many Gram-negative bacteria,C. jejunilacks OxyR and instead possesses the metalloregulator PerR. Despite the important role played by PerR in oxidative stress defense, little is known about the factors influencingperRexpression inC. jejuni. In this study, aperRpromoter-lacZfusion assay demonstrated that iron significantly reduced the level ofperRtranscription, whereas other metal ions, such as copper, cobalt, manganese, and zinc, did not affectperRtranscription. Notably, aperRmutation substantially increased the level ofperRtranscription and intranscomplementation restored the transcriptional changes, suggestingperRis transcriptionally autoregulated inC. jejuni. In theperRmutant, iron did not repressperRtranscription, indicating the iron dependence ofperRexpression results fromperRautoregulation. Electrophoretic mobility shift assays showed that PerR binds to theperRpromoter, and DNase I footprinting assays identified a PerR binding site overlapping the −35 region of the twoperRpromoters, further supportingperRautoregulation at the transcriptional level. Alignment of the PerR binding sequence in theperRpromoter with the regulatory region of other PerR regulon genes ofC. jejunirevealed a 16-bp consensus PerR binding sequence, which shares high similarities to theBacillus subtilisPerR box. The results of this study demonstrated that PerR directly interacts with theperRpromoter and regulatesperRtranscription and thatperRautoregulation is responsible for the repression ofperRtranscription by iron inC. jejuni.

scholarly journals A Novel L-Serine Exporter and Its Positive Regulator in Corynebacterium glutamicum

2019 ◽  
Author(s):  
Xiaomei Zhang ◽  
Yujie Gao ◽  
Ziwei Chen ◽  
Guoqiang Xu ◽  
Xiaojuan Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Transcriptional Regulator ◽  
Deletion Strain ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Amino Acid Transport System ◽  
Positive Regulator ◽  
Green Fluorescent

ABSTRACTExporters play an essential role in the fermentative production of amino acids. In Corynebacterium glutamicum, ThrE, which can export L-threonine and L-serine, is the only identified L-serine exporter so far. In this study, a novel L-serine exporter NCgl0580 was identified and characterized in C. glutamicum ΔSSAAI (SSAAI), and named as SerE (encoded by serE). Deletion of serE in SSAAI led to a 56.5% decrease in L-serine titer, whereas overexpression of serE compensated for the lack of serE with respect to L-serine titer. A fusion protein with SerE and enhanced green fluorescent protein (EGFP) was constructed to confirm that SerE localized at the plasma membrane. The function of SerE was studied by peptide feeding approaches, and the results showed that SerE is a novel exporter for L-serine and L-threonine in C. glutamicum. Subsequently, the interaction of a known L-serine exporter ThrE and SerE was studied, and the results suggested that SerE is more important than ThrE in L-serine export in SSAAI. Probe plasmid and electrophoretic mobility shift assays (EMSA) revealed NCgl0581 as the transcriptional regulator of SerE. Comparative transcriptomics between SSAAI and the NCgl0581 deletion strain showed that NCgl0581 regulated the transcription of 115 genes in C. glutamicum, among which the transcriptional level of NCgl0580 decreased 280-fold in a NCgl0581 deletion strain, indicating that NCgl0581 is a positive regulator of SerE. Thus, this study provides a novel target for L-serine and L-threonine export engineering as well as a novel global transcriptional regulator NCgl0581 in C. glutamicum.ImportanceExporters are gaining increasing attention for improving industrial production of amino acids. This study identified a novel exporter NCgl0580 for L-serine and L-threonine in C. glutamicum, and its positive regulator (NCgl0581), which was shown to be a novel global transcriptional regulator in C. glutamicum. This study provides a new target for engineering efflux of L-serine and L-threonine, expands the exporter and transcriptional regulator family, and enriches our understanding of amino acid transport system in C. glutamicum.

scholarly journals FxkR Provides the Missing Link in the fixL-fixK Signal Transduction Cascade in Rhizobium etli CFN42

2012 ◽  
Vol 25 (11) ◽  
pp. 1506-1517 ◽  
Author(s):  
David Zamorano-Sánchez ◽  
Alma Reyes-González ◽  
Nicolás Gómez-Hernández ◽  
Patricia Rivera ◽  
Dimitris Georgellis ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Transcriptional Control ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Site Directed Mutagenesis ◽  
Rhizobium Etli ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Reading Frame ◽  
Electrophoretic Mobility Shift Assays

Transcriptional control of the fixK gene in Rhizobium etli and R. leguminosarum bv. viciae is governed by a two-component signal transduction system that diverts from the conventional FixL-FixJ cascade that occurs in model rhizobia. Although a fixL gene, encoding a hybrid histidine kinase (hFixL), is present in R. etli, no fixJ, the cognate response regulator, has been identified. In this work, we present evidence that the pRet42f-located open reading frame RHE_PF00530 (fxkR) encodes a novel response regulator indispensable for fixKf activation under microaerobic growth. Moreover, results from complementation assays demonstrate that the activation of fixKf expression requires the presence of both hFixL and FxkR, and that the fxkR ortholog from R. leguminosarum bv. viciae is able to substitute for FxkR transcriptional control in R. etli. In addition, in these two organisms, hFixL- and FxkR-related proteins were identified in other bacteria, located in close proximity to a fixK-related gene. Using reporter fusions, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified the FxkR binding site upstream from the transcriptional start site of fixKf. Similar to our previous observations for fixL and fixKf mutants, a null mutation in fxkR does not affect the symbiotic effectiveness of the strain. Thus, our findings reveal that FxkR is the long-standing missing key regulator that allows the transduction of the microaerobic signal for the activation of the FixKf regulon.

scholarly journals Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum

2021 ◽  
Vol 9 ◽  
Author(s):  
Irene Krahn ◽  
Daniel Bonder ◽  
Lucía Torregrosa-Barragán ◽  
Dominik Stoppel ◽  
Jens P. Krause ◽  
...  
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Pentose Phosphate ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Depth Analysis ◽  
Mutant Strains ◽  
Phosphofructokinase Activity ◽  
Fructose 1,6 Bisphosphate ◽  
Product Accumulation

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

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