scholarly journals Biochemical and Physiological Characterization of the Pyruvate Formate-Lyase Pfl1 of Chlamydomonas reinhardtii, a Typically Bacterial Enzyme in a Eukaryotic Alga

2008 ◽  
Vol 7 (3) ◽  
pp. 518-526 ◽  
Author(s):  
Anja Hemschemeier ◽  
Jessica Jacobs ◽  
Thomas Happe
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Anaerobic Metabolism ◽  
Pyruvate Decarboxylase ◽  
Pyruvate Formate Lyase ◽  
Physiological Characterization ◽  
Bacterial Enzyme ◽  
Genes Encoding ◽  
Oxygen Sensitive ◽  
Biochemical Analyses

ABSTRACT The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.

scholarly journals Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the fhaPromoter of Bordetella pertussis

2001 ◽  
Vol 183 (2) ◽  
pp. 536-544 ◽  
Author(s):  
Philip E. Boucher ◽  
Mei-Shin Yang ◽  
Deanna M. Schmidt ◽  
Scott Stibitz
Keyword(s):  
Bordetella Pertussis ◽  
Response Regulator ◽  
Sequence Specificity ◽  
Binding Region ◽  
Dna Sequence Specificity ◽  
Genes Encoding ◽  
Two Component Signal Transduction ◽  
Biochemical Analyses

ABSTRACT The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (fha) and pertussis toxin (ptx). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA∼P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.

scholarly journals Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii: Discovery of the Betaine Lipid Synthase BTA1Cr

2005 ◽  
Vol 4 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Wayne R. Riekhof ◽  
Barbara B. Sears ◽  
Christoph Benning
Keyword(s):  
Fatty Acid ◽  
Chlamydomonas Reinhardtii ◽  
Active Sites ◽  
Expressed Sequence Tag ◽  
Membrane Lipid ◽  
Flowering Plants ◽  
Site Directed Mutagenesis ◽  
Genes Encoding ◽  
Betaine Lipid

ABSTRACT Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr ) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

scholarly journals Anaerobic Expression of the Ferredoxin-Encoding FDX5 Gene of Chlamydomonas reinhardtii Is Regulated by the Crr1 Transcription Factor

2010 ◽  
Vol 9 (11) ◽  
pp. 1747-1754 ◽  
Author(s):  
Camilla Lambertz ◽  
Anja Hemschemeier ◽  
Thomas Happe
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Binding Sites ◽  
Anaerobic Metabolism ◽  
Response Regulator ◽  
Binding Assays ◽  
Anaerobic Conditions ◽  
Gene Encoding ◽  
Content Type ◽  
Unicellular Green Alga

ABSTRACT The unicellular green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism and reacts to hypoxic or anaerobic conditions with the induced expression of many genes. One gene which is upregulated particularly strongly is the FDX5 gene, encoding one of at least six ferredoxin isoforms in C. reinhardtii. Fdx5 is a typical plant-type 2Fe2S protein that is located in the chloroplast. The FDX5 promoter region contains three GTAC motifs, which are known to be the binding sites for copper response regulator 1 (Crr1) and other SQUAMOSA promoter binding proteins (SBPs). This study shows that two of these GTAC sites are essential to confer oxygen and also copper responsiveness to a reporter gene. The SBP domain of Crr1 is able to bind to both of these GTAC sites in in vitro binding assays. Moreover, in a Crr1-deficient C. reinhardtii strain, FDX5 is not expressed. These results clearly indicate that Crr1 is involved in the transcriptional regulation of the FDX5 gene in the absence of oxygen or copper.

scholarly journals The Bifunctional Pyruvate Decarboxylase/Pyruvate Ferredoxin Oxidoreductase fromThermococcus guaymasensis

Archaea ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Mohammad S. Eram ◽  
Erica Oduaran ◽  
Kesen Ma
Keyword(s):  
Pyruvate Decarboxylase ◽  
Oxidative Decarboxylation ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Ferredoxin Oxidoreductase ◽  
Ph Values ◽  
Keto Acids ◽  
Genes Encoding ◽  
Oxygen Sensitive ◽  
Pyruvate Ferredoxin Oxidoreductase

The hyperthermophilic archaeonThermococcus guaymasensisproduces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized fromT. guaymasensisunder strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of3.8±0.22 U mg−1and20.2±1.8 U mg−1, with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilicβ-keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the correspondingβ-keto acids.

scholarly journals Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1102
Author(s):  
Galina N. Raldugina ◽  
Sergey V. Evsukov ◽  
Liliya R. Bogoutdinova ◽  
Alexander A. Gulevich ◽  
Ekaterina N. Baranova
Keyword(s):  
Choline Oxidase ◽  
Culture Methods ◽  
Gene Encoding ◽  
Oxidase Gene ◽  
Bacterial Enzyme ◽  
Transgenic Lines ◽  
High Regeneration ◽  
Whole Plants ◽  
Implicit Response

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

scholarly journals Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

2021 ◽  
Vol 22 (11) ◽  
pp. 5968
Author(s):  
Egor A. Turovsky ◽  
Maria V. Turovskaya ◽  
Evgeniya I. Fedotova ◽  
Alexey A. Babaev ◽  
Viktor S. Tarabykin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nmda Receptor ◽  
Cortical Neurons ◽  
Target Genes ◽  
Cortical Cell ◽  
Inhibitory System ◽  
Selective Agonist ◽  
Genes Encoding ◽  
Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

scholarly journals In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Amber R Paulson ◽  
Maureen O’Callaghan ◽  
Xue-Xian Zhang ◽  
Paul B Rainey ◽  
Mark R H Hurst
Keyword(s):  
Galleria Mellonella ◽  
Functional Enrichment ◽  
Natural Environments ◽  
Insecticidal Toxin ◽  
Heat Stable ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

scholarly journals Heterodimer Formation of the Homodimeric ABC Transporter OpuA

2021 ◽  
Vol 22 (11) ◽  
pp. 5912
Author(s):  
Patricia Alvarez-Sieiro ◽  
Hendrik R. Sikkema ◽  
Bert Poolman
Keyword(s):  
Abc Transporter ◽  
Conformational Dynamics ◽  
Host Organism ◽  
Affinity Tag ◽  
Practical Applications ◽  
Fundamental Research ◽  
Protein Multimerization ◽  
Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

scholarly journals PRO: Carbapenems should be used for ALL infections caused by ceftriaxone-resistant Enterobacterales

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
David L Paterson ◽  
Burcu Isler ◽  
Patrick N A Harris
Keyword(s):  
Antibiotic Resistance ◽  
Observational Studies ◽  
Randomized Trials ◽  
Antibiotic Resistance Genes ◽  
Definitive Treatment ◽  
In Vitro Activity ◽  
Genes Encoding ◽  
Amoxicillin Clavulanate ◽  
Trial Outcomes

Abstract Ceftriaxone resistance in the Enterobacterales is typically the result of production of ESBLs or AmpC β-lactamases. The genes encoding these enzymes are often co-located with other antibiotic resistance genes leading to resistance to aminoglycosides, quinolones and trimethoprim/sulfamethoxazole. Carbapenems are stable to ESBLs and AmpC giving them reliable in vitro activity against producers of these β-lactamases. In contrast, piperacillin/tazobactam and amoxicillin/clavulanate are compromised by co-production of OXA-1, which is not inhibited by tazobactam or clavulanate. These in vitro findings provide an explanation for the MERINO trial outcomes, where 3.7% (7/191) randomized to meropenem died compared with 12.3% (23/187) randomized to piperacillin/tazobactam as definitive treatment of bloodstream infection due to ceftriaxone-resistant organisms. No randomized trials have yet put cefepime and carbapenems head to head, but some observational studies have shown worse outcomes with cefepime. We argue that carbapenems are the antibiotics of choice for ceftriaxone-resistant Enterobacterales.

scholarly journals HGG-26. H3G34V MUTATION AFFECTS GENOMIC H3K36 METHYLATION IN PEDIATRIC GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii348-iii348
Author(s):  
Tina Huang ◽  
Andrea Piunti ◽  
Elizabeth Bartom ◽  
Jin Qi ◽  
Rintaro Hashizume ◽  
...  
Keyword(s):  
Western Blot ◽  
Allelic Frequency ◽  
Glioma Cell Line ◽  
Fluorescent Imaging ◽  
Wild Type ◽  
Gene Expressions ◽  
Pediatric Glioma ◽  
Genes Encoding ◽  
Normal Human

Abstract BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase), resulting in reduced H3K36me on H3G34V nucleosomes relative to wild-type. This contributes to genomic instability and drives distinct gene expressions associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3G34V on genomic H3K36me3 enrichment in vitro. METHODS Doxycycline-inducible short hairpin RNA (shRNA) against H3F3A was delivered via lentivirus to established H3G34V mutant pediatric glioma cell line KNS42, and H3G34V introduced into H3.3 wild type normal human astrocytes (NHA). Transfections were confirmed by western blot, fluorescent imaging, and flow cytometry, with resulting H3.3WT and H3K36me3 expression determined by western blot. H3.3WT, H3K36me3, and H3G34V ChIP-Seq was performed to evaluate genomic enrichment. RESULTS Complete knockdown of H3G34V was achieved with DOX-induced shRNA, with no change in total H3.3, suggesting disproportionate allelic frequency of genes encoding H3.3 (H3F3A and H3F3B). Modest increase in H3K36me3 occurred after H3F3A-knockdown from KNS42, suggesting H3G34V alone impacts observed H3K36me3 levels. Distinct H3K36me3 genomic enrichment was observed with H3G34V knock-in. CONCLUSIONS We demonstrate that DOX-inducible knockdown of H3F3A in an H3G34V mutant pediatric glioma cells and H3G34V mutation transduction in wild-type astrocytes affects H3K36me3 expression. Further evaluation by ChIP-Seq analysis for restoration of wild-type genomic H3K36me3 enrichment patterns with H3G34V knockdown, and mutant H3K36me3 patterns with H3G34V transduction, is currently underway.

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