D-galactose catabolism in archaea: operation of the DeLey–Doudoroff pathway in Haloferax volcanii

2020 ◽  
Vol 367 (1) ◽  
Author(s):  
Julia-Beate Tästensen ◽  
Ulrike Johnsen ◽  
Andreas Reinhardt ◽  
Marius Ortjohann ◽  
Peter Schönheit
Keyword(s):  
Transcriptional Regulator ◽  
High Specificity ◽  
Comprehensive Analysis ◽  
Haloferax Volcanii ◽  
Knock Out ◽  
Genes Encoding ◽  
Functional Involvement ◽  
Genome Analyses ◽  
Galactose Dehydrogenase ◽  
Substrate Binding Protein

ABSTRACT The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey–Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey–Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey–Doudoroff pathway in the domain of archaea.

scholarly journals Acetate Metabolism in Archaea: Characterization of an Acetate Transporter and of Enzymes Involved in Acetate Activation and Gluconeogenesis in Haloferax volcanii

2020 ◽  
Vol 11 ◽  
Author(s):  
Tom Kuprat ◽  
Ulrike Johnsen ◽  
Marius Ortjohann ◽  
Peter Schönheit
Keyword(s):  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Phylogenetic Analyses ◽  
Haloferax Volcanii ◽  
Acetate Metabolism ◽  
Knock Out ◽  
Functional Involvement ◽  
Acetate Activation ◽  
Growth Experiments

The haloarchaeon Haloferax volcanii grows on acetate as sole carbon and energy source. The genes and proteins involved in uptake and activation of acetate and in gluconeogenesis were identified and analyzed by characterization of enzymes and by growth experiments with the respective deletion mutants. (i) An acetate transporter of the sodium: solute-symporter family (SSF) was characterized by kinetic analyses of acetate uptake into H. volcanii cells. The functional involvement of the transporter was proven with a Δssf mutant. (ii) Four paralogous AMP-forming acetyl-CoA synthetases that belong to different phylogenetic clades were shown to be functionally involved in acetate activation. (iii) The essential involvement of the glyoxylate cycle as an anaplerotic sequence was concluded from growth experiments with an isocitrate lyase knock-out mutant excluding the operation of the methylaspartate cycle reported for Haloarcula species. (iv) Enzymes involved in phosphoenolpyruvate synthesis from acetate, namely two malic enzymes and a phosphoenolpyruvate synthetase, were identified and characterized. Phylogenetic analyses of haloarchaeal malic enzymes indicate a separate evolutionary line distinct from other archaeal homologs. The exclusive function of phosphoenolpyruvate synthetase in gluconeogenesis was proven by the respective knock-out mutant. Together, this is a comprehensive study of acetate metabolism in archaea.

scholarly journals Glucose metabolism and acetate switch in archaea: Analysis of the enzymes involved in Haloferax volcanii

2021 ◽  
Author(s):  
Tom Kuprat ◽  
Marius Ortjohann ◽  
Ulrike Johnsen ◽  
Peter Schönheit
Keyword(s):  
Phylogenetic Analyses ◽  
Comprehensive Analysis ◽  
Haloferax Volcanii ◽  
Phosphoglycerate Mutase ◽  
Deletion Mutants ◽  
Aerobic Growth ◽  
Acetyl Coa ◽  
Functional Involvement ◽  
Growth Experiments

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis and in acetyl-CoA formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase and pyruvate: ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Further, it is shown that H. volcanii - during aerobic growth on glucose - excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway, of anaplerosis, and report first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea. Importance: In this work we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism Haloferax volcanii. The study includes transcriptional analyses, growth experiments with deletion mutants and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl-CoA and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Further, we analyzed the key players involved in the acetate switch, i.e in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects on glucose degradation, anaplerosis and acetate switch in H. volcanii and thus expand our understanding of the unusual sugar metabolism in archaea.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals Regulation of Polysaccharide Utilization Contributes to the Persistence of Group A Streptococcus in the Oropharynx

2007 ◽  
Vol 75 (6) ◽  
pp. 2981-2990 ◽  
Author(s):  
Samuel A. Shelburne ◽  
Nnaja Okorafor ◽  
Izabela Sitkiewicz ◽  
Paul Sumby ◽  
David Keith ◽  
...  
Keyword(s):  
Parental Strain ◽  
Group A Streptococcus ◽  
Transcriptional Regulator ◽  
Human Saliva ◽  
Transcript Levels ◽  
Expression Of Genes ◽  
Rich Media ◽  
Genes Encoding ◽  
Group A ◽  
Mutant Derivative

ABSTRACT Group A Streptococcus (GAS) genes that encode proteins putatively involved in polysaccharide utilization show growth phase-dependent expression in human saliva. We sought to determine whether the putative polysaccharide transcriptional regulator MalR influences the expression of such genes and whether MalR helps GAS infect the oropharynx. Analysis of 32 strains of 17 distinct M protein serotypes revealed that MalR is highly conserved across GAS strains. malR transcripts were detectable in patients with GAS pharyngitis, and the levels increased significantly during growth in human saliva compared to the levels during growth in glucose-containing or nutrient-rich media. To determine if MalR influenced the expression of polysaccharide utilization genes, we compared the transcript levels of eight genes encoding putative polysaccharide utilization proteins in the parental serotype M1 strain MGAS5005 and its ΔmalR isogenic mutant derivative. The transcript levels of all eight genes were significantly increased in the ΔmalR strain compared to the parental strain, especially during growth in human saliva. Following experimental infection, the ΔmalR strain persistently colonized the oropharynx in significantly fewer mice than the parental strain colonized, and the numbers of ΔmalR strain CFU recovered were significantly lower than the numbers of the parental strain CFU recovered. These data led us to conclude that MalR influences the expression of genes putatively involved in polysaccharide utilization and that MalR contributes to the persistence of GAS in the oropharynx.

scholarly journals Catabolism of l-rhamnose in A. nidulans proceeds via the non-phosphorylated pathway and is glucose repressed by a CreA-independent mechanism

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Andrew P. MacCabe ◽  
Elpinickie I. Ninou ◽  
Ester Pardo ◽  
Margarita Orejas
Keyword(s):  
Transcription Factor ◽  
Filamentous Fungus ◽  
Extracellular Enzymes ◽  
Regulatory Mechanisms ◽  
Bioactive Metabolites ◽  
Free Sugar ◽  
Knock Out ◽  
Independent Mechanism ◽  
Genes Encoding ◽  
Specific Uptake

Abstract l-rhamnose (6-deoxy-mannose) occurs in nature mainly as a component of certain plant structural polysaccharides and bioactive metabolites but has also been found in some microorganisms and animals. The release of l-rhamnose from these substrates is catalysed by extracellular enzymes including α-l-rhamnosidases, the production of which is induced in its presence. The free sugar enters cells via specific uptake systems where it can be metabolized. Of two l-rhamnose catabolic pathways currently known in microorganisms a non-phosphorylated pathway has been identified in fungi and some bacteria but little is known of the regulatory mechanisms governing it in fungi. In this study two genes (lraA and lraB) are predicted to be involved in the catabolism of l-rhamnose, along with lraC, in the filamentous fungus Aspergillus nidulans. Transcription of all three is co-regulated with that of the genes encoding α-l-rhamnosidases, i.e. induction mediated by the l-rhamnose-responsive transcription factor RhaR and repression of induction in the presence of glucose via a CreA-independent mechanism. The participation of lraA/AN4186 (encoding l-rhamnose dehydrogenase) in l-rhamnose catabolism was revealed by the phenotypes of knock-out mutants and their complemented strains. lraA deletion negatively affects both growth on l-rhamnose and the synthesis of α-l-rhamnosidases, indicating not only the indispensability of this pathway for l-rhamnose utilization but also that a metabolite derived from this sugar is the true physiological inducer.

scholarly journals Intercalated disc protein Xinβ is required for Hippo-YAP signaling in the heart

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Haipeng Guo ◽  
Yao Wei Lu ◽  
Zhiqiang Lin ◽  
Zhan-Peng Huang ◽  
Jianming Liu ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Cardiac Function ◽  
Heart Development ◽  
Genetic Interaction ◽  
Specific Cell ◽  
Cardiomyocyte Proliferation ◽  
Intercalated Disc ◽  
Knock Out ◽  
Genes Encoding ◽  
Knock Out Mice

Abstract Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinβ, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinβ in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinβ mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinβ knock-out mice—indicating a functional and genetic interaction between Xinβ and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinβ modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.

scholarly journals Transgene-Induced Silencing of the Zoosporogenesis-Specific NIFC Gene Cluster of Phytophthora infestans Involves Chromatin Alterations

2007 ◽  
Vol 6 (7) ◽  
pp. 1200-1209 ◽  
Author(s):  
Howard S. Judelson ◽  
Shuji Tani
Keyword(s):  
Phytophthora Infestans ◽  
Gene Cluster ◽  
Sequence Similarity ◽  
Transcriptional Regulator ◽  
Hairpin Rna ◽  
High Sequence Similarity ◽  
Genes Encoding ◽  
Cognate Gene ◽  
Chromatin Packing ◽  
Nif Proteins

ABSTRACT Clustered within the genome of the oomycete phytopathogen Phytophthora infestans are four genes encoding spore-specific nuclear LIM interactor-interacting factors (NIF proteins, a type of transcriptional regulator) that are moderately conserved in DNA sequence. NIFC1, NIFC2, and NIFC3 are zoosporogenesis-induced and grouped within 4 kb, and 20 kb away resides a sporulation-induced form, NIFS. To test the function of the NIFC family, plasmids expressing full-length hairpin constructs of NIFC1 or NIFC2 were stably transformed into P. infestans. This triggered silencing of the cognate gene in about one-third of transformants, and all three NIFC genes were usually cosilenced. However, NIFS escaped silencing despite its high sequence similarity to the NIFC genes. Silencing of the three NIFC genes impaired zoospore cyst germination by 60% but did not affect other aspects of the life cycle. Silencing was transcriptional based on nuclear run-on assays and associated with tighter chromatin packing based on nuclease accessibility experiments. The chromatin alterations extended a few hundred nucleotides beyond the boundaries of the transcribed region of the NIFC cluster and were not associated with increased DNA methylation. A plasmid expressing a short hairpin RNA having sequence similarity only to NIFC1 silenced both that gene and an adjacent member of the gene cluster, likely due to the expansion of a heterochromatic domain from the targeted locus. These data help illuminate the mechanism of silencing in Phytophthora and suggest that caution should be used when interpreting silencing experiments involving closely spaced genes.

Comprehensive analysis of serum N-glycans as a biomarker for breast cancer.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21031-e21031
Author(s):  
Takayuki Ueno ◽  
Fumiaki Sato ◽  
Nobuhiko Shinkura ◽  
Taichi Aihara ◽  
Masao Fukushima ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
Tumor Size ◽  
Principal Component ◽  
High Specificity ◽  
Comprehensive Analysis ◽  
Her2 Status ◽  
Healthy Group ◽  
Cancer Group ◽  
Healthy Females

e21031 Background: Early detection of breast cancer is an important aspect to improve therapeutic outcome. Serum biomarkers have a number of advantages including less invasiveness and possible application for monitoring. Glycans are involved in many biological processes including cancer progression and promising candidates for cancer biomarker. Methods: Sera were collected from 54 patients with operable breast cancer and 118 healthy volunteers. N-glycans were isolated by BlotGlyco (Sumitomo Bakelite Co., LTD, Japan) and N-glycan profile was obtained by MALDI-TOF MS. The principal component analysis (PCA) was performed by SIMCA-P+ ver.12 (Umetrics). N-glycan compositions were estimated from mass spectra using 'GlycoMod' database. Results: Fifty-four pts (age: 31-78) and 118 healthy females (age: 29-73) were enrolled. The tumor size was between 9 and 107mm (median 29mm). Twenty six pts (48%) had lymph node involvement. Fifty three peaks of N-glycans were observed in more than 95% of the samples and utilized for the analyses. By PCA, no distinction was observed in sera form breast cancer pts in terms of the tumor size, hormone receptor status and HER2 status. The N-glycan signature to identify cancer pts was created based on the PCA. The signature categorized samples into three groups: cancer, healthy and border groups. Half of the samples (even registration number) were used for training and the rest for validation. In the validation cohort, 18 (66.7%) of 27 pts were categorized into the cancer group and 9 were into the border group. Forty-seven (79.7%) of 59 volunteers were categorized into the healthy group and 11 were into the border group. When the border group was included in the cancer group, the sensitivity was 100% (27 of 27 pts in the cancer group) and the specificity was 79.7% (47 of 59 volunteers in the healthy group). Conclusions: The comprehensive analysis of N-glycans in sera enabled the creation of the N-glycan signature to distinguish between breast cancer pts and healthy females with high sensitivity and high specificity. Confirmative studies are warranted.

Transcriptome analysis for identification of indigo biosynthesis pathway genes in Polygonum tinctorium

Biologia ◽  
2015 ◽  
Vol 70 (8) ◽  
Author(s):  
Yoshiko Minami ◽  
Bijaya Ketan Sarangi ◽  
Sanjog Tarachand Thul
Keyword(s):  
De Novo ◽  
Comprehensive Analysis ◽  
Blue Dye ◽  
Biosynthesis Pathway ◽  
Kegg Database ◽  
P450 Monooxygenase ◽  
Blast Search ◽  
Textile Dyeing ◽  
Genes Encoding ◽  
Polygonum Tinctorium

AbstractIndigo is the most important blue dye for textile dyeing and is biosynthesized in Polygonum tinctorium. Some biochemical studies related to biosynthesis are available. However, genomic and transcriptome studies have not received sufficient attention. Here, we report de novo assembly of transcriptome datasets and its comprehensive analysis. A total of 60,395 unigenes were annotated using BLAST search against the different databases. At least 23,721 unigenes mapped onto different pathways using KEGG database. We found that 3,323 genes are involved in biosynthesis of secondary metabolites, 117 phenylalanine, tyrosine and tryptophan biosynthesis and 147 tryptophan metabolisms. Apart from this, indigo biosynthesis pathway genes viz., dioxygenase, monooxygenase, and glucosyltransferase have also been identified. Fourteen genes encoding cytochrome P450 monooxygenase, 26 glucoside dioxygenase, 9 UDP-glucose D-glucosyltransferase and 52 were β-D-glucosidase. These findings provide a foundation for further analysis of this pathway with potential to enhance the synthesis of indican in P. tinctorium

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