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Biosynthetic Genes (biosynthetic + gene)
Terms modified by Biosynthetic Genes Selected AbstractsIdentifying Natural Product Biosynthetic Genes from a Soil Metagenome by Using T7 Phage SelectionCHEMBIOCHEM, Issue 16 2009Keya Zhang Doing the dirty work: Metagenomic DNA was directly isolated from soil bacteria to construct a T7 phage library. Iterative phage selection by protein modification catalyzed by phosphopantetheinyl transferase enriched putative acyl carrier protein and peptidyl carrier protein genes that are associated with natural product biosynthetic enzymes. [source] The AraC/XylS regulator TxtR modulates thaxtomin biosynthesis and virulence in Streptomyces scabiesMOLECULAR MICROBIOLOGY, Issue 3 2007Madhumita V. Joshi Summary Streptomyces scabies is the best studied of those streptomycetes that cause an economically important disease known as potato scab. The phytotoxin thaxtomin is made exclusively by these pathogens and is required for virulence. Here we describe regulation of thaxtomin biosynthesis by TxtR, a member of the AraC/XylS family of transcriptional regulators. The txtR gene is imbedded in the thaxtomin biosynthetic pathway and is located on a conserved pathogenicity island in S. scabies, S. turgidiscabies and S. acidiscabies. Thaxtomin biosynthesis was abolished and virulence was almost eliminated in the txtR deletion mutant of S. scabies 87.22. Accumulation of thaxtomin biosynthetic gene (txtA, txtB, txtC, nos) transcripts was reduced compared with the wild-type S. scabies 87.22. NOS-dependent nitric oxide production by S. scabies was also reduced in the mutant. The TxtR protein bound cellobiose, an inducer of thaxtomin production, and transcription of txtR and thaxtomin biosynthetic genes was upregulated in response to cellobiose. TxtR is the first example of an AraC/XylS family protein regulated by cellobiose. Together, these data suggest that cellobiose, the smallest oligomer of cellulose, may signal the availability of expanding plant tissue, which is the site of action of thaxtomin. [source] Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in StreptomycesMOLECULAR MICROBIOLOGY, Issue 4 2005Neil R. Williamson Summary The biosynthetic pathway of the red-pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP) and the bipyrrole, 4-methoxy-2,2,-bipyrrole-5-carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in-frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC-MS, cross-feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3-acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2). [source] Exploring the Phospholipid Biosynthetic Pathways of Aspergillus fumigatus by Computational Genome AnalysisENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 6 2005H. Do Abstract Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systematic diseases (invasive aspergillosis) with high mortality rates. In recent years, considerable progress in the genome sequencing of this fungus has been made by an international consortium, which includes the Wellcome Trust Sanger Institute (UK) and the Institute for Genome Research (USA). A tenfold whole genome shotgun sequence assembly of A. fumigatus has been made publicly available. In this study, it was attempted to identify the genes related to the phospholipid biosynthesis from the A. fumigatus genome by a gene prediction program (GlimmerM) and to reconstruct the metabolic pathway for phospholipids of A. fumigatus. Fifteen genes related to phospholipid pathway were identified in the A. fumigatus genomic sequence. The open reading frames predicted by GlimmerM showed a high amino acid sequence similarity with the other fungal phospholipid biosynthetic genes and well-conserved functional domains. The obtained results also demonstrated that the reconstructed pathway of A. fumigatus in phospholipid biosynthesis was very similar to that of other fungi such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, and Neurospora crassa. Therefore it is postulated that the antifungal drugs targeted for the biosynthesis of phospholipids could also be effective against A. fumigatus. [source] Proteorhodopsin photosystem gene clusters exhibit co-evolutionary trends and shared ancestry among diverse marine microbial phylaENVIRONMENTAL MICROBIOLOGY, Issue 4 2007Jay McCarren Summary Since the recent discovery of retinylidene proteins in marine bacteria (proteorhodopsins), the estimated abundance and diversity of this gene family has expanded rapidly. To explore proteorhodopsin photosystem evolutionary and distributional trends, we identified and compared 16 different proteorhodopsin-containing genome fragments recovered from naturally occurring bacterioplankton populations. In addition to finding several deep-branching proteorhodopsin sequences, proteorhodopsins were found in novel taxonomic contexts, including a betaproteobacterium and a planctomycete. Approximately one-third of the proteorhodopsin-containing genome fragments analysed, as well as a number of recently reported marine bacterial whole genome sequences, contained a linked set of genes required for biosynthesis of the rhodopsin chromophore, retinal. Phylogenetic analyses of the retinal biosynthetic genes suggested their co-evolution and probable coordinated lateral gene transfer into disparate lineages, including Euryarchaeota, Planctomycetales, and three different proteobacterial lineages. The lateral transfer and retention of genes required to assemble a functional proteorhodopsin photosystem appears to be a coordinated and relatively frequent evolutionary event. Strong selection pressure apparently acts to preserve these light-dependent photosystems in diverse marine microbial lineages. [source] Targeting clusters of transferred genes in Thermotoga maritimaENVIRONMENTAL MICROBIOLOGY, Issue 11 2003Camilla L. Nesbø Summary We screened a Thermotoga sp. strain RQ2 lambda library for genes present in that strain but absent from the closely related completely sequenced relative Thermotoga maritima strain MSB8, by using probes generated in an earlier genomic subtraction study. Five lambda insert fragments were sequenced, containing, respectively, an archaeal type ATPase operon, rhamnose biosynthetic genes, ORFs with similarity to an arabinosidase, a Thermotoga sp. strain RQ2-specific alcohol dehydrogenase and a novel archaeal Mut-S homologue. All but one of these fragments contained additional Thermotoga sp. strain RQ2-specific sequences not screened for, suggesting that many such strain-specific genes will be found clustered in the genome. Moreover, phylogenetic analyses, phylogenetic distribution and/or G + C content suggests that all the Thermotoga sp. strain RQ2 specific sequences in the sequenced lambda clones have been acquired by lateral gene transfer. We suggest that the use of strain-specific small insert clones obtained by subtractive hybridization to target larger inserts for sequencing is an efficient, economical way to identify environmentally (or clinically) relevant interstrain differences and novel gene clusters, and will be invaluable in comparative genomics. [source] Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B. subtilis/E. coli shuttle vector systemFEMS MICROBIOLOGY LETTERS, Issue 2 2002Sascha Doekel Abstract A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins. [source] Adaptive tolerance to oxidative stress and the induction of antioxidant enzymatic activities in Candida albicans are independent of the Hog1 and Cap1-mediated pathwaysFEMS YEAST RESEARCH, Issue 6 2010Pilar Gónzalez-Párraga Abstract In the pathogenic yeast Candida albicans, the MAP-kinase Hog1 mediates an essential protective role against oxidative stress, a feature shared with the transcription factor Cap1. We analysed the adaptive oxidative response of strains with both elements altered. Pretreatment with gentle doses of oxidants or thermal upshifts (28,37 and 37,42 °C) improved survival in the face of high concentrations of oxidants (50 mM H2O2 or 40 mM menadione), pointing to a functional cross-protective mechanism in the mutants. The oxidative challenge promoted a marked intracellular synthesis of trehalose, although hog1 (but not cap1) cells always displayed high basal trehalose levels. Hydrogen peroxide (H2O2) induced mRNA expression of the trehalose biosynthetic genes (TPS1 and TPS2) in the tested strains. Furthermore, oxidative stress also triggered a differential activation of various antioxidant activities, whose intensity was greater after HOG1 and CAP1 deletion. The pattern of activity was dependent on the oxidant dosage applied: low concentrations of H2O2 (0.5,5 mM) clearly induced catalase and glutathione reductase (GR), whereas drastic H2O2 exposure (50 mM) increased Mn-superoxide dismutase (SOD) isozyme-mediated SOD activity. These results firmly support the existence in C. albicans of both Hog1- and Cap1-independent mechanisms against oxidative stress. [source] Identification of direct and indirect targets of the Gln3 and Gat1 activators by transcriptional profiling in response to nitrogen availability in the short and long termFEMS YEAST RESEARCH, Issue 5 2006Bart Scherens Abstract Nitrogen catabolite repression (NCR) consists in the specific inhibition of transcriptional activation of genes encoding the permeases and catabolic enzymes needed to degrade poor nitrogen sources. Under nitrogen limitation or rapamycin treatment, NCR genes are activated by Gln3 or Gat1, or by both factors. To compare the sets of genes responding to rapamycin or to nitrogen limitation, we used DNA microarrays to establishing the expression profiles of a wild type strain, and of a double gln3,,gat1, strain, grown on glutamine, after addition of rapamycin, on proline, or after a shift from glutamine to proline. Analysis of microarray data revealed 392 genes whose expression was dependent on the nitrogen source quality. 91 genes were activated in a GATA factor-dependent manner in all growth conditions, suggesting a direct role of Gln3 and Gat1 in their expression. Other genes were only transiently up-regulated (stress-responsive genes) or down-regulated (genes encoding ribosomal proteins and translational factors) upon nitrogen limitation, and this regulation was delayed in a gln3,,gat1, strain. Repression of amino acid and nucleotide biosynthetic genes after a nitrogen shift did not depend on Gcn4. Several transporter genes were repressed as a consequence of enhanced levels of NCR-responsive permeases present at the plasma membrane. [source] Dothistroma (red-band) needle blight of pines and the dothistromin toxin: a reviewFOREST PATHOLOGY, Issue 3 2004R. E. Bradshaw Summary Dothistroma (red-band) needle blight has been a problem in plantations of exotic pines in the southern hemisphere for many decades. The prevalence of this disease is currently increasing in the northern hemisphere and is now affecting trees in their native ranges. The fungal pathogen Mycosphaerella pini with its anamorph Dothistroma pini, which is responsible for the disease, produces a toxin, dothistromin, that is closely related to the potent carcinogen, aflatoxin. Understandably this has provoked concern about possible effects on the health of forestry workers. This review gives a broad coverage of literature on both disease and toxin. The fungus has a complicated taxonomy with many synonyms and in most countries only the anamorph is found. It is a necrotrophic pathogen that kills needle tissue and completes its life cycle in the lesion thus formed. Dispersal of the disease is normally by rain splash of conidiospores but there is evidence that long range dispersal has occurred by transport of contaminated plant tissue and by wind/cloud dispersal of spores in air currents. The severity of disease is affected by humidity, temperature and light. There is variation in susceptibility of different Pinus species and some achieve increased resistance with age. The current method of control in southern hemisphere plantation forests is through spraying with copper fungicides and, with P. radiata, increased disease resistance has been achieved through a breeding programme. The dothistromin toxin is a difuroanthraquinone and is similar in structure to the aflatoxin precursor versicolorin B. Part of a gene cluster encoding dothistromin biosynthetic genes has been cloned and this has confirmed parallels between the dothistromin and aflatoxin biosynthetic pathways. Dothistromin produces damaging oxygen radicals by reductive oxygen activation rather than by photosensitization, but is also thought to exert its toxic effects on specific cellular targets. Studies have shown that dothistromin is a weak mutagen and clastogen and is therefore a potential carcinogen. Although the risks to forest workers are considered very low it is prudent to avoid unnecessary exposure during periods when dothistromin levels are likely to be at their peak. Résumé La maladie des bandes rouges causée par Dothistroma est un problème dans les plantations de pins exotiques de l'hémisphère sud depuis de nombreuses années. La prévalence de cette maladie est en augmentation dans l'hémisphère nord et affecte maintenant les pins dans leurs régions d'origine. Le champignon pathogène Dothistroma pini, responsable de la maladie, produit une toxine, la dothistromine, proche de l'aflatoxine qui est un puissant carcinogène. Ceci pose donc la question des effets possibles sur la santé des travailleurs forestiers. Cette revue repose sur une large couverture de la littérature concernant aussi bien la maladie que la toxine. Le champignon a une taxonomie complexe avec de nombreux synonymes, et seul l'anamorphe se rencontre dans de nombreux pays. C'est un champignon nécrotrophe qui tue les tissus de l'aiguille et réalise son cycle biologique dans la lésion ainsi formée. La dissémination de la maladie s'effectue principalement par éclaboussures de pluie contenant les conidies mais une dissémination à longue distance a été mise en évidence par transport de matériel contaminé ou par dissémination des spores par le vent ou les nuages dans les courants aériens. La sévérité de la maladie est affectée par l'humidité, la température et la lumière. Il existe des différences de sensibilité entre espèces de Pinus, et certaines présentent une résistance accrue avec l'âge. La méthode actuelle de lutte dans les forêts de plantations de l'hémisphère sud consiste à pulvériser des fongicides à base de cuivre ; dans le cas de Pinus radiata, une augmentation de la résistance a été obtenue grâce à un programme d'amélioration génétique. La toxine dothistromine est une difuroanthraquinone, similaire en structure à la versicolorine B, précurseur de l'aflatoxine. Une partie d'une batterie de gènes comprenant des gènes de biosynthèse de la dothistromine a été clonée, confirmant les analogies entre les voies de biosynthèse de la dothistromine et de l'aflatoxine. La dothistromine produit des radicaux oxygène nocifs par activation de la réduction de l'oxygène plutôt que par photosensibilisation, mais ses effets toxiques s'exercent aussi probablement sur des sites cellulaires spécifiques. Des études montrent que la dothistromine est un mutagène et clastogène faible, et donc potentiellement carcinogène. Bien que les risques pour les ouvriers forestiers soient considérés comme très faibles, il est prudent d'éviter dans la mesure du possible de s'exposer dans les périodes où les niveaux de dothistromine sont supposés élevés. Zusammenfassung Die Dothistroma -Nadelbräune ist in der Südhemisphäre in Plantagen mit exotischen Kiefernarten seit vielen Jahren ein Problem. In der Nordhemisphäre nimmt die Bedeutung dieser Krankheit derzeit zu und sie befällt nun Bäume auch in ihren natürlichen Verbreitungsgebieten. Der Erreger ist der Ascomycet Mycosphaerella pini (Anamorphe: Dothistroma pini). Der Pilz bildet das Toxin Dothistromin, das eng mit dem hochtoxischen Karzinogen Aflatoxin verwandt ist. Daraus ergab sich die Frage nach möglichen Nebenwirkungen dieser Baumkrankheit auf die Gesundheit von Waldarbeitern. Dieser Review fasst die Information über die Krankheit und das Toxin zusammen. Der Pilz hat eine komplizierte Taxonomie mit vielen Synonymen und in den meisten Ländern wurde nur die Anamorphe nachgewiesen. Er ist ein nekrotrophes Pathogen, das Blattgewebe abtötet, und in den so gebildeten Läsionen seinen Lebenszyklus abschliesst. Der normale Ausbreitungsweg der Krankheit erfolgt über Konidiosporen mit Regentropfen, aber es gibt auch Hinweise auf einen Ferntransport mit infiziertem Pflanzenmaterial und über die Verbreitung von Sporen mit dem Wind bzw. Wolken in Luftströmungen. Die Krankheitsintensität wird durch Luftfeuchte, Temperatur und Licht beeinflusst. Es gibt Unterschiede in der Anfälligkeit zwischen verschiedenen Kiefernarten und manche davon werden mit zunehmendem Alter resistenter. Derzeit werden in Plantagen der südlichen Hemisphäre Kupferfungizide zur Kontrolle dieser Krankheit eingesetzt und für Pinus radiata wurde in Züchtungsprogrammen eine erhöhte Resistenz erreicht. Das Toxin Dothistromin ist ein Difuroanthrachinon und ähnelt in seiner Struktur dem Aflatoxin-Präkursor Versicolorin B. Ein Teil des Genclusters, das die Dothistromin-Biosynthese codiert, wurde geklont, und es wurden dabei Parallelen zwischen dem Dothistromin- und dem Aflatoxin-Biosyntheseweg bestätigt. Dothistromin bildet schädliche Sauerstoffradikale (wahrscheinlich eher durch reduktive Sauerstoffaktivierung als durch Photosensibilisierung), es dürfte aber auch auf spezifische Zellkomponenten toxisch wirken. Dothistromin zeigt schwache mutagene und chromosomenschädigende Wirkungen und ist deshalb ein potentielles Karzinogen. Obwohl das Risiko für Waldarbeiter als gering eingeschätzt wird, sollte man in Perioden, in denen der Dothistromingehalt hoch sein dürfte, eine unnötige Exposition vermeiden. [source] The nuclear bile acid receptor FXR as a novel therapeutic target in cholestatic liver diseases: Hype or hope?HEPATOLOGY, Issue 1 2004Michael Trauner M.D. Farnesoid X receptor (FXR) is a bile acid,activated transcription factor that is a member of the nuclear hormone receptor superfamily. FXR-null mice exhibit a phenotype similar to Byler disease, an inherited cholestatic liver disorder. In the liver, activation of FXR induces transcription of transporter genes involved in promoting bile acid clearance and represses genes involved in bile acid biosynthesis. We investigated whether the synthetic FXR agonist GW4064 could protect against cholestatic liver damage in rat models of extrahepatic and intrahepatic cholestasis. In the bile duct ligation and alpha-naphthylisothiocyanate models of cholestasis, GW4064 treatment resulted in significant reductions in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, as well as other markers of liver damage. Rats that received GW4064 treatment also had decreased incidence and extent of necrosis, decreased inflammatory cell infiltration, and decreased bile duct proliferation. Analysis of gene expression in livers from GW4064-treated cholestatic rats revealed decreased expression of bile acid biosynthetic genes and increased expression of genes involved in bile acid transport, including the phospholipid flippase MDR2. The hepatoprotection seen in these animal models by the synthetic FXR agonist suggests FXR agonists may be useful in the treatment of cholestatic liver disease. [source] Identification and characterization of three novel EsaI/EsaR quorum-sensing controlled stewartan exopolysaccharide biosynthetic genes in Pantoea stewartii ssp. stewartiiMOLECULAR MICROBIOLOGY, Issue 4 2009Aurélien Carlier First page of article [source] Transcription regulation of the Saccharomyces cerevisiae PIS1 gene by inositol and the pleiotropic regulator, Ume6pMOLECULAR MICROBIOLOGY, Issue 6 2008Niketa M. Jani Summary In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p. [source] The AraC/XylS regulator TxtR modulates thaxtomin biosynthesis and virulence in Streptomyces scabiesMOLECULAR MICROBIOLOGY, Issue 3 2007Madhumita V. Joshi Summary Streptomyces scabies is the best studied of those streptomycetes that cause an economically important disease known as potato scab. The phytotoxin thaxtomin is made exclusively by these pathogens and is required for virulence. Here we describe regulation of thaxtomin biosynthesis by TxtR, a member of the AraC/XylS family of transcriptional regulators. The txtR gene is imbedded in the thaxtomin biosynthetic pathway and is located on a conserved pathogenicity island in S. scabies, S. turgidiscabies and S. acidiscabies. Thaxtomin biosynthesis was abolished and virulence was almost eliminated in the txtR deletion mutant of S. scabies 87.22. Accumulation of thaxtomin biosynthetic gene (txtA, txtB, txtC, nos) transcripts was reduced compared with the wild-type S. scabies 87.22. NOS-dependent nitric oxide production by S. scabies was also reduced in the mutant. The TxtR protein bound cellobiose, an inducer of thaxtomin production, and transcription of txtR and thaxtomin biosynthetic genes was upregulated in response to cellobiose. TxtR is the first example of an AraC/XylS family protein regulated by cellobiose. Together, these data suggest that cellobiose, the smallest oligomer of cellulose, may signal the availability of expanding plant tissue, which is the site of action of thaxtomin. [source] Deciphering regulatory mechanisms for secondary metabolite production in the myxobacterium Sorangium cellulosum So ce56MOLECULAR MICROBIOLOGY, Issue 6 2007Shwan Rachid Summary Sorangium cellulosum strains produce approximately 50% of the biologically active secondary metabolites known from myxobacteria. These metabolites include several compounds of biotechnological importance such as the epothilones and chivosazols, which, respectively, stabilize the tubulin and actin skeletons of eukaryotic cells. S. cellulosum is characterized by its slow growth rate, and natural products are typically produced in low yield. In this study, biomagnetic bead separation of promoter-binding proteins and subsequent inactivation experiments were employed to identify the chivosazol regulator, ChiR, as a positive regulator of chivosazol biosynthesis in the genome-sequenced strain So ce56. Overexpression of chiR under the control of T7A1 promoter in a merodiploid mutant resulted in fivefold overproduction of chivosazol in a kinetic shake flask experiment, and 2.5-fold overproduction by fermentation. Using quantitative reverse transcription PCR and gel shift experiments employing heterologously expressed ChiR, we have shown that transcription of the chivosazol biosynthetic genes (chiA,chiF) is directly controlled by this protein. In addition, we have demonstrated that ChiR serves as a pleiotropic regulator in S. cellulosum, because mutant strains lack the ability to develop into regular fruiting bodies. [source] Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibioticsMOLECULAR MICROBIOLOGY, Issue 6 2003Nancy A. Buchmeier Summary Mycothiol, MSH or 1d - myo -inosityl 2-(N -acetyl- l -cysteinyl)amido-2-deoxy- , - d -glucopyranoside, is an unusual conjugate of N -acetylcysteine (AcCys) with 1d - myo -inosityl 2-acetamido-2-deoxy-,- d -glucopyranoside (GlcN-Ins), and is the major low-molecular-mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced , 20% of wild-type levels of MSH. Levels of the Rv1170 substrate, GlcNAc-Ins, were elevated, whereas those of the product, GlcN-Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc-Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first-line antituberculosis drugs. [source] GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genesMOLECULAR MICROBIOLOGY, Issue 5 2002Urs A. Ochsner Summary Upon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores, exotoxin, proteases and haemolysin. The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatory genes, such as pvdS, which encodes an alternative sigma factor. This circuit leads to a hierarchical cascade of direct and indirect iron regulation. We used the GeneChip® to analyse the global gene expression profiles in response to iron. In iron-starved cells, the expression of 118 genes was increased at least fivefold compared with that in iron-replete cells, whereas the expression of 87 genes was decreased at least fivefold. The GeneChip® data correlated well with results obtained using individual lacZ gene fusions. Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport. A low-iron milieu led to increased expression of the genes encoding TonB, alkaline protease, PrpL protease, exotoxin A, as well as fumarase C, Mn-dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases. Iron-controlled regulatory genes included seven alternative sigma factors and five other transcriptional regulators. Roughly 20% of the iron-regulated genes encoded proteins of unknown function and lacked any conclusive homologies. Under low-iron conditions, expression of 26 genes or operons was reduced in a ,pvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes. The GeneChip® proved to be a very useful tool for rapid gene expression analysis and identification of novel genes controlled by Fur or PvdS. [source] Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the ,R regulonMOLECULAR MICROBIOLOGY, Issue 4 2001Mark S. B. Paget In the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2), the thiol-disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, ,R, and its cognate anti-sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for ,R, thereby releasing ,R to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for ,R target promoters to identify 27 new ,R target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin-like protein and enzymes involved in the biosynthesis of the low-molecular-weight thiol-containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the ,R targets. Three ,R target genes encode ribosome-associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another ,R target gene was found to be a novel RNA polymerase-associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the ,R homologue in the actinomycete pathogen Mycobacterium tuberculosis. [source] Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in ArabidopsisNEW PHYTOLOGIST, Issue 4 2008Elena Loreti Summary ,,Anthocyanins are secondary metabolites, which play an important role in the physiology of plants. Both sucrose and hormones regulate anthocyanin synthesis. Here, the interplay between sucrose and plant hormones was investigated in the expression of sucrose-regulated genes coding for anthocyanin biosynthetic enzymes in Arabidopsis seedlings. ,,The expression pattern of 14 genes involved in the anthocyanin biosynthetic pathway, including two transcription factors (PAP1, PAP2), was analysed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in Arabidopsis seedlings treated with sucrose and plant hormones. ,,Sucrose-induction of the anthocyanin synthesis pathway was repressed by the addition of gibberellic acid (GA) whereas jasmonate (JA) and abscisic acid (ABA) had a synergic effect with sucrose. The gai mutant was less sensitive to GA-dependent repression of dihydroflavonol reductase. This would seem to prove that GAI signalling is involved in the crosstalk between sucrose and GA in wild-type Arabidopsis seedlings. Conversely, the inductive effect of sucrose was not strictly ABA mediated. Sucrose induction of anthocyanin genes required the COI1 gene, but not JAR1, which suggests a possible convergence of the jasmonate- and sucrose-signalling pathways. ,,The results suggest the existence of a crosstalk between the sucrose and hormone signalling pathways in the regulation of the anthocyanin biosynthetic pathway. [source] HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis thalianaNEW PHYTOLOGIST, Issue 3 2008Tamara Gigolashvili Summary ,,In a previous transactivation screen, two Arabidopsis thaliana R2R3-MYB transcription factors, HAG2/MYB76 and HAG3/MYB29, along with the already characterized HAG1/MYB28, were identified as putative regulators of aliphatic glucosinolate biosynthesis. ,,Molecular and biochemical characterization of HAG2/MYB76 and HAG3/MYB29 functions was performed using transformants with increased or repressed transcript levels. Real-time PCR assays, cotransformation assays and measurements of glucosinolate contents were used to assess the impact of both MYB factors on the steady-state level of glucosinolate biosynthetic genes and accumulation of aliphatic glucosinolates. ,,Both HAG2/MYB76 and HAG3/MYB29 were shown to be positive regulators of aliphatic glucosinolate biosynthesis. Expression of promoter-,-glucuronidase (GUS) fusions indicated GUS activities in both vegetative and generative organs, with distinct characteristics for each MYB factor. HAG1/MYB28, HAG2/MYB76 and HAG3/MYB29 reciprocally transactivated each other in the control of aliphatic glucosinolate biosynthesis and downregulated the expression of genes involved in the control of indolic glucosinolate biosynthesis, pointing to a reciprocal negative regulation of these two pathways. ,,All three HAG transcription factors exert a coordinated control on aliphatic glucosinolate biosynthesis. [source] Relative contributions of nine genes in the pathway of histidine biosynthesis to the control of free histidine concentrations in Arabidopsis thalianaPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2009Jonathan D. Rees Summary Despite the functional importance of histidine (His) as an essential amino acid in proteins and as a metal-coordinating ligand, comparatively little is known about the regulation of its biosynthesis in plants and the potential for metabolic engineering of this pathway. To investigate the contribution of different steps in the pathway to overall control of His biosynthesis, nine His biosynthetic genes were individually over-expressed in Arabidopsis thaliana to determine their effects on free amino acid pools. Constitutive, CaMV 35S -driven over-expression of the cDNAs encoding either isoform of ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme in the pathway, was sufficient to increase the pool of free His by up to 42-fold in shoot tissue of Arabidopsis, with negligible effect on any other amino acid. In contrast, over-expression of cDNAs for seven other enzymes in the biosynthetic pathway had no effect on His content, suggesting that control of the pool of free His resides largely with ATP-PRT activity. Over-expression of ATP-PRT and increased His content had a negative pleiotropic effect on plant biomass production in 35S:PRT1 lines, but this effect was not observed in 35S:PRT2 lines. In the presence of 100 µM Ni, which was inhibitory to wild-type plants, a strong positive correlation was observed between free His content and biomass production, indicating that the metabolic cost of His overproduction was outweighed by the benefit of increased tolerance to Ni. His-overproducing plants also displayed somewhat elevated tolerance to Co and Zn, but not to Cd or Cu, indicating chemical selectivity in intracellular metal binding by His. [source] Inulin metabolism in dicots: chicory as a model systemPLANT CELL & ENVIRONMENT, Issue 6 2002A. Van Laere Abstract Fructans are fructose-based oligo- and polymers that serve as reserve carbohydrates in many plant species. The original Edelman and Jefford model for GFn inulin type fructan biosynthesis was confirmed by the de novo synthesis of fructans in vitro as well as by heterologous expression of the respective cDNAs in non-fructan plants. Now the model can be extended for the biosynthesis of Fn inulin type fructans. Recent progress has now succeeded in elucidating the biochemistry and molecular biology of fructan biodegradation in chicory, an economically important species used for commercial inulin extraction. Unlike fructan biosynthetic genes that originated from vacuolar type invertase, plant fructan exohydrolases seem to have evolved from a cell wall invertase ancestor gene that later obtained a low iso-electric point and a vacuolar targeting signal. Using chicory as a model system, expression analysis revealed that fructan enzymes are mainly controlled at the transcriptional level. [source] Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in ArabidopsisTHE PLANT JOURNAL, Issue 6 2009Sébastien Baud Summary The WRINKLED1 (WRI1) protein is an important regulator of oil accumulation in maturing Arabidopsis seeds. WRI1 is a member of a plant-specific family of transcription factors (AP2/EREBP) that share either one or two copies of a DNA-binding domain called the AP2 domain. Here, it is shown that WRI1 acts as a transcriptional enhancer of genes involved in carbon metabolism in transgenic seeds overexpressing this transcription factor. PKp-,1 and BCCP2, two genes encoding enzymes of the glycolysis and fatty acid biosynthetic pathway, respectively, have been chosen to investigate the regulatory action exerted by WRI1 over these pathways. Using the reporter gene uidA, it was possible to demonstrate in planta that WRI1 regulates the activity of both PKp-,1 and BCCP2 promoters. Electrophoretic mobility-shift assays and yeast one-hybrid experiments showed that WRI1 was able to interact with the BCCP2 promoter. To further elucidate the regulatory mechanism controlling the transcription of these genes, functional dissections of PKp-,1 and BCCP2 promoters were performed. Two enhancers, of 54 and 79 bp, respectively, have thus been isolated that are essential to direct the activity of these promoters in oil-accumulating tissues of the embryo. A consensus site is present in these enhancers as well as in other putative target promoters of WRI1. Loss of this consensus sequence in the BCPP2 promoter decreases both the strength of the interaction between WRI1 and this promoter in yeast and the activity of the promoter in planta. [source] Recent developments in bisintercalator natural productsBIOPOLYMERS, Issue 9 2010Olga E. Zolova Abstract The bisintercalator natural products are a family of nonribosomal peptides possessing a range of biological properties that include antiviral, antibiotic, and anticancer activities. The name bisintercalator is derived from the ability to directly bind to duplex DNA through two planar intercalating moieties. Although 19 members of this family of compounds have been identified over the past 50 years, the biosynthetic genes responsible for the formation of four of these molecules (thiocoraline, SW-163, triostin A, and echinomycin) were identified only recently. This recent progress opens an avenue towards understanding how Nature produces these bisintercalating products and provides the potential to develop and identify novel potent analogous lead compounds for clinical applications. This review discusses the mode of action of bisintercalators and summarizes recent genetic and biochemical insights into their biosynthetic production, analog formation, and possible mechanisms by which resistance to these compounds is achieved by their producing organisms. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 777,790, 2010. [source] Metabolic engineering of Escherichia coli for the production of putrescine: A four carbon diamineBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Zhi-Gang Qian Abstract A four carbon linear chain diamine, putrescine (1,4-diaminobutane), is an important platform chemical having a wide range of applications in chemical industry. Biotechnological production of putrescine from renewable feedstock is a promising alternative to the chemical synthesis that originates from non-renewable petroleum. Here we report development of a metabolically engineered strain of Escherichia coli that produces putrescine at high titer in glucose mineral salts medium. First, a base strain was constructed by inactivating the putrescine degradation and utilization pathways, and deleting the ornithine carbamoyltransferase chain I gene argI to make more precursors available for putrescine synthesis. Next, ornithine decarboxylase, which converts ornithine to putrescine, was amplified by a combination of plasmid-based and chromosome-based overexpression of the coding genes under the strong tac or trc promoter. Furthermore, the ornithine biosynthetic genes (argC-E) were overexpressed from the trc promoter, which replaced the native promoter in the genome, to increase the ornithine pool. Finally, strain performance was further improved by the deletion of the stress responsive RNA polymerase sigma factor RpoS, a well-known global transcription regulator that controls the expression of ca. 10% of the E. coli genes. The final engineered E. coli strain was able to produce 1.68,g,L,1 of putrescine with a yield of 0.168,g,g,1 glucose. Furthermore, high cell density cultivation allowed production of 24.2,g,L,1 of putrescine with a productivity of 0.75,g,L,1,h,1. The strategy reported here should be useful for the bio-based production of putrescine from renewable resources, and also for the development of strains capable of producing other diamines, which are important as nitrogen-containing platform chemicals. Biotechnol. Bioeng. 2009; 104: 651,662 © 2009 Wiley Periodicals, Inc. [source] The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricinCELLULAR MICROBIOLOGY, Issue 8 2008Pradeep K. Gupta Summary Diphthamide is a post-translational derivative of histidine in protein synthesis elongation factor-2 (eEF-2) that is present in all eukaryotes with no known normal physiological role. Five proteins Dph1,Dph5 are required for the biosynthesis of diphthamide. Chinese hamster ovary (CHO) cells mutated in the biosynthetic genes lack diphthamide and are resistant to bacterial toxins such as diphtheria toxin. We found that diphthamide-deficient cultured cells were threefold more sensitive than their parental cells towards ricin, a ribosome- inactivating protein (RIP). RIPs bind to ribosomes at the same site as eEF-2 and cleave the large ribosomal RNA, inhibiting translation and causing cell death. We hypothesized that one role of diphthamide may be to protect ribosomes, and therefore all eukaryotic life forms, from RIPs, which are widely distributed in nature. A protective role of diphthamide against ricin was further demonstrated by complementation where dph mutant CHO cells transfected with the corresponding DPH gene acquired increased resistance to ricin in comparison with the control-transfected cells, and resembled the parental CHO cells in their response to the toxin. These data show that the presence of diphthamide in eEF-2 provides protection against ricin and suggest the hypothesis that diphthamide may have evolved to provide protection against RIPs. [source] | ||||||||||||||||||||||||||||