Activity of NAD-dependent isocitrate dehydrogenase,isocitrate lyase,and malate dehydrogenase in <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of γ-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of γ-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP⁺ to NAD⁺ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.     

Riboflavin production by <Emphasis Type="Italic">Ashbya gossypii</Emphasis>

Riboflavin is an important nutrient for humans and animals. Industrial production has shifted completely from chemical synthesis to microbial fermentation. First generation riboflavin production was improved by a combination of traditional mutagenesis and genetic engineering, and isolated strains have been used in industry. As the DNA genome of riboflavin producers has the potential to reveal new technologies, DNA microarray, proteomic and metabolic analyses have been applied to the analysis of hyper-riboflavin producers. In this review, disparity mutagenesis technology is introduced as a means of improving riboflavin production by Ashbya gossypii. DNA microarray, proteomic and metabolic analyses of this high riboflavin producer are discussed, as well as recent riboflavin production trends, costs and future improvements.     

Kinetic model of functioning and regulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> isocitrate dehydrogenase

To describe published experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH), a Rapid Equilibrium Random Bi Ter mechanism involving the formation of two dead-end enzyme complexes is proposed and a kinetic model of enzyme functioning is constructed. The enzyme is shown to be regulated through reversible phosphorylation by IDH kinase/phosphatase; the latter, in its turn, is controlled by IDH substrates and also by a number of central metabolites—pyruvate, 3-phosphoglycerate, and AMP—reflecting the energy demand of the cell. Using the model, it is shown that an increase in the concentration of the above effectors raises the fraction of active IDH and thus enhances the Krebs cycle flux. The ratio between the free and the phosphorylated forms of IDH is more sensitive to AMP, NADP, and isocitrate than to pyruvate and 3-phosphoglycerate. The model also predicts changes in the ratio between phosphorylated and active forms of IDH in the Krebs cycle that occur with a change in the energy and biosynthetic loads on E. coli cells.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Adaptation of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to ethanol

Resistance of the yeast Yarrowia lipolytica to ethanol stress was studied under different ethanol concentrations and treatment duration. Cell viability was shown to increase in the variants including preliminary treatment with small doses of ethanol, oxidants, or soft thermal exposure. The study of the respiratory activity under ethanol stress revealed the involvement of an alternative cyanide-resistant oxidase in the adaptive response of the cells. The level of intracellular cAMP decreased in response to the action of ethanol, which correlated with increased activity of the antioxidant systems (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, glutathione reductase) and NAD⁺-dependent alcohol dehydrogenase.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Isopentenyl-diphosphate isomerase is essential for viability of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Homozygosity for a mutation in the idi-1 gene of Caenorhabditis elegans results in paralysis during the first larval stage, followed by an arrest of growth and development late in the first larval stage. Apoptotic corpses, which are apparently the result of normal programmed cell death, persist in the arrested larvae. In genetic mosaics, an additional defect becomes evident upon examination with Nomarski optics: cells that are genotypically mutant enlarge, and their cytoplasm becomes dimpled. Electron microscopy indicates that the dimpling reflects an accumulation of many enlarged lysosomes and autophagosomes. The mosaics demonstrate that the lethal mutation acts cell autonomously with respect to this vesicular abnormality and that there is a maternal effect with respect to the time of developmental arrest of mutant progeny. Cloning of the gene reveals that it is the only gene in C. elegans for isopentenyl-diphosphate isomerase, an enzyme that is important for the synthesis of lipophilic molecules, including farnesyl and geranyl diphosphates.     

Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

Characterization of the <Emphasis Type="Italic">AXDH</Emphasis> gene and the encoded xylitol dehydrogenase from the dimorphic yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD⁺ or NADH as coenzymes.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Structural and transcriptional analysis of<Emphasis Type="Italic"> S</Emphasis>-locus F-box genes in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S₂, very similar to S₂-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S₂ also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S₁S₅ and S₂S₄, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S₂-specific primers, TAC clones containing both AhSLF-S₂ and its homologs were subsequently identified (S₂TAC, S₅TACa, S₄TAC, and S₁TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S₂, -S₁, -S₄ and -S₅) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S₂ were identified. In total, three F-box genes (AhSLF-S₂, -S₂A and -S₂C) in S₂TAC (51 kb), three (AhSLF-S₄, -S₄A and -S₄D) in S₄TAC (75 kb), two (AhSLF-S₅ and -S₅A) in S₅TACa (55 kb), and two (AhSLF-S₁ and -S₁E) in S₁TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S₂C, -S₄D and -S₁E) were specifically expressed in pollen, similar to AhSLF-S₂, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535