Biotransformation of racemic diisophorone by <Emphasis Type="Italic">Cephalosporium aphidicola</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Racemic diisophorone (500 mg) was converted by Cephalosporium aphidicola and Neurospora crassa over 10 days at 25 °C to 8β-hydroxydiisophorone in yields of 10% (52 mg) and 20% (103 mg), respectively. The structure was established by IR, specific rotation, mass spectral, 1D and 2D-NMR studies.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 8 April 2005 and 10 May 2005     

Molecular and biochemical characterization of squalene synthase from <Emphasis Type="Italic">Siraitia grosvenorii</Emphasis>

Objectives

To clone and characterize the squalene synthase from Siraitia grosvenorii (SgSQS).

Results

The gene encoding SgSQS was cloned. SgSQS has 417 amino acid residues with an pI of 7.3. There are 32 phosphorylation sites in its sequence: S⁴⁸ as well as S¹⁹⁶ play important roles in regulation of enzyme activity. The enzyme is a monomeric protein with a cave-like active center formed by α helixes and has two transmembrane domains at its C-terminus. SgSQS mRNA expression in stem and root were about twice as much as that in leaf and peel. Full-length SgSQS with measurable catalytic activity was expressed in Escherichia coli. SgSQS activity was optimal at 37 °C and pH 7.5 respectively.

Conclusion

SgSQS gene was cloned, and the molecular structure and biochemical function of SgSQS were characterized.

     

Identification of a type-D feruloyl esterase from<Emphasis Type="Italic"> Neurospora crassa</Emphasis>

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri–food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.     

Effect of oxylipins on <Emphasis Type="Italic">Neurospora crassa</Emphasis> growth and differentiation

The effect of the natural oxylipins 3(R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE) and 18-hydroxy-(9Z,12Z)-octadecadienoic acids (18-HODE) on the growth and hypha aggregation, as well as on some light-depending processes, such as carotenoid biosynthesis, protoperithecia formation (sexual cycle), and conidiation (asexual cycle), of the ascomycete Neurospora crassa was studied. Hypha aggregation and growth slowdown were induced by 3-HETE, 18-HODE, and linoleic acid. At concentrations from 5 to 50 μM, these compounds had no significant effect on the light-induced carotenogenesis. At the same time, these 3-HETE and 18-HODE concentrations, unlike linoleic acid, induced the formation of protoperithecia in the dark. At the concentration of 5 μM, an additive effect of oxylipins and light was revealed. The studied oxylipins had different effects on the asexual reproduction of N. crassa: 3-HETE induced conidiation in the dark, whereas 18-HODE induced conidiation in the light. The possible involvement of oxylipins in the regulation of the processes of sexual and asexual reproduction of N. crassa is discussed.     

Tip growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis> under glucose deprivation

Growth parameters of vegetative hyphae and isolated tip fragments of the mycelial fungus N. crassa were studied after complete substitution of an easily metabolized carbon source (glucose) for a non-metabolized one (sorbitol). The images of growing tips were recorded at 20–30-min intervals. Using original image processing software, geometrical parameters of the hyphal trees (length and number of branches, area of convex polygons circumscribed about the hyphal trees, etc.) were determined and growth characteristics, such as rate of tip elongation (V) and the ratio of the total hyphal length to the number of growing tips (termed “hyphal growth unit”, HGU), were calculated. It is shown that after 4–5-h growth in sorbitol-enriched media growth characteristics of intact hyphae did not differ significantly from the corresponding parameters of hyphae growing in glucose-enriched media. In isolated tip fragments (about 800-μ m long), the values of V were lower than those in intact hyphae but did not depend on the carbon source in the nutrient media. However, in such fragments growing in sorbitol-enriched media the number of branches decreased, while the HGU value and the number of large intracellular vacuoles increased. Staining of cells with a standard chitin probe, Calcofluor White (10 μg/ml), did not reveal any considerable differences in hyphal cell walls and septa in tip fragments grown in the presence of different carbon sources. Possible mechanisms of the dependence of the tip growth parameters on the glucose deficiency are discussed.     

Dynamics of mitochondria during the <Emphasis Type="Italic">Neurospora crassa</Emphasis> tip growth

Tip growth of the mycelial fungus N. crassa vegetative hyphae is realized owing to the combined activities of tens of the cells and diverse intracellular structures, such as microvesicles, microtubules, microfilaments, mitochondria, etc. Using a vital mitochondrial probe Mitotracker Red (10 μM, 10 min) we have found that the same mitochondria can move hundreds of microns along the hyphae within several hours. Analysis of the mitochondria distribution along 100 μm of the tips in intact hyphae as well as in the isolated apical fragments has shown that the congregation of mitochondria in the growing tips can correlate with the rate of elongation. These data together with the earlier electrophysiological estimations of the membrane potential gradients along the hyphal tips suggest that the electrical gradients along the hyphal apical part can be involved in the regulation of the energy supply of the tip growth.     

Molecular genetics of the<Emphasis Type="Italic"> Alhambra</Emphasis> (<Emphasis Type="Italic"> Drosophila AF10</Emphasis>) complex locus of<Emphasis Type="Italic"> Drosophila</Emphasis>

The Alhambra ( Alh) gene is the Drosophila homologue of the human AF10 gene. AF10 has been identified as a fusion partner of MLL, a human homologue of the fly gene trithorax, in infant leukemias. The endogenous function of human AF10 is not known, but may be vital to its role in acute leukemia. This prompted us to analyse Alh function. We describe here the genetic organisation of the Alh locus in D. melanogaster. We show that an independent lethal complementation group encoding a muscle protein ( Mlp84B) is located within an Alh intron. We have already shown that the leucine zipper (LZ) domain of ALH activates several Polycomb group-responsive elements. We further demonstrate that the LZ domain on its own bears the Alh vital function, since it is necessary and sufficient for rescue of Alh mutant lethality. Finally, we demonstrate that, in contrast to a previous report, Alh does not affect position-effect variegation.Communicated by G. Reuter     

Molecular analysis of the<Emphasis Type="Italic"> CRINKLY4</Emphasis> gene family in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The maize (Zea mays L.) CRINKLY4 (CR4) gene encodes a serine/threonine receptor-like kinase that controls an array of developmental processes in the plant and endosperm. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The available genome sequence of rice (Oryza sativa L.) encodes a CR4 ortholog, OsCR4, and four CRR proteins: OsCRR1, OsCRR2, OsCRR3 and OsCRR4, not necessarily orthologous to the Arabidopsis CRRs. A phylogenetic study showed that AtCRR1 and AtCRR2 form a clade closest to the CR4 group while all the other CRRs form a separate cluster. The five Arabidopsis genes are differentially expressed in various tissues. A construct formed by fusion of the ACR4 promoter and the GUS reporter, ACR4::GUS, is expressed primarily in developing tissues of the shoot. The ACR4 cytoplasmic domain functions in vitro as a serine/threonine kinase, while the AtCRR1 and AtCRR2 kinases are not active. The ability of ACR4 to phosphorylate AtCRR2 suggests that they might function in the same signal transduction pathway. T-DNA insertions were obtained in ACR4, AtCRR1, AtCRR2, AtCRR3 and AtCRK1. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function.     

High constitutive peroxidase activity and constitutive thermotolerance in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

 During the isolation of mutations in the heat-inducible hsp70-1 gene of Neurospora crassa by RIP (repeat-induced point mutations), several transformants were generated by electroporation of conidia with a plasmid harboring an incomplete copy of this gene. One isolate, designated E-45, containing ectopically integrated hsp70-1 DNA, exhibited a slow growth rate, low-temperature sensitivity, constitutive thermotolerance (without prior heat shock), and high constitutive peroxidase activity. The constitutive form of peroxidase (CP) was distinguishable from the heat-inducible form (HIP) by immunoinactivation employing polyclonal antiserum against the latter enzyme and by electrophoretic resolution in nondenaturing polyacrylamide gels. This enzyme was purified to near homogeneity and some of its properties examined. The relative molecular mass of native CP was in the range of 118–136 kDa, as estimated by gel filtration analysis on size exclusion matrices, whereas SDS-PAGE analysis yielded a size of ∼37 kDa for the polypeptide. Substrate saturation kinetics studies were conducted using ABTS [2,2′-azino-bis (3-ethylbenzthiazole-6-sulfonic acid)] and H₂O₂ as substrates: K _m, V _max, and K _cat values for H₂O₂ were ∼22 μM, ∼447 nmol mg⁻¹, and 0.33 s⁻¹, respectively, and those for ABTS were ∼55 μM, ∼453 nmol mg⁻¹, and 0.3 s⁻¹, respectively. Guaiacol was not used as a substrate by this enzyme. CP peroxidase was shown to be a heme-containing enzyme, stable at temperatures up to 58°C. Received: August 5, 2002 / Accepted: January 22, 2003 Acknowledgments This work was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to M.K.). The financial support provided to A. M. in the form of a graduate studentship award by the AHFMR (Alberta Heritage Foundation for Medical Research) and of a graduate teaching assistantship to A. S. by the Department of Biological Sciences, University of Calgary, is gratefully acknowledged. Correspondence to:M. Kapoor     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

Exploring the<Emphasis Type="Italic"> Penicillium marneffei</Emphasis> genome

Penicillium marneffei is a dimorphic fungus that intracellularly infects the reticuloendothelial system of humans and bamboo rats. Endemic in Southeast Asia, it infects 10% of AIDS patients in this region. The absence of a sexual stage and the highly infectious nature of the mould-phase conidia have impaired studies on thermal dimorphic switching and host-microbe interactions. Genomic analysis, therefore, could provide crucial information. Pulsed-field gel electrophoresis of genomic DNA of P. marneffei revealed three or more chromosomes (5.0, 4.0, and 2.2 Mb). Telomeric fingerprinting revealed 6-12 bands, suggesting that there were chromosomes of similar sizes. The genome size of P. marneffei was hence about 17.8-26.2 Mb. G+C content of the genome is 48.8 mol%. Random exploration of the genome of P. marneffei yielded 2303 random sequence tags (RSTs), corresponding to 9% of the genome, with 11.7, 6.3, and 17.4% of the RSTs having sequence similarity to yeast-specific sequences, non-yeast fungus sequences, and both (common sequences), respectively. Analysis of the RSTs revealed genes for information transfer (ribosomal protein genes, tRNA synthetase subunits, translation initiation, and elongation factors), metabolism, and compartmentalization, including several multi-drug-resistance protein genes and homologues of fluconazole-resistance gene. Furthermore, the presence of genes encoding pheromone homologues and ankyrin repeat-containing proteins of other fungi and algae strongly suggests the presence of a sexual stage that presumably exists in the environment.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase,PKSIII<Emphasis Type="Italic">Nc</Emphasis> in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIIINc, a functionally and structurally characterized type III PKS from the fungus, Neurospora crassa, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIIINc through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIIINc catalysis. Site-directed mutagenesis of Cys120 and Ser186 to Ser and Cys, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIIINc product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIIINc products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of Cys120 to bulky Phe side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIIINc protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.