Identification and characterisation of structural maintenance of chromosome 1 (smc1) mutants of Coprinopsis cinerea

A Coprinopsis cinerea homokaryotic fruiting strain was mutagenised, identifying a mutant that exhibited a hyphal growth temperature sensitive defect and hyphal knot development defect at an early fruiting stage, even at the hyphal growth permissive temperature. Microscopic observation suggested that the mutant nuclei exhibited defects in the metaphase to anaphase transition at the restrictive temperature. The gene in which the mutation occurred was cloned, sequenced and determined to be homologous to smc1. Sequence analyses of the mutant revealed deletion of 28 base pairs in the 19th intron of the Cc.smc1 gene, resulting in complete failure of splicing of that intron and in insertion of 14 amino acids in the C-terminal region of the Cc.Smc1 protein. We isolated eight hyphal growth revertants and identified four intragenic suppressors. All were the result of amino acid substitutions in the C-terminal region. Three of the suppressors caused reversion of the arrest in an early fruiting stage. One of the suppressors exhibited cold sensitivity and failed to suppress the fruiting defect, suggesting that flexibility of a lobe in the C-terminal region is important for proper function of Cc.Smc1.     

Isolation and characterization of developmental variants in fruiting using a homokaryotic fruiting strain ofCoprinus cinereus

Developmental variants in fruiting ofCoprinus cinereus were induced by mutagenizing oidia of the homokaryotic fruiting strain CopD5-12 with UV light. Through screening of 2,696 isolates, 1,018 strains exhibited defects in fruiting and were classified into 8 groups: (1) knotless variants, which fail to form hyphal knots, the first visible sign of fruiting; (2) primordiumless variants, which form hyphal knots but fail to develop fruit-body primordia; (3) maturationless variants, which form fruit-body primordia but do not form mature fruit bodies; (4) elongationless variants, which form mature fruit bodies with short stipes; (5) expansionless variants, which form mature fruit bodies with unexpanded pilei; (6) sporeless variants, which fail to produce black basidiospores, resulting in fruit bodies with white pilei after maturation; (7) compound type, which includes variants exhibiting several of the phenotypes described above; (8) others, including variants that produce a “dark stipe” even under in light/dark conditions, which is formed under continuous darkness in the wild-type. Two elongationless variants were characterized histologically.     

MFE1, a member of the peroxisomal hydroxyacyl coenzyme A dehydrogenase family,affects fatty acid metabolism necessary for morphogenesis in Dictyostelium spp

Beta-oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or D-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB, encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Such a persistent accumulation was not detected in wild-type cells or axenically grown mutant cells. Bacterial phosphatidylethanolamine that contains abundant cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while dipalmitoyl phosphatidylethanolamine did not. These results suggest that MFE1 protects the cells from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes cellular lipid composition for proper development. Hence, we propose that this enzyme plays an irreplaceable role in the survival strategy of Dictyostelium cells to form spores for their efficient dispersal in nature.     

巴氏蘑菇子实体不同发育阶段的转录组分析

为探讨巴氏蘑菇子实体不同发育阶段基因的表达情况,本研究对巴氏蘑菇子实体不同发育时期（原基、采收期和开伞期）进行转录组测序,以本实验室已获得的巴氏蘑菇JA菌株的不育单孢菌株JA-15036基因组为参考基因组研究原基与采收期及开伞期样本间差异表达基因,并对差异表达基因进行了GO功能和Pathway富集分析。GO功能分析结果显示,差异表达基因主要富集在跨膜转运、碳水化合物代谢途径和膜组分,它们协同调控为子实体生长发育提供稳定的内环境。KEGG富集分析结果表明,原基期上调的差异表达基因主要富集在核糖体蛋白和DNA复制,表明原基期细胞代谢旺盛,其中核糖体蛋白基因上调为后期蛋白质合成提供重要场所;采收期和开伞期子实体时期差异表达基因主要富集在碳水化合物代谢、脂肪酸降解和氨基酸代谢等途径,为巴氏蘑菇子实体的生长发育与成熟提供营养与能量。     

Influence of activated<Emphasis Type="Italic"> A</Emphasis> and<Emphasis Type="Italic"> B</Emphasis> mating-type pathways on developmental processes in the basidiomycete<Emphasis Type="Italic"> Coprinus cinereus</Emphasis>

The A and B mating type pathways in Coprinus cinereus monokaryons can be activated by transformation with cloned genes from strains of compatible mating types. The presence of heterologous A mating-type genes (Aon) induces production of submerged chlamydospores, hyphal knots and sclerotia in cultures kept in the dark. Upon illumination of transformants of certain strains (218), fruiting body primordia may develop that arrest before karyogamy. Furthermore, formation of aerial spores (oidia) is repressed by the action of A mating type genes in the dark, but light overrides this repression. Heterologous B mating type genes enhance the effects of the A genes on developmental processes, and partially repress the negative action of light on A-mediated regulation of development. Most notably, A-induced fruiting occurs more efficiently and earlier when the B mating type pathway is also active (Bon). However, activation of the B pathway alone is not sufficient to induce fruiting. Unlike A-activated transformants, A+ B-activated transformants of monokaryon 218 form mature fruiting bodies. Therefore, the B genes control fruiting body maturation at the stage of karyogamy. Basidia within the fruiting bodies that were analysed contained four spores in a typical post-meiotic arrangement. In the absence of an activated A mating type pathway, B mating type genes cause deformation and hyperbranching of vegetative hyphae, a reduction in aerial mycelium, and invasion of the agar substrate - a phenotype resembling the "flat" phenotype known from B-activated Schizophyllum commune strains. B-activated transformants usually show enhanced production of chlamydospores and hyphal knots, but maturation of sclerotia is variably efficient. Activation of the B mating type pathway in monokaryons blocked acceptance of nuclei, but not activation of the A mating type pathway.     

Defect in peroxisomal multifunctional enzyme MFE1 affects cAMP relay in Dictyostelium

We have previously reported that cells of Dictyostelium discoideum lacking the fatty acid oxidation enzyme MFE1 accumulate excess cyclopropane fatty acids from ingested bacteria. Cells in which mfeA(-) is disrupted fail to develop when grown in association with bacteria but form normal fruiting bodies when grown in axenic media. Bacterially grown mfeA(-) cells express the genes for the cyclic AMP (cAMP) receptor (carA) and adenylyl cyclase (acaA) but fail to respond to a cAMP pulse by synthesis of additional cAMP which normally relays the signal. Moreover, they do not accumulate the adhesion protein, gp80, which is encoded by the cAMP-induced gene, csaA. As a consequence, they do not acquire developmentally regulated EDTA-resistant cell-cell adhesion. When mutant cells are mixed with wild-type cells and allowed to develop together, they co-aggregate and differentiate into both spores and stalk cells. Thus, most of the developmental consequences of excess cyclopropane fatty acids appear to result from impaired cAMP relay.     

A mutation in the Cc.ubc2 gene affects clamp cell morphogenesis as well as nuclear migration for dikaryosis in Coprinopsis cinerea

The formation and proliferation of the dikaryon in the agaricomycete Coprinopsis cinerea is controlled by the mating type genes, A and B. The B genes, which encode pheromones and pheromone receptors, control nuclear migration for dikaryosis as well as the fusion of the clamp cell with the subterminal cell while the A genes, which encode two classes of homeodomain proteins, control conjugate nuclear division associated with clamp connection development. We characterized the mutant, B28, which was newly isolated as a strain that fails to form a primary hyphal knot, the first visible sign toward fruiting, from a homokaryotic fruiting strain after REMI mutagenesis. Detailed phenotypic analysis revealed that strain B28 exhibits, in addition to the fruiting defect, a defect in A-regulated clamp cell morphogenesis as well as a defect in B-regulated nuclear migration for dikaryosis. The mutant clamp cells are unique in that they continue growing like branches without fusing with the subterminal cells, in contrast to the unfused pseudoclamp which are normally formed in A-on B-off strains, providing evidence for the existence of an as yet unidentified mechanism for the growth suppression of the clamp cell. Molecular analysis revealed that the gene responsible for the phenotypes, designated Cc.ubc2, encodes a protein similar to Ubc2, an adaptor protein for filamentous growth, pheromone response and virulence in the smut fungus Ustilago maydis. In addition, western blot analysis demonstrated that the Cc.ubc2-1 mutation blocks phosphorylation of a presumptive MAP kinase.     

LRP1, a gene expressed in lateral and adventitious root primordia of arabidopsis.

We describe a gene that is expressed in lateral and adventitious root primordia of Arabidopsis. The gene was identified by expression of a transposon-borne promoterless beta-glucuronidase gene in lateral root primordia. The gene, designated LRP1 for lateral root primordium 1, and its corresponding cDNA were cloned and sequenced. The expression pattern of the gene in lateral root primordia was confirmed by in situ hybridization with LRP1 cDNA probes. The LRP1 gene encodes a novel protein. LRP1 expression is activated during the early stages of root primordium development and is turned off prior to the emergence of lateral roots from the parent root. Insertion of the transposon in the LRP1 gene disrupted its expression. To evaluate the homozygous insertion line for a mutant phenotype, several aspects of wild-type lateral root development were analyzed. A mutant phenotype has not yet been identified in the insertion line; however, there is evidence that the gene belongs to a small gene family. LRP1 provides a molecular marker to study the early stages of lateral and adventitious root primordium development.     

Cell Differentiation during Sexual Development of the Fungus Sordaria macrospora Requires ATP Citrate Lyase Activity

During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrospora developmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of the acl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL expression suggests that ACL is specifically induced at the beginning of the sexual cycle and produces acetyl-CoA, which most probably is a prerequisite for fruiting body formation during later stages of sexual development. We discuss the contribution of ACL activity to the life cycle of S. macrospora.     

An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction

We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.     

Basidiocarp development inCoprinus macrorhizus

The dikaryon #5026 (A2B2)+#5132 (A7B7) of the basidiomyceteCoprinus macrorhizus was treated with ultraviolet light (UV),N-methyl-N′-nitro-N-nitrosoguanidine (NG), and 5-bromouracil (BU) in order to induced developmental variations in fruiting. Among a total of 11,383 isolates, 742 were homokaryons. Fruiting test was made with the remaining 10,641 dikaryotic isolates, of which 1,594 showed abnormality in basidiocarp development. Both UV and NG were very efficient in inducing such variations, but BU was not. The induced variations were classified into seven basic types as follows: 1) “knotless”, in which hyphal knots, the first sign of fruiting, do not differentiate; 2) “primordiumless,” in which hyphal knots are formed, but no further development occurs; 3) “maturationles”, in which primordia are formed, but the maturation of primordia into adult fruit bodies does not occur; 4) “elongationless”, in which the elongation of stipe is blocked, and a basidiocarp with a very short stipe is produced; 5) “expansionless,” in which pilei do not expand normally; 6) “sporeless,” in which the formation of basidiospores is blocked and albinic pilei bearing none or only a small amount of spores are produced; and 7) “autolysisless,” in which the autolysis of pilei does not appear to occur. It has been noticed that the four steps of maturation, i.e. stipe elongation, pileus expansion, spore formation, and perhaps pileus autolysis, can proceed independently. Compound types of variations such as “elongation-expansionless,” “elongation-sporeless,” “expansion-sporeless,” and “elongation-expansion-sporeless” were also induced. UV treatment induced maturationless at the highest rate, while NG treatment sporeless. Contributions from the Division of Plant Morphology, Department of Biology, Faculty of Science, Okayama University. No. 117.     

Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli.

The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12?h light/12?h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.     

茄科雷尔氏菌脂酰CoA脱饱和酶和环丙烷脂肪酸合成酶的鉴定

茄科雷尔氏菌(Ralstonia solanacearum)是一种危害严重的土传植物致病菌,其宿主范围广泛,在世界各地严重影响重要经济作物的生产.研究茄科雷尔氏菌的生理特性,探索其致病机理,有利于研发防治青枯病的技术与方法.脂肪酸是细菌细胞重要的组成物质,但是茄科雷尔氏菌脂肪酸合成的机制尚不清晰.本文以茄科雷尔氏菌GMI1000为材料,鉴定了该菌的脂酰Co A脱饱和酶和环丙烷脂肪酸合成酶,并分析了这两种酶在不饱和脂肪酸和环丙烷脂肪酸合成中的作用.结果显示,茄科雷尔氏菌RSc2450编码脂酰Co A脱饱和酶,参与其不饱和脂肪酸合成,但是该菌还存在其他不饱和脂肪酸合成途径.同时发现在茄科雷尔氏菌编码两个可能的环丙烷脂肪酸合成酶蛋白质中,仅有Cfa1(RSc0776)参与了该菌环丙烷脂肪酸的合成,并在低p H和高渗透压的耐受中起作用.该研究结果为深入研究茄科雷尔氏菌脂肪酸合成代谢特点及致病机理奠定了基础.     

Antibiofilm and antifungal activities of medium-chain fatty acids against Candida albicans via mimicking of the quorum-sensing molecule farnesol

Candida biofilms are tolerant to conventional antifungal therapeutics and the host immune system. The transition of yeast cells to hyphae is considered a key step in C. albicans biofilm development, and this transition is inhibited by the quorum-sensing molecule farnesol. We hypothesized that fatty acids mimicking farnesol might influence hyphal and biofilm formation by C. albicans. Among 31 saturated and unsaturated fatty acids, six medium-chain saturated fatty acids, that is, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and lauric acid, effectively inhibited C. albicans biofilm formation by more than 75% at 2 µg ml⁻¹ with MICs in the range 100–200 µg ml⁻¹. These six fatty acids at 2 µg ml⁻¹ and farnesol at 100 µg ml⁻¹ inhibited hyphal growth and cell aggregation. The addition of fatty acids to C. albicans cultures decreased the productions of farnesol and sterols. Furthermore, down-regulation of several hyphal and biofilm-related genes caused by heptanoic or nonanoic acid closely resembled the changes caused by farnesol. In addition, nonanoic acid, the most effective compound diminished C. albicans virulence in a Caenorhabditis elegans model. Our results suggest that medium-chain fatty acids inhibit more effectively hyphal growth and biofilm formation than farnesol.     

Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain. Received: 11 May 1998 / Accepted: 15 July 1998