Mutant Strains Of Aspergillus Nidulans Research Articles

Efeito da Aloe arborescens Miller e da Aloe barbadensis Miller sobre o desenvolvimento vegetativo em Aspergillus nidulans

RESUMOA pesquisa de produtos naturais benéficos à saúde humana vem crescendo nos últimos 20 anos. Considerando que as plantas de Aloe são amplamente utilizadas pela população humana, em geral de maneira terapêutica, o objetivo deste estudo foi avaliar os efeitos de Aloearborescens Miller e Aloe barbadensis Miller, sobre o desenvolvimento vegetativo de linhagens normais e mutantes de Aspergillus nidulans. Conídios da linhagem biA1methG1, MSE e CLB3 de A. nidulans, foram inoculados em meio completo sem (Controle) e com extratos das duas espécies incubados por 2, 4, 6 e 8 horas a 37ºC, no escuro. Foi analisado em microscópio óptico, 200 conídios de cada tratamento. Para o desenvolvimento das colônias, as linhagens foram inoculadas no centro das placas juntamente com o meio de cultura sólido e sobre a membrana de diálise, visando a medição do diâmetro e do peso. A análise estatística foi baseada no teste de Tukey e todos os procedimentos experimentais foram conduzidos em triplicata. Todas as linhagens apresentaram interferências positivas quando expostas às plantas de Aloe, porém, de maneira variada. Ambas as espécies aceleraram a germinação em todas as linhagens testadas e atuaram na redução significativa de conídios mortos e/ou malformados. Em relação ao desenvolvimento vegetativo, todos os dados referentes ao peso úmido e diâmetro corrigido dos tratamentos demonstraram progressos, contudo, a razão diâmetro/peso apresentou somente na linhagem MSE, ação favorável dos tratamentos naturais. As informações deste estudo sugerem benefícios de A. arborescens e A. barbadensis, justificando a importância e continuidade da investigação, para melhor elucidar os mecanismos de ação dessas plantas.

Colony patterning and collective hyphal growth of filamentous fungi

Colony morphology of wild and mutant strains of Aspergillus nidulans at various nutrient and agar levels was investigated. Two types of colony patterning were found for these strains. One type produced uniform colonies at all nutrient and agar levels tested, and the other exhibited morphological change into disordered ramified colonies at low nutrient levels. Both types showed highly condensed compact colonies at high nutrient levels on low agar media that was highly diffusive. Disordered colonies were found to develop with low hyphal extension rates at low nutrient levels. To understand basic pattern selection rules, a colony model with three parameters, i.e., the initial nutrient level and the step length of nutrient random walk as the external parameters, and the frequency of nutrient uptake as an internal parameter, was constructed. At low nutrient levels, with decreasing nutrient uptake frequency under diffusive conditions, the model colony exhibited onsets of disordered ramification. Further, in the growth process of A. nidulans, reduction of hyphal extension rate due to a population effect of hyphae was found when hyphae form three-dimensional dense colonies, as compared to the case in which hyphal growth was restricted into two-dimensional space. A hyphal population effect was introduced in the colony model. Thickening of colony periphery due to the population effect became distinctive as the nutrient diffusion effect was raised at high nutrient levels with low hyphal growth rate. It was considered that colony patterning and onset of disorder were strongly governed by the combination of nutrient diffusion and hyphal growth rate.

A mutant strain of Aspergillus nidulans is hypersensitive to cycloheximide

We routinely use a strain derived from the master strain A513 (FGSC) for mitotic linkage studies in our laboratory. This strain carries the ActA1 mutation on linkage group III, which should confer resistance to actidione (trade name for cycloheximide). However, we have observed that our strain 513s is hypersensitive to actidione in comparison with a wild-type strain at the ActA1 locus (our collection).

Disruption of the 3? phosphoglycerate kinase (pgk) gene in Aspergillus nidulans

We report the isolation of a pgk- mutant strain of Aspergillus nidulans by means of a gene disruption strategy, and demonstrate that the pgk gene is located on chromosome VIII. The pgk- mutant conidiates poorly, will only grow on media supplemented with both a glycolytic and a gluconeogenic carbon source, and is inhibited by hexoses.

Thiophene carboxamide fungicides: Structure-activity relationships with the succinate dehydrogenase complex from wild-type and carboxin-resistant mutant strains of Aspergillus nidulans

Carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and the thiophene compound 3-methylthiophene-2-carboxanilide (I), inhibit succinate oxidation (succinate-ubiquinone reductase; complex II) in mitochondria from a wild-type strain and three mutant, carboxin-resistant strains of Aspergillus nidulans. Studies by White et al. [ Pestic. Biochem. Physiol. 9, 165–182 (1978)] showed that certain oxathiin carboxamide structures were selectively active against particular mutated succinate dehydrogenase complexes (SDCs) of A. nidulans, significantly lowering the level of resistance. Although no oxathiin carboxamides were found to be negatively correlated to carboxin with respect to their effect on SDCs from wild-type and mutant strains of A. nidulans, several could distinguish between moderately and highly carboxin-resistant SDCs and, as shown in the present study, between the two non-allelic highly carboxin-resistant mutants cbx B-28 and cbx C-34. Variation in the molecular structure of thiophene carboxamides can also affect the phenotypic expression of mutations to carboxin resistance in the SDC of A. nidulans, with certain structures being capable of differentiating between moderately and highly carboxin-resistant mutated SDCs. With a moderately carboxin-resistant mutant, cbx A-17, a wide structural variety of thiophene carboxamides, e.g., the 2′-methyl, 2′-benzoyl, 3′-phenoxy, 4′- nbutyl and the N-nhexyl derivatives of (I), did exhibit negative activity correlation to the parent anilide (I). However, with the possible exception of the 4′- nbutyl and 4′- noctyloxy analogs of (I), thiophene carboxamides showed no negative activity correlation to carboxin or (I) for the highly carboxin-resistant mutants cbx B-28 and cbx C-34. As with carboxin-resistant mutants of Ustilago maydis [ Pestic. Biochem. Physiol. 14, 26–40 (1980)], molecular selectivity for mutated carboxin-resistant SDCs of A. nidulans can be markedly influenced by substitution of an oxathiin with a thiophene heterocyclic ring. None of the thiophene carboxamides were considerably toxic to mycelial growth of the wild-type and carboxin-resistant strains of A. nidulans with permeability rather than affinity for the SDC appearing to be the limiting factor. For certain derivatives such as the 5-amino analog of (I), SDC activity and cell growth were inhibited similarly. Several thiophene carboxamides [2′-phenyl, 4′-phenoxy, and N-ndecyl analogs of (I)] showed specificity for the highly carboxin-resistant mutants cbx B-28 and cbx C-34. Thiophene carboxamide structures have been identified which inhibit spore germination of non-Basidiomycete plant pathogens, particularly Phytophthora infestans and Verticillium dahliae. In vivo experiments with late blight ( P. infestans) on tomato plants have shown that a few thiophene carboxamides, e.g., the 3′- nbutyl analog of (I) give satisfactory protectant activity.

Characterization and genetical analysis of an Aspergillus nidulans strain that produces multinucleate conidia

A mutant strain of Aspergillus nidulans, in addition to forming the uninucleate conidia normally occurring in the species, also produces binucleate and trinucleate conidia at constant proportions when the same environmental conditions are maintained. Genetical analysis showed that the trait is controlled by a single gene, denoted bncA, mapped on linkage group IV at 227 units from the methG1 locus. The trait under study has non-autonomous expression in heterokaryons and is affected by temperature, suggesting the presence of a heat-sensitive component involved in the migratory progress of nuclei from the vesicle towards the first branches. Analysis of multinucleate conidia from heterokaryons permitted the conclusion that these nuclei may be of different origins.

The regulation of NADP-linked isocitrate dehydrogenase in Aspergillus nidulans.

The regulation of NADP-linked isocitrate dehydrogenase (NADP-IDH) has been studied in wild-type and mutant strains of Aspergillus nidulans. In the wild-type strain studied, the levels of NADP-IDH vary in a similar way to those of acetamidase, acetyl-CoA synthase, isocitrate lyase and malate synthase under all growth conditions used. Similarly, fac mutants, which are altered in the regulation of these enzymes of acetate utilization, are affected in NADP-IDH levels in a parallel fashion, as are cre mutants, which show altered carbon catabolite repression of this group of enzymes. Possible functions of the NADP-IDH enzyme are considered.

Nickel requirement of a urease-deficient mutant in Aspergillus nidulans.

The addition of nickel ions restored urease activity in vivo and ability to grow on urea in a mutant strain of Aspergillus nidulans otherwise unable to utilize urea. This train carries a mutation in the ureD locus, one of four loci involved in urea utilization. No other urease-deficient strains tested responded to the presence of nickel ions. The analogous characteristics of the ureD mutant and the nitrate reductase and xanthine dehydrogenase associated cnxE mutants in Aspergillus nidulans are discussed. It is postulated that the ureD locus is in some way involved in the production or incorporation of a nickel cofactor essential for urease activity.

The Relevance of a Study of a Temperature-sensitive Ballooning Mutant of Aspergillus nidulans Defective in Mannose Metabolism to our Understanding of Mannose as a Wall Component and Carbon/Energy Source

SUMMARY: A temperature-sensitive mutant strain of Aspergillus nidulans has been isolated which failed to grow normally on minimal agar medium at 43 °C unless mannose was supplied as a sole carbon source. The mutant has been given the symbol mnr A455 for mannose relief. Under restrictive conditions (43 °C) the mutant produced extensive areas of swollen hyphae, called balloons, and wall preparations from cultures grown at 40 °C had approximately one-third of the mannose found in walls of control cultures. The mnrA455 strain produced a more thermolabile phosphomannose mutase than the wild-type strain, so it is suggested that the mutation is in the structural gene coding for this enzyme. The mnrA455 strain failed to grow on minimal agar medium containing 0·1% (w/v) mannose and 0·9% (w/v) glucose at 43 °C. This property was used to obtain revertants, one of which was a double mutant mnrA455 manAl. Growth of the double mutant in media containing [14C]mannose at 43 °C showed that 84% of the label found in the wall occurred in mannose in comparison with 33% in the wild-type control. Autoradiography of cultures of the double mutant using [3H]mannose showed predominant incorporation of label into the tip region of growing hyphae. A manAl strain, isolated following haploidization of a diploid, was unable to grow on media containing glucose or mannose alone. The manAl strain had reduced phosphomannose isomerase (EC 5.3.1.8) activity. The manAl mutation, which is epistatic to mnrA455, has been located to linkage group VIII and the mnrA455 mutation to linkage group V. A pathway is presented for the utilization of mannose in A. nidulans.

Pyridoxine and its relation to lipids. Studies with pyridoxineless mutants of Aspergillus nidulans.

The effect of pyridoxine deficiency on fat metabolism was studied using mutant strains of Aspergillus nidulans requiring pyridoxine for growth. Under pyridoxine deficiency the mutants exhibited increased levels of total lipid, sterols, phospholipids, and triacylglycerols. Total fatty acids were found to decrease with pyridoxine deficiency. An increase in saturated fatty acids and decrease in unsaturated fatty acids were seen with deficiency. Pyridoxine deficiency also increased lower carbon chain fatty acids. A possible involvement of pyridoxine in the elongation of fatty acid chain and in the desaturation of fatty acids in Aspergillus nidulans is suggested.

The Genetic Control of Molybdoflavoproteins in Aspergillus nidulans. A Xanthine Dehydrogenase I Half-Molecule in cnx- Mutant Strains of Aspergillus nidulans

The cnx- group of mutants of Aspergillus nidulans lacks xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) and nitrate reductase (EC 1.6.6.3) activities and are thought to be defective in the synthesis of a molybdenum-containing cofactor, 'cnx', common to xanthine dehydrogenase and nitrate reductase [Pateman, J.A., Rever, B.M., Cove, D.J. and Roberts, D.B. (1964) Nature (Lond.) 201, 58-60]. The cnx cofactor has a role in maintaining the aggregated multimeric structure of nitrate reductase [MacDonald, D.W., Cove, D.J. and Coddington, A. (1974) Mol. Gen. Genet. 128, 187-199]. We report here that, in cnx- mutants grown under conditions inducing xanthine dehydrogenase I, a species cross-reacting with antisera to the native enzyme and of half its molecular weight is present, together with cross-reacting molecules of similar molecular weight to the native enzyme. This suggests that the cnx cofactor has a role in maintaining the aggregated structure of xanthine dehydrogenase I. Both cross-reacting species are capable of passing reducing equivalents from NADH to a tetrazolium salt, showing that the cnx cofactor is not necessary for enzymic activity towards NADH.

Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans.

The antimitotic compound methyl benzimidazol-2-yl carbamate (MBC) formed a complex in vitro with a protein present in mycelial extracts of fungi. The binding protein of Aspergillus nidulans showed a set of properties which is unique for tubulin. Binding occurred rapidly at 4 degrees C and was competitively inhibited by oncodazole and colchicine. Other inhibitors of microtubule function such as podophyllotoxin, vinblastine sulfate, melatonin, and griseofulvin did not interfere with binding of MBC. Electrophoretic analysis of partially purified preparations of the binding protein revealed the presence of proteins with similar mobilities as mammalian tubulin monomers. Hence it is concluded that the binding protein is identical with fungal tubulin. The effect of MBC on mycelial growth of mutant strains of A. nidulans was positively correlated with the affinity of the binding sites for this compound. The apparent binding constant for MBC and tubulin from a wild type was estimated at 4.5 X 10(5), from a resistant strain at 3.7 X 10(4), and from a strain with increased sensitivity to MBC at 1.6 X 10(6) liters/mol. Mutants showing resistance and increased sensitivity to MBC are candidates to have alterations in tubulin structure. Affinity of tubulin for MBC is probably a common mechanism of resistance to this compound in fungi. Low affinity of tubulin for MBC is probably a common mechanism of resistance binding constant of 2.5 X 10(3) liters/mol.

Genetic aspects of resistance to imazalil in Aspergillus nidulans

Mutant strains of Aspergillus nidulans have been isolated which display a low level of resistance to imazalil, a recently developed systemic fungicide. Agar growth tests showed that A. nidulans is about three times as sensitive to imazalil when growing on supplemented minimal medium (SM) as compared with complete medium. This effect was reduced by adding glutamic acid to the SM.

Cytochrome-c reductases from wild-type and mutant strains of Aspergillus nidulans.

The sedimentation coefficients of the NADPH: cytochrome-c oxidoreductase enzymes from wild-type and mutant strains of Aspergillus nidulans have been estimated by sucrose density gradient centrifugation. In the wild-type, two species of cytochrome-c reductase were found, with sedimentation coefficients of 13.7s and 7.6s respectively. The 13.7s species did not appear to be associated with the enzymes of nitrate reduction, whereas the 7.6s species was closely associated with NADPH: nitrate oxidoreductase. In mutant strains lacking nitrate reductase, a thir species of cytochrome-c reductase with a sedimentation coefficient of 4.5s was found. There is some evidence that this 4.5s cytochrome-c reductase is a subunit or breakdown product of nitrate reductase and a model is presented for the role of this 4.5s cytocnorome-c reductase in the assembly of the intact nitrate reductase molecule.

Colony and specific growth rates of normal and mutant strains of Aspergillus nidulans

Colony and specific growth rates of normal and mutant strains of Aspergillus nidulans

The induction and repression of the enzymes of purine breakdown in Aspergillus nidulans

The enzymes of purine breakdown have been assayed in wild-type and various mutant strains of Aspergillus nidulans. Most of these enzymes are markedly inducible and these data show which metabolites are involved in the induction of each enzyme. Uric acid induces xanthine dehydrogenase and urate oxidase, and the existance of mutants non-inducible for both these enzymes suggests that uric acid may require either enzymic conversion to the real intracellular effector or transport to an effective location within the cell. Allantoinase requires both uric acid and allantoin for maximal induction. Allantoicase is induced by allantoin. Ureidoglycollase and urease show no marked induction. Ammonium reduces the levels of activity of all these enzymes.

Accumulation of S-sulphocysteine by a mutant strain of Aspergillus nidulans.

RECENT work in this laboratory1,2, together with earlier observations by Hockenhull3, have provided strong evidence indicating that in Aspergillus nidulans cysteine is synthesized from inorganic sulphur sources via S-sulphocysteine (cysteine-S-sulphonate) as the immediate precursor. This latter compound has, however, not yet been reported to occur in micro-organisms. In the present communication we report that a mutant strain of A. nidulans actually accumulates radioactive S-sulphocysteine when grown in a medium containing sulphate labelled with sulphur-35. The mutant used, strain 793, has been shown to be genetically blocked in the reaction by which S-sulphocysteine is converted to cysteine1,2.

Biochemical Genetical Studies on the Pathway of Sulphate Assimilation in Aspergillus nidulans

SUMMARY: Mutant strains of Aspergillus nidulans requiring methionine for growth were isolated and their growth responses to inorganic and organic sulphur sources studied. It is suggested that methionine is synthesized in this mould from inorganic sulphate through sulphite, thiosulphate and cysteine. The mutant strains genetically blocked in the reaction from thiosulphate to cysteine could be divided into two genetically different groups by means of the heterokaryosis test; heterokaryons formed between these two groups were able to grow on sulphate as sole sulphur source because of the syntrophic action of two different types of nuclei. It was further shown that cysteine-S-sulphonate supported good growth of one of the two groups, whereas the other group showed no growth on this compound. It was concluded that the metabolic conversion of thiosulphate to cysteine involves cysteine-S-sulphonate as an intermediate. Mutant strains responded to sulphide in the same way as to thiosulphate. From this and other evidence, it is suggested that sulphide is utilized by way of thiosulphate.

Cysteine-S-sulphonate as an intermediate in microbiol synthesis of cysteine.

THE synthesis of cysteine in micro-organisms from inorganic sulphur sources has been studied for some time, and the available evidence suggests that sulphate sulphur is reduced to the thiosulphate or sulphide state prior to its introduction into the carbon chain of cysteine1–3, although some investigators have postulated that the formation of sulphur–carbon linkage occurs at the sulphite stage4. Working with mutant strains of Aspergillus nidulans, Hockenhull2 suggested that thiosulphate is the form in which sulphate sulphur combines with the carbon skeleton. Based on the observations that cysteine-S-sulphonate supported good growth of all the thiosulphate-requiring mutants tested and that growth of these strains on thiosulphate was enhanced by the addition of serine, he concluded that thiosulphate first reacts with serine to form cysteine-S-sulphonate as an intermediate and cysteine is formed from the latter by hydrolysis. The same possibility was also postulated by Shepherd4, although the involvement of thiosulphate in the main route of sulphate assimilation was denied by this investigator. Schlossmann and Lynen5, on the other hand, reported the occurrence in yeast of a pyridoxal phosphate-dependent enzyme catalysing the condensation of hydrogen sulphide with serine to form cysteine. The present communication describes more convincing evidence in favour of the cysteine-S-sulphonate pathway in A. nidulans.

Acriflavine-resistant mutants of Aspergillus nidulans.

SUMMARY: In three independently obtained mutant strains of Aspergillus nidulans, resistant in different degrees to acriflavine, resistance is due, in each case, to mutation in a single gene. Two of the mutant alleles, ACR1 and ACR3, are semi-dominant and either allele confers a high degree of resistance. These alleles are located about 23 units distal to the w (white conidia) locus and are presumably allelic. A cross involving these two alleles in repulsion gave 0.1% sensitives. A third mutant allele (acr2) is also located on the w chromosome, but on the other arm about 25 units distal to the ad1 locus and over 100 units distant from the ACR1 and ACR3 loci. This allele, which confers relatively slight resistance, is almost completely recessive. Diploid strains which carry any allele for resistance in heterozygous condition give, by vegetative segregation, haploid and homozygous diploid resistant types which are preferentially selected on medium with acriflavine. The use of this technique for the automatic selection of vegetative segregants provides an additional tool for analyses through the parasexual cycle.