Specificity and transformations of the trisporic acid series of fungal sex hormones

Chromatographic and spectroscopic characterizations and comparative bioassay data are given for trisporic acid A, the separate 9-cis- and 9-trans-isomers of trisporic acids B and C, and trisporol C, all obtained from 'mated' (plus and minus) cultures of Blakeslea trispora. All five acids show comparable levels of hormone activity on both the mating types of Mucor mucedo, whereas natural trisporol C more specifically affects a plus strain and the laboratory-derived methyl esters are minus-specific. Similarly plus and minus strains of B. trispora convert trisporol C and the esters into trisporic acids at different rates, and they effect different transformations of administered methyl ¹⁴C-trisporate C.     

Sex specificity of hormone synthesis in Mucor mucedo

Sex specificity is observed in the mating types of the fungus Mucor mucedo with respect to the production of 4-hydroxy methyltrisporates (plus mating type) and trisporins (minus mating type), and in the conversion of these metabolites to trisporic acids by the mating partner. These compounds induce zygophores on the opposite mating type only.     

A comparative developmental pattern of enzymes of carbon metabolism and pentose phosphate pathway in mungbean and lentil nodules

The activities of enzymes of pentose phosphate pathway (PPP) viz. glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carbon metabolism viz. phosphoenol pyruvate carboxylase, NADP- isocitrate dehydrogenase and NADP-malic enzyme were measured in the plant and bacteroid fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules along with the developing roots for comparison. The enzymes of pentose phosphate pathway in legume cytosol had higher activities at a stage of maximum nitrogenase activity and higher sucrose metabolism. However, bacteroids had only limited capacity for this pathway. The specific activities of these enzymes were greater in ureide than in amide exporter. CO₂ fixation via higher activity of phosphoenolpyruvate carboxylase in the plant part of the nodules in lentil might have been due to the greater synthesis of four carbon amino acids for amide export. The peak of NADP-isocitrate dehydrogenase in both legumes coincided with the pentose phosphate pathway enzymes at the time of high rates of sucrose metabolism and nitrogen fixation. Higher activities of NADP-malic enzyme were obtained in mungbean than in the lentil nodules. These findings are consistent with the role of these enzymes in providing reductant (NADPH) and substrates for energy yielding metabolism of bacteroids and carbon skeletons for ammonia assimilation.     

(−) Mating Type-Specific Mutants ofPhycomycesDefective in Sex Pheromone Biosynthesis

Sutter, R. P., Grandin, A. B., Dye, B. D., and Moore, W. R. 1996. (−) Mating type-specific mutants ofPhycomycesdefective in sex pheromone biosynthesis.Fungal Genetics and Biology20,268–279. We have isolated the first mating type-specific mutants in mucoraceous fungi. Both mutants inPhycomyces blakesleeanusappear to be defective in the same gene. The gene, present in both mating types, is necessary only in cultures of the (−) mating type. The gene codes for an enzyme in sex pheromone biosynthesis. The pheromone precursor made by the mutants is detectable only in cross-feeding experiments. The biological and solubility properties of the precursor suggest the precursor is 4-dihydrotrisporin, a metabolite of β-carotene. Separate studies with β-carotene-deficient mutants and Compound-P, a new chemically synthesized precursor of the pheromones, imply the constitutive level of enzymes for pheromone biosynthesis inPhycomycesis extremely low. In comparison, the level of enzymes for pheromone conversion to trisporic acid is higher. The mating type-specific mutants also catalyze the conversion of (+) pheromone to trisporic acid. This finding was unexpected because literature models predicted this reaction was catalyzed by the same enzyme which catalyzed the conversion of 4-dihydrotrisporin to (−) pheromone—a reaction missing in the (−) mating type-specific mutants. Thus, we propose a revised model for trisporic acid biosynthesis.     

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.     

Sexual differentiation in Mucor: Trisporic acid response mutants and mutants blocked in zygospore development

Spores of a minus strain of Mucor mucedo (Bref.) were treated with 1-methyl-[3-nitro]-1-nitro-soguanidine and mutants were isolated either by testing for zygophore induction with externally supplied trisporic acids (TA) or by mating with wild type plus colonies. Mutants were found defective (Tar^?) or temperature-sensitive (Tar-Ts) in their reaction towards trisporic acids, blocked or temperature-sensitive in their mating with plus strain (Mat^? or Mat-Ts) or temperature-sensitive in zygospore development (Zyg-Ts). The inability to react against externally supplied trisporic acids was not necessarily coupled with an inability to mate with plus strain (phenotype Tar^? Mat⁺). This indicated that the diffusion and uptake of trisporic acids is not a necessary prerequisite to the sexual interaction of Mucor mating types.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Coexpression of Elo-like enzyme and Δ5, Δ4-desaturases derived from Thraustochytrium aureum ATCC 34304 and the production of DHA and DPA in Pichia pastoris

Thraustochytrium aureum ATCC 34304 produces a high level of polyunsaturated fatty acids (PUFAs), which are typically synthesized by strings of reactions catalyzed by desaturase and elongase enzymes. In this study, the genes related to the biosynthesis of PUFAs were investigated and targeted to enable optimization of the production of PUFAs. To the best of our knowledge, this is first study to evaluate the co-expression of genes TaElo, Tad5, and Tad4genes derived from T. aureum. We found that C22 PUFAs such as docosapentaenoic acid (DPA, C22:5n–6) and docosahexaenoic acid (DHA, 22:6n–3) were synthesized from γ-linolenic acid (GLA, C18:3n–6) and stearidonic acid (SDA, C18:4n–3), respectively, as exogenous substrates via a series of reactions catalyzed by an Elo-like enzyme and Δ5, Δ4-desaturase enzymes. In addition, the results of this study revealed that the TaElo gene could synthesize the Δ6-and Δ5-elongation products. Taken together, these results confirmed that the Elo-like enzyme was involved in multiple reactions leading to the production of PUFAs and that the TaElo, Tad5, and Tad4 genes were capable of functioning together to produce DPA and DHA using GLA and SDA.     

The effects of recessive lethal Notch mutations of Drosophila melanogaster on flavoprotein enzyme activities whose inhibitions cause Notch-like phenocopies

The biochemical action of the Notch locus whose mutants cause morphological aberrations in flies, viz., notches of wings and bristle multiplication, has been analyzed (1) by the addition to the food medium of enzyme inhibitors causing phenocopies of Notch and (2) by comparison of enzyme activity patterns of Notch mutants with different degrees of phenotypic expression. Notch phenocopies were induced by inhibitors of enzyme activities in two biochemical pathways: (1) the de novo pyrimidine synthesis by 5-methylorotate (inhibitor of dihydroorotate dehydrogenase) and (2) the choline shunt by amobarbital (inhibits choline dehydrogenase) and methoxyacetate (inhibits sarcosine dehydrogenase). The inhibition of de novo pyrimidine synthesis prevents the production of deoxyuridine-5-phosphate, the substrate for the synthesis of thymidine-5-phosphate via thymidylate synthase, whereas the inhibition of the choline shunt prevents the production of HCHO groups and glycine, both of which are involved in the synthesis of 5,10-methylenetetrahydrofolate, which is a cofactor of thymidylate synthase. It was already known that the inhibition of the latter enzyme in vivo induces Notch phenocopies. Notch mutants with a strong morphological expression show low enzyme activities for dihydroorotate dehydrogenase and choline dehydrogenase. Both are flavoprotein enzymes linked to the respiratory chain. The correspondence between the low enzyme activities in Notch mutants with a strong morphological expression and the phenocopying effect of antimetabolites on these enzymes in the two biochemical pathways involved strongly suggests that the morphological effects of Notch on flies are a consequence of lowered activities of choline dehydrogenase and dihydroorotate dehydrogenase.     

Rhodothermus marinus: physiology and molecular biology

Rhodothermus marinus has been the subject of many studies in recent years. It is a thermohalophilic bacterium and is the only validly described species in the genus Rhodothermus. It is not closely related to other well-known thermophiles and is the only thermophile within the family Crenotrichaceae. R. marinus has been isolated from several similar but distantly located geothermal habitats, many of which are subject to large fluctuations in environmental conditions. This presumably affects the physiology of R. marinus. Many of its enzymes show optimum activity at temperatures considerably higher than 65°C, the optimum for growth, and some are active over a broad temperature range. Studies have found distinguishing components in the R. marinus electron transport chain as well as in its pool of intracellular solutes, which accumulate during osmotic stress. The species hosts both bacteriophages and plasmids and a functional intein has been isolated from its chromosome. Despite these interesting features and its unknown genetics, interest in R. marinus has been mostly stimulated by its thermostable enzymes, particularly polysaccharide hydrolysing enzymes and enzymes of DNA synthesis which may be useful in industry and in the laboratory. R. marinus has not been amenable to genetic analysis until recently when a system for gene transfer was established. Here, we review the current literature on R. marinus.     

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l⁻¹) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h⁻¹. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h⁻¹. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD⁺, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H₂ removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H₂ scavenging and acetate producing microorganisms.     

Morphological and molecular evidence for hybridization and introgression in Central European Arctium (Asteraceae)

Three species of burdock (Arctium lappa, A. tomentosum, and A. minus; Asteraceae), which commonly occur in sympatry in Central Europe, are thought to hybridize frequently, hence raising questions about their integrity. We surveyed mixed, monotypic, and/or single plant stands of these species in Central Germany to assess their extent of hybridization and introgression, and also the likelihood of insect host shifts between them. Individual plants were characterized by multivariate analyses of morphological and RAPD variation in combination with RAPD-based character-index-scores. All analyses recognized the three species as distinct units, but also identified a limited number of first (or early) generation hybrids between sympatric A. lappa and either A. tomentosum or A. minus, respectively, as well as one introgressant of A. tomentosum into A. lappa. While pre-zygotic isolation may partly account for the rarity of first generation crosses (F₁'s), the near absence of later generation hybrid segregants or introgressants likely reflects post-zygotic isolation due to the disruption of co-adapted gene complexes and/or habitat-mediated superiority shared by the ruderal parentals. As the tephritid fly Tephritis bardanae oviposits on both A. tomentosum (T) and A. minus (M), but not on A. lappa (L), the occurrence of L × M and L × T hybrids and/or introgressants may have important evolutionary consequences regarding the acquisition of a new host, i.e. A. lappa, by these insects.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Regulation of the synthesis of methanol oxidizing enzymes inKloeckera sp. 2201 andHansenula polymorpha,a comparison

A comparative study was made of the regulation of the synthesis of methanol dissimilating enzymes inkloeckera sp. 2201 andHansenula polymorpha using chemostat and batch growth conditions and methanol or glucose as carbon sources. During growth in methanol-limited chemostat cultures similar enzyme patterns for alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two yeasts were found. When growing in batch culture with glucoseH. polymorpha, but notKloeckera sp. 2201, was found to produce ethanol which might affect the synthesis of these enzymes.     

Regulation of amino acid and glucose dissimilation in so-called ammonifiers and in other soil microorganisms

Pseudomonas acidovorans and P. putida, isolated from an enrichment culture with casein hydrolysate, and Agrobacterium radiobacter and Torulopsis sp., isolated from a glucose enrichment, were compared with respect to the physiology of ammonification. Decreasing ammonifying ability as well as increasing repression of the synthesis of amino acid degrading enzymes by glucose were found in the above order of organisms. In degradation sequences, observed with P. putida and A. radiobacter as test organisms, substances dissimilated prior to others had both, enhancing and repressing effects on the oxidation of the other compounds. This fact was parallelled by the observation, that in these two bacteria, glucose and single amino acids, when added to the same medium, exerted mutual repression of the synthesis of catabolic enzymes of their partners. The ecological significance of this type of regulation has been discussed.     

Immunogold localization of glutamine synthetase and NADP-glutamate dehydrogenase of Laccaria laccata in Douglas fir ectomycorrhizas

Using antibodies raised against glutamine synthetase (GS) and NADP-glutamate dehydrogenase (NADP-GDH) from Laccaria laccata, we examined tissular localization of GS and NADP-GDH in symbiotic tissues of Douglas fir/L. laccata ectomycorrhizas by immunogold labeling. Thin sections of mycorrhizal roots were first treated either with an anti-GS- or antiNADP-GDH-specific antibody and then with a colloidal gold marker. Both enzymes appeared to be cytoplasmic. Our results also indicated the presence of GS in some fungal cells in dense cytoplasmic patches. It also appeared that GS is more abundant than NADP-GDH. The distribution of these nitrogen-assimilating enzymes in the fungal Hartig net and the sheath did not differ significantly. No labelling was observed in host cells.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.