Michael J. Hynes

The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth.

Penicillium marneffei is the only known species of its genus that is dimorphic. At 25°C, P. marneffei exhibits true filamentous growth and undergoes asexual development producing spores borne on complex structures called conidiophores. At 37°C, P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. We have cloned a homologue of the Aspergillus nidulans abaA gene encoding an ATTS/TEA DNA‐binding domain transcriptional regulator and shown that it is involved in both these developmental programs. Targeted deletion of abaA blocks asexual development at 25°C before spore production, resulting in aberrant conidiophores with reiterated terminal cells. At 37°C, the abaA deletion strain fails to switch correctly from multinucleate filamentous to uninucleate yeast cells. Both the transitional hyphal cells, which produce the yeast cells, and the yeast cells themselves contain multiple nuclei. Expression of the abaA gene is activated during both conidiation and the hyphal–yeast switch. Interestingly, the abaA gene of the filamentous monomorphic fungus A. nidulans can complement both conidiation and dimorphic switching defects in the P. marneffei abaA mutant. In addition, ectopic overexpression of abaA results in anucleate yeast cells and multinucleate vegetative filamentous cells. These data suggest that abaA regulates cell cycle events and morphogenesis in two distinct developmental programmes.

TupA, the Penicillium marneffei Tup1p homologue, represses both yeast and spore development

Fungal pathogenesis is frequently associated with dimorphism – morphological changes between yeast and filamentous forms. Penicillium marneffei, an opportunistic human pathogen, exhibits temperature‐dependent dimorphism, with growth at 25°C as filamentous multinucleate hyphae switching at 37°C to uninucleate yeast cells associated with intracellular pathogenesis. The filamentous hyphae also undergo asexual development generating uninucleate spores, the infectious propagules. Both processes require a switch to coupled nuclear and cell division. Homologous regulators, including Tup1p/GROUCHO‐related WD40 repeat transcription factors, control dimorphism in Candida albicans and asexual development in Aspergillus nidulans. Unlike these fungi, P. marneffei has both developmental programmes allowing examination of common and programme‐specific controls. We show that deletion of tupA, the P. marneffei TUP1 homologue, confers reduced filamentation and inappropriate yeast morphogenesis at 25°C, in stark contrast to constitutive filamentation observed when C. albicans TUP1 is deleted. Deletion of tupA also confers premature brlA‐dependent asexual development, unlike reduced asexual development in the corresponding A. nidulans rcoA deletion mutant. Furthermore, the A. nidulans rcoA deletion mutant is self‐sterile, and we show that tupA from P. marneffei, which lacks an apparent sexual cycle, complements both the asexual and sexual development phenotypes. Therefore, TupA coordinates cell fate by promoting filamentation and repressing both spore and yeast morphogenetic programmes.

Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans.

Aspergillus niger grows poorly on acetamide as a nitrogen or carbon source and lacks sequences detectably homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans. We have taken advantage of these observations to develop a transformation system for A. niger using the amdS gene as a dominant heterologous marker for selecting transformants on the basis of acetamide utilization. Transformants varied in their ability to grow on amide media and the number of integrated copies of the amdS plasmid ranged from 1 or 2 to greater than 100. Southern analysis of transformants revealed that the multiple copies were integrated into the chromosome in tandem arrays. This result indicates that transformation of A. niger is more similar to mammalian cells than to yeast. Analysis of enzyme activity levels and RNA levels showed that most of the copies of amdS were expressed. Mitotic stabilities of transformants were found to be high. A transformant containing greater than 100 copies of the amdS gene was impaired in omega‐amino acid utilization, a result that has also been found in A. nidulans. Since, in A. nidulans, omega‐amino acids induce acetamidase via a characterizied regulatory gene (amdR/intA) this observation implies that titration of an analogous A. niger regulatory gene product by multiple amdS copies has occurred. Additional evidence suggested that the amdS gene is regulated in A. niger. It has also been shown that an unselected plasmid can be co‐transformed with the amdS plasmid into A. niger.

Anion–π interaction augments halide binding in solution

1H NMR spectroscopic data and complementary theoretical predictions suggest that a designed receptor exhibits the anion-pi interaction in solution.

EQNMR: a computer program for the calculation of stability constants from nuclear magnetic resonance chemical shift data

A computer program has been elaborated which uses the complexation-induced displacements of NMR chemical shifts to calculate the stability constants for the general reaction (i) which gives the generalised stability constant (ii). The program can deal with data from a wide variety of reactions including proton mM +nL +jH ⇌ MmHjLn(i), βmjn=[MmHjLn]/[M]m[H]j[L]n(ii) equilibria, metal-ion hydrolysis and metal–ligand interactions. It can also deal with situations where both ligand proton equilibria and complex-formation reactions must be considered.

Pleiotropic mutants ofAspergillus nidulans altered in carbon metabolism

SummaryMutants altered in carbon catabolite regulation have been isolated by selecting for mutants of theareA217 strain capable of using acetamide as the sole nitrogen source in the presence of sucrose. In addition tocreA mutants described previously by Arst and Cove, strains with mutations in two new genes,creB andcreC, have been found. ThecreB andcreC mutants grow poorly on some sole carbon sources and have low levels of some enzymes of carbon catabolism e.g. β-galactosidase and D-quinate dehydrogenase. ThecreB andcreC mutants are hypersitive to fluoroacetate, fluoroacetamide and allyl alcohol in the presence of glucose or sucrose but not glycerol; and the enzymes, acetamidase, and alcohol dehydrogenase, are less sensitive to carbon catabolite repression than the wild-type strain. Extracellular protease and α-glucosidase enzyme activities are elevated increB andcreC mutants, while L-proline and L-glutamate uptake capacities are lower in both the presence and absence of glucose. Interactions betweencreA, B and C mutations have been investigated in double mutants, and the dominance properties ofcreB andcreC mutants determined. The results indicate that thecreB andcreC genes may have a regulatory role in the control of carbon catabolism.

The kinetics and mechanisms of the reaction of iron(III) with gallic acid, gallic acid methyl ester and catechin.

The kinetics and mechanisms of the reactions of a number of pyrogallol-based ligands with iron(III) have been investigated in aqueous solution at 25 degrees C and ionic strength 0.5 M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are generally consistent with a mechanism in which the 1:1 complex that is formed initially when the metal reacts with the ligand subsequently decays through an electron transfer reaction. There was also some evidence for the formation of a 1:2 ligand-to-metal complex at higher pH values. The kinetics of complex formation were investigated with either the ligand or metal in pseudo-first-order excess. Rate constants for k(1) of 2.83(+/-0.09)x10(3), 1.75(+/-0.045)x10(3) and 3300(+/-200) M(-1) s(-1) and k(-1) of 20(+/-6.0), 35(+/-13) and 25+/-7.6 M(-1) s(-1) have been evaluated for the reaction of Fe(OH)(2+) with gallic acid, gallic acid methyl ester and catechin, respectively. The stability constant of each [Fe(L)](+) complex has been calculated from the kinetic data. The iron(III) assisted decomposition of the initial iron(III) complex formed was investigated. Analysis of the kinetic data yielded both the equilibrium constants for protonation of the iron(III) complexes initially formed together with the rate constants for the intramolecular electron transfers for gallic acid and gallic acid methyl ester. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time-dependent spectra.

Genetic manipulation of Aspergillus nidulans: meiotic progeny for genetic analysis and strain construction.

The multicellular microbial eukaryote Aspergillus nidulans is an excellent model for the study of a wide array of biological processes. Studies in this system contribute significantly to understanding fundamental biological principles and are relevant for biotechnology and industrial applications, as well as human, animal and plant fungal pathogenesis. A. nidulans is easily manipulated using classical and molecular genetics. Here, we describe the storage and handling of A. nidulans and procedures for genetic crossing, progeny analysis and growth testing. These procedures are used for Mendelian analysis of segregation of alleles to show whether a mutant phenotype segregates as a single gene and independent assortment of genes to determine the linkage relationship between genes. Meiotic crossing is used for construction of multiple mutant strains for genetic analysis. Genetic crossing and analysis of progeny can be undertaken in 2–3 weeks and growth testing takes 2–3 days.

Comparison and cross‐species expression of the acetyl‐CoA synthetase genes of the ascomycete fungi, Aspergillus nidulans and Neurospora crassa

The genes encoding the acetate‐inducible enzyme acetyl‐coenzyme A synthetase from Neurospora crassa and Aspergillus nidulans (acu‐5 and facA, respectively) have been cloned and their sequences compared. The predicted amino acid sequence of the Aspergillus enzyme has 670 amino acid residues and that of the Neurospora enzyme either 626 or 606 residues, depending upon which of the two possible initiation codons is used. The amino acid sequences following the second alternative AUG show 86% homology between the two species; the extended N‐terminal sequences show no homology. The Neurospora protein is characterized by the appearance of the S(T)PXX sequence motif where the amino acid homologies break down. The codon usage is biased in both genes, with a marked deficiency, especially in Neurospora, of codons with A in the third position.

AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR.

ABSTRACT CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapCgene, encoding a protein with significant similarity to Hap3p ofSaccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the geneshapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae,Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB,hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression ofamdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on γ-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdSand gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.