Identification of the sites of action for regulatory genes controlling the amdS gene of Aspergillus nidulans.

The amdS gene of Aspergillus nidulans, which encodes an acetamidase enzyme, is positively regulated by the trans-acting genes amdR, facB, amdA, and areA. Sequence changes in several cis-acting mutations in the 5' region of the gene which specifically affect amdS regulation were determined. The amdI9 mutation, which results in increased facB-dependent acetate induction, is due to a single-base change at base pair -210 relative to the start point of translation. The amdI93 mutation, which abolishes amdR-dependent omega-amino acid induction, is a deletion of base pairs -181 to -151. The amdI66 mutation, which causes increased gene activation in strains carrying amdA regulatory gene mutations, is a duplication of base pairs -107 to -90. Transformation of A. nidulans can generate transformants containing multiple integrated copies of plasmid sequences. When these plasmids carry a potential binding site for a regulatory gene product, growth on substrates whose catabolism requires genes activated by that regulatory gene can be reduced, apparently because of titration of the regulatory gene product. Introduction of 5' amdS sequences via cotransformation into strains of various genotypes was used to localize sequences apparently involved in binding of the products of the amdR, amdA, and facB genes. The position of these sequences is in agreement with the positions of the specific cis-acting mutations. Consistent with these results, a transformant of A. nidulans derived from a plasmid deleted for sequences upstream from -111 was found to have lost amdR- and facB-mediated control but was still regulated by the amdA gene. In addition, amdS expression in this transformant was still dependent on the areA gene.     

Structural and functional analysis of the amdR regulatory gene of Aspergillus oryzae.

We have isolated the Aspergillus oryzae homologue of the amdR regulatory gene of Aspergillus nidulans by cross hybridization. Sequence analysis and functional studies have shown that the amdR genes are highly conserved and functionally interchangeable between the two species. The homology between the two genes extends throughout most of the coding sequences, including sequences encoding the DNA-binding domain and putative activation domains. Two regions of nonconserved sequence were also identified. Studies using various amdS::lacZ fusion constructs indicate that the A. oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A. nidulans protein. Inactivation of the A. oryzae gene results in the inability to grow on gamma-amino-butyric acid (GABA) as a carbon and/or nitrogen source indicating that GABA utilization is amdR-dependent in A. oryzae as it is in A. nidulans.     

Identification of amdX, a new Cys-2–His-2 (C2H2) zinc-finger gene involved in the regulation of the amdS gene of Aspergillus nidulans

The acetamidase-encoding amdS gene of Aspergillus nidulans has been shown to be controlled by multiple regulatory genes. A new gene, amdX , involved in amdS regulation was identified during the characterization of a translocation affecting amdS control. The amdX gene is predicted to encode a 1150-amino-acid polypeptide which contains two Cys-2–His-2 (C2H2) zinc finger DNA-binding motifs. Insertional inactivation of amdX and the phenotypes of transformants containing multiple copies of the amdX gene show that it is an activator of amdS expression. A fusion protein containing the AmdX zinc fingers was found to bind to sequences in the 5' region of amdS which overlap binding sites for the CreA and AmdA regulatory proteins. Evidence is presented for AmdX acting at these sites in vivo .     

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.     

Characterization of the Amdr-Controlled Lama and Lamb Genes of Aspergillus Nidulans

Four Aspergillus nidulans genes are known to be under the control of the trans-acting regulatory gene amdR. We describe the isolation and initial characterization of one of these amdR-regulated genes, lamA. The lam locus, however, was found to consist of two divergently transcribed genes, the lamA gene, and a new gene, also under amdR control, which we have designated lamB. Using recombinant DNA techniques we have constructed a strain of A. nidulans lacking a functional lamB gene. Experiments conducted with this strain demonstrate that lamB, like lamA, is involved in utilization of 2-pyrrolidinone in A. nidulans. Metabolism of a related compound, gamma-amino butyric acid (GABA) is not affected. We also provide evidence that the conversion of exogenous 2-pyrrolidinone to endogenous GABA requires a functional lamB gene. The expression of both lamA and lamB is subject to carbon and nitrogen metabolite repression in addition to amdR-mediated induction by omega-amino acids.     

The Aspergillus nidulans homoaconitase gene lysF is negatively regulated by the multimeric CCAAT-binding complex AnCF and positively regulated by GATA sites

In beta-lactam-antibiotic-producing fungi, such as Aspergillus (Emericella) nidulans, L-alpha-aminoadipic acid is the branching point of the lysine and penicillin biosynthesis pathways. To obtain a deeper insight into the regulation of lysine biosynthesis genes, the regulation of the A. nidulans lysF gene, which encodes homoaconitase, was studied. Band-shift assays indicated that the A. nidulans multimeric CCAAT-binding complex AnCF binds to two of four CCAAT motifs present in the lysF promoter region. AnCF consists at least of three different subunits, designated HapB, HapC, and HapE. In both a delta hapB and a delta hapC strain, the expression of a translational lysF-lacZ gene fusion integrated in single copy at the chromosomal argB gene locus was two to three-fold higher than in a wild-type strain. These data show that AnCF negatively regulates lysF expression. The results of Northern blot analysis and lysF-lacZ expression analysis did not indicate a lysine-dependent repression of lysF expression. Furthermore, mutational analysis of the lysF promoter region revealed that two GATA sites matching the GATA consensus sequence HGATAR positively affected lysF-lacZ expression. Results of Northern blot analysis also excluded that the global nitrogen regulator AreA is the responsible trans-acting GATA-binding factor.     

The Aspergillus nidulans multimeric CCAAT binding complex AnCF is negatively autoregulated via its hapB subunit gene

Cis-acting CCAAT elements are frequently found in eukaryotic promoter regions. Many of them are bound by conserved multimeric complexes. In the fungus Aspergillus nidulans the respective complex was designated AnCF (A. nidulans CCAAT binding factor). AnCF is composed of at least three subunits designated HapB, HapC and HapE. Here, we show that the promoter regions of the hapB genes in both A. nidulans and Aspergillus oryzae contain two inversely oriented, conserved CCAAT boxes (box alpha and box beta). Electrophoretic mobility shift assays (EMSAs) using both nuclear extracts and the purified, reconstituted AnCF complex indicated that AnCF binding in vitro to these boxes occurs in a non-mutually exclusive manner. Western and Northern blot analyses showed that steady-state levels of HapB protein as well as hapB mRNA were elevated in hapC and hapE deletion mutants, suggesting a repressing effect of AnCF on hapB expression. Consistently, in a hapB deletion background the hapB-lacZ expression level was elevated compared with the expression in the wild-type. This was further supported by overexpression of hapB using an inducible alcA-hapB construct. Induction of alcA-hapB expression strongly repressed the expression of a hapB-lacZ gene fusion. However, mutagenesis of box beta led to a fivefold reduced expression of a hapB-lacZ gene fusion compared with the expression derived from a wild-type hapB-lacZ fusion. These results indicate that (i) box beta is an important positive cis-acting element in hapB regulation, (ii) AnCF does not represent the corresponding positive trans-acting factor and (iii) that AnCF is involved in repression of hapB.     

An amdS-lacZ fusion for studying gene regulation in Aspergillus

A translational fusion has been constructed between the amdS gene of Aspergillus nidulans and the lacZ gene of Escherichia coli. Sequencing across the fusion junction confirmed the generation of an in-frame fusion at amino acid 34 of amdS and a novel protein has been detected in transformants carrying the fusion plasmid. Transformants of A. nidulans and Aspergillus niger carrying the fusion plasmid were obtained by co-transformation with a second selectable plasmid. These transformants were readily identified on media containing XGal. The intensity of the reaction on XGal media was indicative of the number of copies of the fusion plasmid carried by the transformants. The growth of highly expressing strains of A. nidulans was inhibited on XGal media. The fusion plasmid was used to develop a two-step gene replacement strategy in which the resident amdS gene was replaced with the fusion gene free of vector sequences. Plate tests and in vitro assays of the beta-galactosidase enzyme confirmed that expression of the fusion gene was regulated by amdS flanking sequences and trans-acting regulatory genes.