alcA Promoter Research Articles

Proliferation of intrahyphal hyphae caused by disruption of csmA, which encodes a class V chitin synthase with a myosin motor-like domain in Aspergillus nidulans.

We have found that the Aspergillus nidulans csmA gene encodes a novel protein which consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain (M. Fujiwara, H. Horiuchi, A. Ohta, and M. Takagi, Biochem. Biophys. Res. Commun. 236:75-78, 1997). To clarify the roles of csmA in fungal morphogenesis, we constructed csmA null mutants. The growth rate of the mutant colonies was almost the same as that of the wild-type strain, but hyphal growth was severely inhibited when a chitin-binding reagent, Calcofluor white or Congo red, was added to the medium. Moreover, morphological abnormalities in tip growth and septum formation were identified microscopically. Proliferation of intracellular new hyphae, called intrahyphal hyphae, which behaved as intrinsic hyphae, was the most striking phenotypic feature among them. These phenotypes were not suppressed when the only chitin synthase domain of csmA was expressed under the control of the alcA promoter, whereas they were suppressed when the intact form of csmA was expressed. Therefore, it was concluded that the product of csmA (CsmA) has important roles in polarized cell wall synthesis and maintenance of cell wall integrity and that the myosin motor-like domain is indispensable for these functions.

Structural Requirements for in Vivo Myosin I Function in Aspergillus nidulans

We have investigated the minimal requirements of the tail region for myosin I function in vivo using the filamentous fungus Aspergillus nidulans. The CL3 strain (McGoldrick, C. A., Gruver, C., and May, G. S. (1995) J. Cell Biol. 128, 577-587) was transformed with a variety of myoA constructs containing mutations in the IQ, TH-1-like, SH3, and proline-rich domains by frameshift or in-frame deletions of the tail domains. The resulting strains contained wild type myoA driven by the alcA promoter and a mutant myoA driven by its endogenous promoter. This strategy allowed for selective expression of the wild type and/or mutant form of MYOA by the choice of growth medium. Proper septation and hyphal branching were found to be dependent on the interaction of the IQ motifs with calmodulin, as well as, the presence of its proline-rich domain. Additionally, a single proline-rich motif was sufficient for nearly wild type MYOA function. Most surprisingly, the SH3 domain was not essential for MYOA function. These studies expand our previous knowledge of the function of MYOA to include roles in hyphal morphogenesis, septal wall formation, and cell polarity, laying the groundwork for more detailed investigations on the function of the various tail domains in MYOA.

The CreA Repressor Is the Sole DNA-binding Protein Responsible for Carbon Catabolite Repression of the alcA Gene in Aspergillus nidulans via Its Binding to a Couple of Specific Sites

Carbon catabolite repression is mediated in Aspergillus nidulans by the negative acting protein CreA. The CreA repressor plays a major role in the control of the expression of the alc regulon, encoding proteins required for the ethanol utilization pathway. It represses directly, at the transcriptional level, the specific transacting gene alcR, the two structural genes alcA and aldA, and other alc genes in all physiological growth conditions. Among the seven putative CreA sites identified in the alcA promoter region, we have determined the CreA functional targets in AlcR constitutive and derepressed genetic backgrounds. Two different divergent CreA sites, of which one overlaps a functional AlcR inverted repeat site, are largely responsible for alcA repression. Totally derepressed alcA expression is achieved when these two CreA sites are disrupted in addition to another single site, which overlaps the functional palindromic induction target. The fact that derepression is always associated with alcA overexpression is consistent with a competition model between AlcR and CreA for their cognate targets in the same region of the alcA promoter. Our results also indicate that the CreA repressor is necessary and sufficient for the total repression of the alcA gene.

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans.

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp, produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfps as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.

Improved gene expression inAspergillus nidulans

A variety of gene expression systems have been developed that utilize the promoter and transcriptional regulatory sequences derived from carbon-catabolite repressed genes for the expression of heterologous genes. The alcA expression system of Aspergillus nidulans utilizes the promoter and regulatory sequences derived from the alcohol dehydrogenase I (alcA) gene. Expression of the alcA gene is repressed by a DNA-binding protein (CreA) in the presence of glucose and induced by ethanol under glucose-depleted conditions. One problem encountered during the expression of therapeutic proteins in A. nidulans is the coexpression of secreted proteases at the time of maximal secretion of heterologous product. To avoid the proteases we created an alcA promoter variant that is no longer sensitive to glucose repression hence could drive expression at earlier time points during the fermentation. The use of this promoter variant in the expression of recombinant interleukin-6 is discussed. A second problem encountered during the expression of high-quality human therapeutic proteins in Aspergillus is aberrant glycosylation. Lower eukaryotic systems, such as Aspergillus, tend to add highly branched mannosidic chains to heterologous secreted protein products. N-Glycans can be important for both the structure and function of specific glycoproteins, hence efforts are being made to in vivo alter the type and complexity of N-glycans substituted by A. nidulans. Key words: Aspergillus, gene expression, alcohol dehydrogenase, glycosylation.

A novel Arabidopsis type 1 protein phosphatase is highly expressed in male and female tissues and functionally complements a conditional cell cycle mutant of Aspergillus

Type 1 protein phosphatases are very highly conserved throughout eukaryotes where they regulate a number of key metabolic and morphogenetic processes. A cDNA, AtPP1bg, representing a new member of the type 1 protein phosphatase gene family in Arabidopsis has been isolated on the basis of hybridization with the Aspergillus bimG protein phosphatase gene. The AtPP1bg gene potentially encodes a 37 kDa protein very closely related to PP1 but with divergent N- and C-termini. The predicted amino acid sequence shows 71% identity to the ORF of the bimG gene. When expressed in Aspergillus under the alcA promoter, this phosphatase complements the temperature-sensitive bimG11 mutation allowing nearly normal vegetative growth at 37 degrees C (but not at 42 degrees C). Notably, the plant PP1 does not support morphogenesis (conidiation) at 37 degrees C. This may indicate that conidophore formation has particular phosphatase requirement(s) which the plant PP1 cannot supply. The pattern of expression of the AtPP1bg transcript has been studied during development of the plant. In situ hybridization of Arabidopsis with antisense probes shows that this phosphatase gene is expressed at a low level throughout the plant, but is strongly upregulated within developing flowers, especially in the tapetum, the developing and mature pollen and in the ovaries. This implies that the AtPP1bg either has a specialized role in the formation of these organs, or that there is an increased requirement for protein phosphatase 1 at these stages. It was found that the level of AtPP1bg transcript, as judged by the relative intensity of staining in different cells within the floral meristems, did not vary during the cell cycle.

Identification of developmental regulatory genes in Aspergillus nidulans by overexpression.

Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [alcA(p)] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA(p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA(p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA(p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA(p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA(p)::brlA induction. Sequence analyses of the DNA fragments under alcA(p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA(p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.

Overexpression of two penicillin structural genes in Aspergillus nidulans.

We have placed two different penicillin structural genes from Aspergillus nidulans, ipnA (encoding isopenicillin N synthetase, IPNS) and acyA (encoding acyl-CoA:6-aminopenicillanic acid acyltransferase, AAT), under the control of the strong alcA promoter [alcA(p)]. Single copies of these transcriptional fusions were targeted to the same chromosomal location and conditions have been worked out which simultaneously allow induction of the alcA(p) and support penicillin biosynthesis. Transcriptional induction of the chimeric genes alcA(p)::ipnA or alcA(p)::acyA(cdna) in the relevant recombinant strains results in 10-fold higher levels of the ipnA or acyA transcripts than those resulting from transcription of the corresponding endogenous genes. This increase causes a 40-fold rise in IPNS activity or a 8-fold rise in AAT activity. Despite this rise in enzyme levels, forced expression of the ipnA gene results in only a modest increase in levels of exported penicillin, whereas forced expression of the acyA gene reduces penicillin production, showing that neither of these enzymes is rate-limiting for penicillin biosynthesis in A. nidulans. A genomic version of the alcA(p)::acyA fusion in which the acyA gene is interrupted by three small introns, is inducible by threonine to a lesser extent (as determined by both acyA mRNA levels and AAT enzyme levels) than the corresponding cDNA version, suggesting that processing of the introns present in the primary transcript may limit acyA expression.

Molecular cloning and sequence analysis of the cellobiohydrolase I gene from Trichoderma koningii G-39.

The cellobiohydrolase I gene, cbh1, has been cloned from an enhanced cellulase-producing strain, Trichoderma koningii G-39. Sequence comparisons show that T. koningii cbh1 is identical to that of T. reesei with the exception of 6 bp--two causing silent substitutions in the coding region, three differing in one of the introns, and one in 5'-noncoding region. Thus, it should encode an identical CBHI to that of T. reesei despite the differences in morphological characters of the two species. Analysis of approximately 1.4 kb of the 5' flanking region shows a number of surprisingly interesting putative regulatory features. There are no unusual features within about 600 bp upstream of the translation start ATG. However, prior to the 600-bp region, there are seven CAAT sequences, a number of direct and inverted repeats, and two C/T-rich regions. Also, there are five consensus 5'-(G/C)PyGGGG-3' sequences that have been identified to be carbon catabolite repressor binding sites of Aspergillus nidulans CREA and Saccharomyces cerevisiae MIG1 repressors. The structural organization around these consensus sequence regions is similar to those of A. nidulas alcR and alcA promoters. While the production of large amounts of CBHI by T. koningii upon induction apparently correlates with the large number of CAAT boxes in the 5' upstream untranslated region of cbh1, the presence of five CREA/MIG1 repressor-binding consensus sequences in the region suggests the wide-domain carbon catabolite repression regulatory system that controls the A. nidulans ethanol regulon, and yeast GAL genes transcription might also be operative and responsible for regulation of T. koningii cbh1 transcription.

Relationship between zinc content and DNA-binding activity of the DNA-binding motif of the transcription factor ALCR in Aspergillus nidulans

The transcription factor ALCR of the ethanol utilisation pathway in Aspergillus nidulans contains a zinc binuclear motif (CysX2CysX6CysX16CysX2CysX6Cys), within the DNA-binding domain located in the N-terminal region of the ALCR protein. Specific targets have been localised in the promoter of the alcR gene, involved in the autoregulation process, and in the promoter of the structural gene alcA (encoding alcohol dehydrogenase I), which is also under the control of ALCR. The DNA-binding domain has been expressed in-Escherichia coli as a GST-ALCR (7-58*) fusion protein and also obtained as an ALCR (7-58*) peptide. Both the ALCR fusion protein and the ALCR peptide are able to bind 65Zn(II) in vitro, if reduction of cysteines occurs prior to the addition of zinc. Competition experiments showed that Cd(II), Co(II) and Cu(II) are efficient competitors for the zinc binding sites. The ALCR DNA-binding domain was shown to contain 2 mol of tightly bound Zn(II) per mole of fusion protein. Removal of the intrinsic Zn(II) requires treatment with Chelex. This treatment abolishes the ability of the protein to bind to the targets of ALCR located in the alcA and alcR promoters. The apo-ALCR DNA-binding motif could be reconstituted with Zn(II) or Cd(II), restoring specific DNA binding to both types of targets. Thus a direct relationship was shown to exist between the zinc content of ALCR and its DNA-binding activity.

A glucose-derepressed promoter for expression of heterologous products in the filamentous fungus Aspergillus nidulans.

We describe a putative binding sequence (GCGGGGC) for the glucose-responsive repressor protein CreA at two positions upstream of the transcription start site of the alcohol dehydrogenase I (alcA) gene of Aspergillus nidulans. To positively identify the putative binding sites as CreA-specific, the GCGGGGC blocks were mutated at five internal nucleotide positions to GTACTAC and reintroduced into the wild type alcA promoter driving expression of the endogenous alcohol dehydrogenase I gene. This CreA-binding site variant was then transformed into an AlcR constitutive A. nidulans host strain (T2625) and growth was monitored in the presence of the non-metabolized glucose analogue, 2-deoxyglucose. Positive transformants were selected by their ability to grow using ethanol as a carbon source in the presence of 2-deoxyglucose. Similar CreA binding site variant alcA promoters should permit the alcA-driven expression of heterologous genes in A. nidulans in the presence of glucose, the preferred carbon source for biomass accumulation and provides a model for controlling carbon-catabolite regulated expression in other expression systems.

Essential roles for calcium and calmodulin in G2/M progression in Aspergillus nidulans.

nimT encodes a protein in Aspergillus nidulans that is required for tyrosine dephosphorylation of p34cdc2 and has a strong homology to cdc25-type proteins. Conditional mutation of nimT (nimT23 mutation) arrests cells in G2 at the restrictive temperature. After release of the temperature-sensitive nimT23 block, p34cdc2 undergoes tyrosine dephosphorylation and we showed that as cells entered mitosis, a rapid increase in calmodulin was observed. The increase in calmodulin and progression into mitosis were prevented by reducing extracellular Ca2+ levels to 2 nM. The calmodulin gene of a nimT23-containing strain was replaced with a hybrid gene in which calmodulin transcription was regulated by the alcA promoter (AlcCaM/T23). This allowed experimental manipulation of the level of intracellular calmodulin by the carbon source in the medium. When either extracellular Ca2+ or intracellular calmodulin levels were reduced at the nimT23 G2 arrest point, p34cdc2 remained tyrosine phosphorylated but the mitotic NIMA kinase encoded by nimA was not activated. Release of the temperature sensitive nimT23 arrest when either extracellular Ca2+ or calmodulin concentrations were low blocked tyrosine dephosphorylation of p34cdc2, activation of NIMA and progression of cells into mitosis. However, reduced levels of either Ca2+ or calmodulin had no effect on the increase in histone H1 kinase activity associated with p13 beads or the degree of phosphorylation of the majority of MPM-2-reacting proteins following release of the nimT23 mutation. These results demonstrate that both Ca2+ and calmodulin are important for progression into mitosis from the nimT23 arrest point in a pathway involving activation of both NIMA and p34cdc2 protein kinases.

Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures.

An inducible expression system for the production of human lactoferrin in Aspergillus nidulans

The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase ( alcA) promoter. Recombinant hLF (rehLF) is produced at levels up to 5 μg/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.

Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans.

ALCR is the specific activator of the Aspergillus nidulans ethanol-utilization pathway, mediating the induction of its own transcription and that of the structural genes alcA and aldA, encoding respectively, alcohol dehydrogenase I and aldehyde dehydrogenase. ALCR is a DNA binding protein in which 6 cysteines are coordinated in a zinc binuclear cluster. This domain was fused to glutathione-S-transferase (GST) and isolated as a GST-ALCR(7-58*) fusion protein from Escherichia coli. Mobility shift assays showed that the ALCR fusion protein binds at sites upstream of the alcA promoter. DNaseI protection footprinting experiments revealed three specific binding sites, two that are direct repeats and one that is an inverted repeat with the same half-site 5'-CCGCA-3'. The half-sites are separated by a variable number of nucleotides in both types of target. The interaction of the ALCR fusion protein with direct and inverted repeats were examined by using interference and protection footprinting assays. In both binding sites, modification of the guanines in the half-sites interfered with the formation of the DNA complex, but the adjacent ones did not. Our results suggest that the ALCR protein makes contact in the major groove of the DNA helix of the half-sites. The functionality of two out of three binding sites of the GST-ALCR protein was demonstrated after their deletion. Therefore, the region encompassing these binding sites is a cis-acting element involved in the full induction of the alcA gene.

Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans.

Calcium and calmodulin have been widely implicated in the control of cell proliferation. We have created a strain of the genetically tractable filamentous fungus, Aspergillus nidulans, that is conditional for calmodulin expression. This was accomplished by replacing the unique endogenous calmodulin gene with one regulated by the inducible alcohol dehydrogenase (alcA) gene promoter by homologous recombination. This strain cannot grow when the cells are incubated in medium containing a carbon source that represses the alcA promoter. Characterization of the arrested cells shows that 83% are blocked in the G2 phase of the cell cycle. The block is due to very low levels of calmodulin and is fully reversible upon changing to medium that contains an inducer of the alcA promoter. The rate of cell proliferation in this strain is dependent upon both the intracellular calmodulin and extracellular Ca2+ concentrations. Raising the calmodulin concentration by inducing the alcA promoter not only causes the cells to enter the proliferative cycle more quickly and to grow faster, but also decreases the concentration of extracellular Ca2+ required to support growth by 10-fold, as compared with cells grown in noninducing medium. Thus both the intracellular calmodulin and extracellular Ca2+ concentrations are important and interactive factors in regulating the nuclear division cycle of Aspergillus nidulans.