Cytochrome c is not essential for viability of the fungus Aspergillus nidulans.

The filamentous fungus Aspergillus nidulans is an obligate aerobe, which is capable of anaerobic survival, but not anaerobic growth. Since cytochrome c forms an essential part of the oxidative respiratory pathway it was expected that mutants lacking this component would be non-viable. Gene replacement of one homologue of the cycA (cytochrome c) gene was carried out in a diploid strain. Benomyl-induced haploidisation of this diploid yielded all cycA+ haploid colonies, initially suggesting that loss of cycA was indeed lethal. However, use of an alternative unbiased method to recover haploids yielded viable, but slow-growing, cycA- mutants. Replacement of the cycA locus in the cycA- mutants was verified by Southern blotting. Spectral analysis confirmed the absence of detectable levels of cytochrome c, and respiratory insensitivity to cyanide suggested the absence of cytochrome c-dependent respiration. Growth parameters were consistent with those expected of a CycA- mutant. Compared to the wild type, the mutants grew slowly on fermentable carbon sources, did not grow on non-fermentable carbon sources, and produced higher levels of ethanol. To our knowledge, this is the first report of a filamentous fungus that remains viable after complete elimination of a functional cytochrome c gene. We propose that the mutants are viable due to their ability to ferment and to use alternative respiratory pathways.

A mutualistic fungal symbiont of perennial ryegrass contains two different pyr4 genes, both expressing orotidine-5'-monophosphate decarboxylase.

A fragment of the Claviceps purpurea pyr4 gene, encoding orotidine-5'-monophosphate decarboxylase (OMP decarboxylase), was used to screen a genomic library from an isolate of a fungus, Acremonium sp. (designated Lp1), which grows as an endophyte in perennial ryegrass (Lolium perenne). Three positive clones, lambda MC11, lambda MC12 and lambda MC14, were isolated. Two of these clones, lambda MC12 and lambda MC14, were overlapping clones from the same locus, while lambda MC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other Pyrenomycete fungi. The pyr4 gene from lambda MC12 and lambda MC14 was designated pyr4-1 and that from lambda MC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved, with 97.5% identity at the nucleotide level, the 5' non-coding sequences were the least conserved with 88.5% identity and the 3' non-coding sequences had 93.0% identity. RT-PCR analysis of total RNA from Lp1 demonstrated that transcripts from the two genes were present at similar levels, and hybridisation of pyr4-1 to Northern blots of total RNA from Lp1 showed that full-length transcripts were being produced. Genomic fragments containing pyr4 were transformed into a strain of Aspergillus nidulans which has a mutation in pyrG (encoding OMP decarboxylase). Both pyr4-1 and pyr4-2 complemented the pyrG mutation in A. nidulans, indicating that both encode functional OMP decarboxylases. It has been proposed [Schardl et al., Genetics 136 (1994) 1307-1317] that the two pyr4 in Lp1 arose by interspecific hybridisation, most likely between the ryegrass choke pathogen, Epichloë typhina, and another endophyte from perennial ryegrass, Acremonium lolii. Analysis by PCR amplification and direct sequencing of the variable 5' non-coding regions of pyr4, from possible ancestors to Lp1 supports this hypothesis. Comparisons of these sequences to the 5' non-coding sequences from pyr4-1 and pyr4-2 demonstrated that E. typhina and A. lolii were the most likely ancestors of the two pyr4 found in Lp1.

Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans.

The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced. The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues. The nucleotide sequence of the A. nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1). The S. cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A. nidulans. The A. nidulans cytochrome c sequence contains two small introns. One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced. Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximately tenfold in the presence of oxygen and three- to fourfold under heat-shock conditions.

Isolation and nucleotide sequence of the 5-aminolevulinate synthase gene from Aspergillus nidulans.

The structural gene for 5-aminolevulinate (ALA) synthase has been cloned and sequenced from the filamentous fungus Aspergillus nidulans using an oligonucleotide probe based on a highly conserved-amino-acid sequence found in ALA synthase genes of a wide range of species. The cloned gene, hemA, has a 5' untranslated mRNA of 92 nucleotides (nt) and one intron (64 nt). The deduced protein sequence (648 amino acids) shows 64% identity to the yeast ALA synthase in the C-terminal region of 453 amino acids. The N-terminal region is typical of ALA synthase proteins in that the specific amino-acid sequence is not conserved but consists of a "leader" region rich in basic amino acids, believed to be involved in mitochondrial targeting, followed by a stretch of largely hydrophobic residues which may allow interaction with the inner mitochondrial membrane. Under the conditions used the transcription of hemA was unaffected by dextrose repression, heat shock, or oxygen levels.

Isolation and nucleotide sequence of the ribosomal protein S16-encoding gene from Aspergillus nidulans.

A genomic clone has been isolated from Aspergillus nidulans which is homologous to the ribosomal (r) protein S16-encoding gene of Saccharomyces cerevisiae (S16A) and the r-protein S19-encoding gene of rat (S19). The amino acid (aa) sequences, deduced from nucleotide (nt) sequence analysis, show that in both cases more than 63% of the aa are conserved. The proposed A. nidulans r-protein S16 gene (rps16) differs from that of S. cerevisiae in that it occurs as a single copy in the haploid genome (rather than two copies as in yeast) and contains two putative introns (rather than one). The mRNA leader is long compared to many Aspergillus genes, commencing 293 nt upstream from the coding region, and contains an open reading frame of 13 codons.

Heat shock and stationary phase induce transcription of the Saccharomyces cerevisiae iso-2 cytochrome c gene.

Transcription of the iso-2 cytochrome c gene of Saccharomyces cerevisiae (CYC7) is strongly induced by stationary-growth phase, heat shock and low cAMP levels. CYC1, the iso-1 cytochrome c gene, is strongly repressed in stationary phase and unaffected by heat shock and cAMP levels. Heat shock-induced transcription of CYC7 occurs both aerobically (4-6 fold) and anaerobically (30 fold).

Aspects of genetic interaction in hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion.

Hybrids were produced by protoplast fusion between strains of Aspergillus rugulosus and mitotic master strains of Aspergillus nidulans with a genetic marker on each linkage group. Analysis of segregants induced by growth on benomyl revealed recombination between every pair of unlinked markers. Parental combinations of markers were often recovered at significantly higher frequencies than expected. This aberrant segregation was not correlated with any particular pair of linkage-groups and was attributed to inter-species incompatibility. The segregation of genetic markers of A. rugulosus from the hybrids suggested that A. nidulans and A. rugulosus may differ in haploid chromosome number and chromosome size. In sexual crosses between A. nidulans and strains containing chromosomes of mixed parental origin recombinants were recovered. The results support the classification of A. nidulans and A. rugulosus as separate species.