The effects of specific auxotrophic mutations on the virulence of Aspergillus nidulans for mice

The paper describes studies of the influence of a number of genetically-mapped auxotrophic mutations on the virulence ofAspergillus nidulans for DBA/2J male mice. Specific mutations were found to be either neutral in regard to the character of virulence, i.e., strains carrying such mutations had a virulence similar to that of prototrophic strains, or were associated with significantly decreased virulence or avirulence. These data provide the first extensive information concerning the effects of auxotrophic mutation on the virulence of a mycotic pathogen of animals.     

A morphologic mutation in Aspergillus nidulans associated with increased virulence for mice

Studies of the virulence for inbred mice of a morphologic mutant ofAspergillus nidulans and the morphologically-normal strain from which it was isolated as a spontaneous variant are described. The study demonstrates that the single gene difference known to exist between the two strains is associated with increased virulence. This is the first example of a mutation inA. nidulans associated with increased virulence.     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

Diploid Strains of the Pathogenic Basidiomycete Cryptococcus neoformans Are Thermally Dimorphic

Sia, R. A., Lengeler, K. B., and Heitman, J. 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATα and MATa strains. At 37°C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATα and MATa loci. At 24°C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.     

Virulence genetics of Aspergillus nidulans Eidam: A review

Studies of the influence of genotypic alterations on the murine virulence of Aspergillus nidulans Eidam are reviewed to emphasize the potential of this fungus for genetic studies of virulence.The filamentous ascomycete Aspergillus nidulans Eidam produces a fatal systemic mycosis characterized by signs referable to the central nervous system after its intravenous inoculation into male DBA/2J mice. The time and dosage of conidia required to produce a fatal infection are functions of the specific virulence of the strain. In previous investigations we showed that the murine virulence of A. nidulans was strongly dependent on genotype (21–24). In this paper the effects of a number of different genotypic alterations on the virulence of A. nidulans will be reviewed to emphasize the potential of A. nidulans as a model for genetic studies of fungal virulence. The paucity of genetic information regarding the virulence of mycotic zoopathogens makes such studies highly desirable.     

Molecular genetics ofAspergillus pathogenicity

Aspergillus fumigatus is the most frequent cause of Invasive Pulmonary Aspergillosis (IPA), a life-threatening disease of immunosuppressed patients. In addition to a number of general physiological attributes of this fungus, it has been suggested that extracellular elastase and toxins might facilitate its growth in lung tissue. We have investigated the roles of two extracellular proteins, an alkaline protease with elastase activity (AFAlp), and the ribotoxin restrictocin in murine models of IPA. Gene disruption was used to create stable null mutant strains of the fungus lacking one or other protein, and their virulence and histopathological features were compared with an isogenic parental strain in steroid-treated and neutropenic mice. We have been unable to demonstrate any significant differences between the three strains, which shows that, considered independently, these proteins are not important virulence determinants. We are also interested in identifying fungal-specific gene products involved in general metabolism and which are required for growth in the lung, because these could represent new targets for antifungal drugs. For this work a model of murine IPA involvingAspergillus nidulans was established, to take advantage of the many well characterised mutations affecting metabolic pathways. Pathogenicity tests with strains carrying one of two auxotrophic mutations,lysA2 andpabaA1, have shown while lysine biosynthesis is not essential for the fungus to cause pulmonary disease, biosynthesis ofp-aminobenzoic acid is essential. We are now in the process of cloning theA. fumigatus pabaA homologue to determine its function and whether this gene is required for growth of the clinically important species in the lung.     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

The Spoilage Yeast Zygosaccharomyces bailii Forms Mitotic Spores: a Screening Method for Haploidization

Zygosaccharomyces bailii ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content. Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants. Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of Z. bailii. This new set of Z. bailii strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies.     

Recombinagenic effect of cryptolepine in uvsH+//uvsH+ and uvsH//uvsH diploid strains of Aspergillus nidulans

Recombinagenic potential of the alkaloid cryptolepine was evaluated in two diploid strains ofAspergillus nidulans—a wild type strain (uvsH ⁺//uvsH ⁺) and a DNA-repair-deficient one (uvsH//uvsH). Treatment of both strains with cryptolepine failed to alter colony growth in culture; its recombinagenic potential was determined by the homozygotization index (in which events of mitotic exchange may cause expression of genes previously masked by the dominant allele). Mitotic crossing-overs were induced by 7 and 14 mg/L doses of cryptolepine in both diploidA. nidulans strains.     

Most of the yeast genomic sequences are not essential for cell growth and division

To determine the fraction of the yeast Saccharomyces cerevisiae genome that is required for normal cell growth and division, we constructed diploid strains that were heterozygous for random single disruptions. We monitored the effects of approximately 200 independent disruptions by sporulating the diploids and examining the phenotype of the resulting haploid strains. We found that only 12% of the disruptions were haploid-lethal, 14% resulted in slow growth, and an additional 4% were associated with some other new phenotype (such as an auxotrophic requirement). No obvious new phenotype was detected for 70% of the disruptions.     

Alternation of nuclear phase in the filamentous basidiomycete,Helicobasidium mompa

Variation in the number of nuclei and cellular ploidy were observed in eight strains ofHelicobasidium mompa. The basidiospores, single-spore isolates and field-isolated strains were all dikaryons. The cellular ploidy, which was assessed by analyzing the fluorescence emitted by DAPI-stained nuclei, was unstable: monokaryotic strains derived from the original dikaryotic strains by successive subcultures were mainly tetraploid, although the original dikaryon was in most cases diploid. On the other hand, a dikaryotic strain derived by treatment with benomyl was haploid. These results suggest that diploid dikaryon is a normal nuclear phase ofH. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.     

Effect of auxotrophic starvation on mitochondrial marker transmission in the cdc8 mutant of Saccharomyces cerevisiae

Summary Crosses were made using strains of S. cerevisiae which carried mitochondrial markers conferring resistance to erythromycin and chloramphenicol. The effect of auxotrophic starvation of one parent prior to mating on the transmission of its mitochondrial markers was studied in different crosses relative to the presence of the cdc8 nuclear mutation (a temperature-sensitive DNA replication).In crosses between two cdc8 mutant strains, auxotrophic starvation of one of the haploid parental strains prior to mating caused a marked decrease of its mitochondrial marker transmission to the diploid progeny of the cross. The transmission decreased as a function of the time of starvation. This effect was not observed in the cross between two wild type strains and in crosses of starved cdc8 phenotypic revertants with cdc8 mutant strains. Only a small, if any, effect of starvation on mitochonrial marker transmission was observed when starved cdc8 mutant strains were crossed either with their phenotypic revettants or with the wild-type strains.In one of the haploid parental strains the starvation increased the frequency of petites as a function of starvation time, while in the other this effect was not observed.In the progeny of cdc8xcdc8 crosses (both in starvation experiments and in control crosses) an increased frequency of diploid petite cells accompanied by a decreased frequency of recombination between mitochondrial markers was noticed.The influence of the cdc8 mutation on the transmission of mitochondrial markers is discussed in terms of high frequency of ^– molecule formation in cdc8 strains.     

Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic

Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATalpha and MATa strains. At 37 degrees C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATalpha and MATa loci. At 24 degrees C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

Penicillin production and its mode of inheritance inAspergillus nidulans

Previous workers have shown that some strains ofAspergillus nidulans produce penicillin-like substances. In the present studies, shake-flask cultures of 101 wild-type strains ofA. nidulans, representatives of 18 different heterokaryon-compatible groups, were examined and filtrates of most found to inhibit the growth of a strain ofBacillus subtilis sensitive to penicillin, although members of two of these groups had no detectable antibiotic activity. Five strains with antibacterial properties were chosen for detailed investigation as well as two genetically labelled derivatives obtained from one of these after ultraviolet light treatments; one derivative had a similar antibiotic yield to its original wild-type parent but the other was selected as having increased antibiotic yield. The antibiotic produced by these seven strains was by all tested criteria, including chromatographic and electrophoretic behaviour, indistinguishable from penicillin. A heterokaryon test between the two mutants indicated that antibiotic productivity was under nuclear control.     

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate

A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.     

The spoilage yeast Zygosaccharomyces bailii forms mitotic spores: a screening method for haploidization

Zygosaccharomyces bailii ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content. Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants. Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of Z. bailii. This new set of Z. bailii strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies.     

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains ofSaccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in theG ₁ stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.     

Mitotic recombination accelerates adaptation in the fungus Aspergillus nidulans

Understanding the prevalence of sexual reproduction in eukaryotes is a hard problem. At least two aspects still defy a fully satisfactory explanation, the functional significance of genetic recombination and the great variation among taxa in the relative lengths of the haploid and diploid phases in the sexual cycle. We have performed an experimental study to explore the specific advantages of haploidy or diploidy in the fungus Aspergillus nidulans. Comparing the rate of adaptation to a novel environment between haploid and isogenic diploid strains over 3,000 mitotic generations, we demonstrate that diploid strains, which during the experiment have reverted to haploidy following parasexual recombination, reach the highest fitness. This is due to the accumulation of recessive deleterious mutations in diploid nuclei, some of which show their combined beneficial effect in haploid recombinants. Our findings show the adaptive significance of mitotic recombination combined with flexibility in the timing of ploidy level transition if sign epistasis is an important determinant of fitness.