Genetic analysis in fungi using restriction-enzyme-mediated integration.

Restriction-enzyme-mediated integration (REMI), a method for generating nonhomologous integration of transforming DNA into the chromosomes of eukaryotic cells, has been used for insertion mutagenesis and other genetic studies in diverse organisms. Insertion mutations generated by REMI have facilitated the genetic dissection of developmental pathways in Dictyostelium discoidium and the isolation of virulence factors in several plant pathogenic fungi. Recent work indicates that REMI occurs by nonhomologous end joining.

Role of a Candida albicans P1-type ATPase in resistance to copper and silver ion toxicity.

Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels. To identify Candida albicans genes that are involved in resistance to copper ion toxicity, a library containing inserts of C. albicans genomic DNA was used to complement the copper sensitivity phenotype of a Saccharomyces cerevisiae cup1Delta strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance. A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned. The gene encoding this pump was termed CRD1 (for copper resistance determinant). A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termed CRD2. Transcription of the CRD1 gene was found to increase upon growth with increasing copper levels, while the CRD2 mRNA was expressed at a constant level. Strains with the CRD1 gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 microM CuSO(4). These crd1 strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport. Although strains with the CRD2 gene disrupted showed reduced growth rate with increasing copper concentration, the crd2 mutants eventually attained wild-type levels of growth, demonstrating that CRD2 is less important for resistance to copper ion toxicity. Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence of C. albicans as a nosocomial pathogen.

Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system.

Streptomycete antibiotic synthesis is coupled to morphological differentiation such that antibiotics are produced as a colony sporulates. Streptomyces coelicolor produces several structurally and genetically distinct antibiotics. The S. coelicolor absA locus was defined by four UV-induced mutations that globally blocked antibiotic biosynthesis without blocking morphological differentiation. We show that the absA locus encodes a putative eubacterial two-component sensor kinase-response regulator system. All four mutations lie within a single open reading frame, designated absA1, which is predicted to encode a sensor histidine kinase. A second gene downstream of absA1, absA2, is predicted to encode the cognate response regulator. In marked contrast to the antibiotic-deficient phenotype of the previously described absA mutants, the phenotype caused by disruption mutations in the absA locus is precocious hyperproduction of the antibiotics actinorhodin and undecylprodigiosin. Precocious hyperproduction of these antibiotics is correlated with premature expression of XylE activity in a transcriptional fusion to an actinorhodin biosynthetic gene. We propose that the absA locus encodes a signal transduction mechanism that negatively regulates synthesis of the multiple antibiotics produced by S. coelicolor.

Intestinal lesions associated with disseminated candidiasis in an experimental animal model.

In human patients, disseminated candidiasis, a life-threatening disease for immunocompromised patients, is often associated with intestinal lesions. In this study, we demonstrate that immunosuppressed gnotobiotic (IGB) piglets orally inoculated with wild-type Candida albicans developed extensive intestinal lesions and disseminated infection. Severe ulceration of the ileal mucosa was observed overlying regions of colonization and necrosis of the gut-associated lymphoid tissue. Despite the high susceptibility of IGB piglets to many microbial pathogens, an avirulent mutant strain of C. albicans failed to produce intestinal lesions and exhibited poor dissemination, demonstrating that these effects required virulent organisms. It is likely that in IGB piglets, as in human patients, intestinal lesions provide the mechanism for escape of C. albicans from the gastrointestinal tract. Multinucleated giant cells containing fungal organisms were observed within lymph nodes and lymphatic vessels, and as with other pathogens, such cells could provide a mechanism for dissemination of C. albicans.

Invasive lesions containing filamentous forms produced by a Candida albicans mutant that is defective in filamentous growth in culture.

A Candida albicans efg1 cph1 double mutant is nonfilamentous under standard laboratory conditions and avirulent in mice. However, this mutant produced filaments in the tongues of immunosuppressed gnotobiotic piglets and when embedded in agar, demonstrating that an Efg1p- and Cph1p-independent pathway for promotion of filamentous growth exists.

The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development.

The chsB gene from Aspergillus nidulans encodes a class III chitin synthase, an enzyme class found in filamentous fungi but not in yeast-like organisms. Using a novel method, we isolated haploid segregants carrying a disrupted chsB allele from heterozygous diploid disruptants. The haploid disruptants grow as minute colonies that do not conidiate. Hyphae from the disruptants have enlarged tips, a high degree of branching, and disorganized lateral walls. The mycelium is not deficient in chitin content and shows no evidence of lysis. The disruptant phenotype is not remedied by osmotic stabilizers. The results indicate that chitin synthesized by the chsB-encoded enzyme does not substantially contribute to the rigidity of the cell wall but is necessary for normal hyphal growth and organization. The properties of the A. nidulans disruptant are similar to those for Neurospora crassa strains with a disrupted chs-1 gene, which also encodes a class III chitin synthase. The morphology of an A. nidulans heterokaryon containing both the wild-type and the disrupted chsB alleles indicates that chsB acts in local areas of the mycelium. The heterokaryon produces conidia of both parental genotypes in nearly equal numbers, indicating that the wild-type chsB gene is not necessary for conidium formation. In addition, we identified and sequenced a second, previously undescribed, homolog of chsB from the closely related opportunistic pathogen, A. fumigatus. The finding of two class III chitin synthase genes in A. fumigatus and a single gene of this class in A. nidulans illustrates limitations of using A. nidulans as a genetic model for A. fumigatus.

The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis.

Two chitin synthase genes, chsD and chsE, were identified from the filamentous ascomycete Aspergillus nidulans. In a region that is conserved among chitin synthases, the deduced amino acid sequences of chsD and chsE have greater sequence identity to the polypeptides encoded by the Saccharomyces cerevisiae CHS3 gene (also named CSD2, CAL1, DIT101, and KTI1) and the Candida albicans CHSE gene than to other chitin synthases. chsE is more closely related to the CHS3 genes, and this group constitutes the class IV chitin synthases. chsD differs sufficiently from the other classes of fungal chitin synthase genes to constitute a new class, class V. Each of the wild-type A. nidulans genes was replaced by a copy that had a substantial fraction of its coding region replaced by the A. nidulans argB gene. Hyphae from both chsD and chsE disruptants contain about 60-70% of the chitin content of wild-type hyphae. The morphology and development of chsE disruptants are indistinguishable from those of wild type. Nearly all of the conidia of chsD disruption strains swell excessively and lyse when germinated in low osmotic strength medium. Conidia that do not lyse produce hyphae that initially have normal morphology but subsequently lyse at subapical locations and show ballooned walls along their length. The lysis of germinating conidia and hyphae of chsD disruptants is prevented by the presence of osmotic stabilizers in the medium. Conidiophore vesicles from chsD disruption strains frequently swell excessively and lyse, resulting in colonies that show reduced conidiation. These properties indicate that chitin synthesized by the chsD-encoded isozyme contributes to the rigidity of the walls of germinating conidia, of the subapical region of hyphae, and of conidiophore vesicles, but is not necessary for normal morphology of these cells. The phenotypes of chsD and chsE disruptants indicate that the chitin synthesized by each isozyme serves a distinct function. The propensity of a chsD disruptant for osmotically induced lysis was compared to that of strains carrying two other mutations (tsE6 and orlA::trpC) which also result in reduced chitin content vegetative cell lysis. The concentration of osmotic stabilizer necessary to remedy the lysis of strains carrying the three mutations is inversely related to the chitin content of each strain. This finding directly demonstrates the importance of chitin to the integrity of the cell wall and indicates that agents that inhibit the chsD-encoded chitin synthase could be useful anti-Aspergillus drugs.