Mutants of Aspergillus nidulans blocked at an early stage of sporulation secrete an unusual metabolite

Mutants of Aspergillus nidulans defective in conidiation (asexual sporulation) can be classified according to whether they are blocked before or after induction of conidiation. Mutants blocked before induction (preinduction mutants) appear to be unable to respond to the inducing stimulus and thus are defective in one of the earliest events in the sporulation process. Three preinduction mutants have been isolated and characterized. Each was found to exhibit the same pleiotropic phenotype: they also were defective in sexual sporulation and secreted a set of phenolic metabolites at a level much higher than did wild type or mutants blocked at later stages of conidiation. One of the metabolites has been identified as the antibiotic diorcinal (3,3'-dihydroxy-5,5'-dimethyldiphenyl ether) which is known to be involved in the synthesis of certain farnesyl phenols of unknown function. These results suggest that preinduction mutants are blocked in a phenolic metabolic pathway, one or more product of which participates in the initiation of sporulation.     

Suppression of defective-sporulation phenotypes by mutations in transcription factor genes of Bacillus subtilis.

Mutations in the Bacillus subtilis major RNA polymerase sigma factor gene (rpoD/crsA47) and a sensory receiver gene (spoOA/rvtA11) are potent intergenic suppressors of several stage 0 sporulation mutations (spoOB, OE, OF & OK). We show here that these suppressors also rescue temperature-sensitive sporulation phenotypes (Spots) caused by mutations in RNA polymerase, ribosomal protein, and protein synthesis elongation factor EF-G genes. The effects of the crsA and rvtA suppressors on RNA polymerase and ribosomal protein spots mutations are similar to those previously described for mutations in another intergenic suppressor gene rev. We have examined the effects of rvtA and crsA mutations on the expression of sporulation-associated membrane proteins, including flagellin and penicillin binding protein 5* (PBP 5*). Both suppressors restored sporulation and synthesis of PBP 5* in several spoO mutants. However, only rvtA restored flagellin synthesis in spoO suppressed backgrounds. The membrane protein phenotypes resulting from the presence of crsA or rvtA suppressors in spoO strains suggests that these suppressors function via distinct molecular mechanisms. The rvtA and crsA mutations are also able to block the ability of ethanol to induce spoO phenocopies at concentrations of ethanol which prevent sporulation in wild type cells. The effects of ethanol on sporulation-associated membrane protein synthesis in wild type and suppressor containing strains have been examined.     

Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis: isolation and phenotype identification of partial revertants

Single-step mutants of Bacillus subtilis blocked at stage zero of sporulation (spoO mutants) are pleiotropic. They are impaired in several traits, including increased sensitivity to polymyxin B, when compared to the wild type. Revertants for one or another of the traits associated in this pleiotropy have been isolated and classified according to their phenotypes. Attention was centered on partial revertants (spoO still), of which at least three classes could be recognized. Class I partial revertants recovered a high level of resistance to polymyxin and, in a constant order, a variable number of the other traits; some kind of hierarchy is thus revealed to exist among the traits affected in the original pleiotropy. Partial revertants in the other two classes are still hypersensitive to polymyxin. In class II revertants, ability to excrete exoenzymes and antibiotic(s) is recovered to various extents. Only inducibility of nitrate reductase by nitrate is recovered in class III mutants. The possible significance of these observations is discussed.     

Developmental defects resulting from arginine auxotrophy in Aspergillus nidulans

A mutant of Aspergillus nidulans, isolated for inability to form asexual spores (conidia) on complete medium, was found to regain the ability to conidiate if the medium was supplemented with arginine. On minimal medium the mutant required arginine for growth but at a much lower concentration than that required for conidiation. This mutant, designated argB12, thus defines a phase-critical gene, i.e. a gene whose function is in greater demand for development than for growth. In addition to its aconidial phenotype, the mutant also exhibited (depending on the medium) aberrant sexual development and a low efficiency of conidial germination. In crosses, each of these developmental phenotypes segregated with arginine auxotrophy. Genetic and biochemical analyses showed the argB12 mutation to be an allele of the previously described argB locus, mutants of which lack ornithine transcarbamylase. Arginine-requiring mutants at at least two other loci were also found to be defective in asexual sporulation, but the germination defect appears to be specific to argB mutants.     

Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis. Genetic study of polymyxin resistant partial revertants

Summary A class of suppressor mutations restores, in pleiotropic sporulation mutants of B. subtilis (SPO mutants), the wild type level of resistance to Polymyxin, and, most often, other properties of the wild strain as well, but never the ability to sporulate. These suppressors, extracistronic, are active on mutations occurring in any one of the 5 genes in which SPO mutations have been found. The phenotype of the suppressed strains is dependent on both the suppressed (SPO) and the suppressive mutations. All these suppressors are located in a single locus and some of them are thermosensitive. The evidence suggests that a physiological compensation is at work in the partial revertants, so that the locus at which the suppressors are located was called cps X. Two hypotheses are discussed that might account for these observations.     

An endogenous inducer of sexual development in Aspergillus nidulans

During development of the homothallic ascomycete Aspergillus nidulans, asexual sporulation is followed by sexual sporulation. We report here the detection of a solvent-extractable activity which inhibits asexual sporulation and stimulates premature sexual sporulation. This activity, called precocious sexual inducer (psi), is overproduced by certain mutants that are blocked in both modes of sporulation. Using partially purified preparations of psi, biological response could be elicited with as little as 50 ng of material. We suggest that psi is a hormone-precursor which is converted to a hormone by normal sporulating strains that respond to psi, but not by the asporogenous mutants that overproduce psi. The stability of psi activity gives promise that the compound can be purified and identified.     

Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea.

cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Allelspezifische suppressormutationen vonSchizosaccharomyces pombe

By comparing the intragenic distribution of suppressor sensitive mutants in fine structure maps, 13 allele specific suppressor mutations (isolated from revertants in adenine dependent mutants of constitutionad ₇) have been analyzed for their allele specific patterns of action in three different groups of mutants blocked in adenine biosynthesis. The 13 suppressor mutations, which have resulted from mutations at seven different suppressor loci, are characterized by four different suppression patterns. Three of these patterns, which partially overlap, are not locus specific since they include sensitive mutants at each of the three lociad ₇, ad₆ andad ₁ studied. The relative frequency of mutants sensitive to one or the other of the suppressors of this type, the absence of osmotic-remedial strains among the suppressor sensitive mutants, and the polarized complementation behaviour of one suppressiblead ₆ mutant and two suppressiblead ₁ mutants capable of interallelic complementation, suggest that the suppression mechanism involves misreading of a mutant triplet of the nonsense type.     

Identification and characterization of sporulation gene spoVS from Bacillus subtilis.

We report the identification and characterization of an additional sporulation gene from Bacillus subtilis called spoVS, which is induced early in sporulation under the control of sigma H. We show that spoVS is an 86-codon-long open reading frame and is capable of encoding a protein of 8,796 Da which exhibits little similarity to other proteins in the databases. Null mutations in spoVS have two contrasting phenotypes. In otherwise wild-type cells they block sporulation at stage V, impairing the development of heat resistance and coat assembly. However, the presence of a spoVS mutation in a spoIIB spoVG double mutant (which is blocked at the stage [II] of polar septation) acts as a partial suppressor, allowing sporulation to advance to a late stage. The implications of the contrasting phenotypes are discussed in the context of the formation and maturation of the polar septum.     

Dominant spore color mutants of Aspergillus nidulans defective in germination and sexual development.

The ascomycete Aspergillus nidulans produces green conidia (asexual spores). Recessive mutants which produce yellow conidia have been previously isolated from haploid strains and have been shown to be deficient in laccase (diphenol oxidase), an enzyme that requires copper for activity. Using a diploid parent strain, we isolated dominant yellow conidial mutants which, in the haploid state, produced even less laccase activity than a recessive mutant. Three isolates of such mutants behaved similarly and define a single complementation group (yB) on chromosome VIII distinct from the yA locus on chromosome I defined by recessive mutants. Unlike yA mutants, whose only discernable phenotype is their conidial color, yB mutants are pleiotropic: conidial germination was delayed relative to the wild type, and sexual development was blocked at an early stage. The three phenotypes of yB mutants were expressed on yeast extract-glucose medium containing 1.6 microM of added copper. When copper was added to above 5 microM, all three phenotypes were remediated, and near wild-type levels of laccase were produced. We conclude that yB mutants have a reduced availability of copper. The dominance of yB mutants could result, for example, from an alteration in transport or storage of copper. Using an immunological assay, we detected no laccase antigenic cross-reacting material in yB mutants grown on medium of low copper content. We conclude that either the synthesis or the stability of laccase is copper dependent.     

NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa

We have proposed that reactive oxygen species (ROS) play essential roles in cell differentiation. Enzymes belonging to the NADPH oxidase (NOX) family produce superoxide in a regulated manner. We have identified three distinct NOX subfamilies in the fungal kingdom and have shown that NoxA is required for sexual cell differentiation in Aspergillus nidulans. Here we show that Neurospora crassa NOX-1 elimination results in complete female sterility, decreased asexual development, and reduction of hyphal growth. The lack of NOX-2 did not affect any of these processes but led instead to the production of sexual spores that failed to germinate, even in the presence of exogenous oxidants. The elimination of NOR-1, an ortholog of the mammalian Nox2 regulatory subunit gp67(phox), also caused female sterility, the production of unviable sexual spores, and a decrease in asexual development and hyphal growth. These results indicate that NOR-1 is required for NOX-1 and NOX-2 functions at different developmental stages and establish a link between NOX-generated ROS and the regulation of growth. Indeed, NOX-1 was required for the increased asexual sporulation previously observed in mutants without catalase CAT-3. We also analyzed the function of the penta-EF calcium-binding domain protein PEF-1 in N. crassa. Deletion of pef-1 resulted in increased conidiation but, in contrast to what occurs in Dictyostelium discoideum, the mutation of this peflin did not suppress the phenotypes caused by the lack of NOX-1. Our results support the role of ROS as critical cell differentiation signals and highlight a novel role for ROS in regulation of fungal growth.     

The role of pleiotropy in the maintenance of sex in yeast

In facultatively sexual species, lineages that reproduce asexually for a period of time can accumulate mutations that reduce their ability to undergo sexual reproduction when sex is favorable. We propagated Saccharomyces cerevisiae asexually for approximately 800 generations, after which we measured the change in sexual fitness, measured as the proportion of asci observed in sporulation medium. The sporulation rate in cultures propagated asexually at small population size declined by 8%, on average, over this time period, indicating that the majority of mutations that affect sporulation rate are deleterious. Interestingly, the sporulation rate in cultures propagated asexually at large population size improved by 11%, on average, indicating that selection on asexual function effectively eliminated most of the mutations deleterious to sporulation ability. These results suggest that pleiotropy between mutations' effects on asexual fitness and sexual fitness was predominantly positive, at least for the mutations accumulated in this experimental evolution study. A positive correlation between growth rate and sporulation rate among lines also provided evidence for positive pleiotropy. These results demonstrate that, at least under certain circumstances, selection acting on asexual fitness can help to maintain sexual function.     

The GanB Galpha-protein negatively regulates asexual sporulation and plays a positive role in conidial germination in Aspergillus nidulans

We isolated the ganB gene encoding the Galpha-protein homolog from Aspergillus nidulans. To investigate the cellular function of GanB, various mutant strains were isolated. Deletion of constitutively inactive ganB mutants showed conidiation and derepressed brlA expression in a submerged culture. Constitutive activation of GanB caused a reduction in hyphal growth and a severe defect in asexual sporulation. We therefore propose that GanB may negatively regulate asexual sporulation through the BrlA pathway. In addition, deletion or constitutive inactivation of GanB reduced germination rate while constitutive activation led to precocious germination. Furthermore, conidia of a constitutively active mutant could germinate even without carbon source. Taken together, these results indicated that GanB plays a positive role during germination, possibly through carbon source sensing, and negatively regulates asexual conidiation in A. nidulans.     

Use of Constructed Double Mutants for Determining the Temporal Order of Expression of Sporulation Genes in Bacillus subtilis

Double mutants containing two Spo mutations concerned with different stages of sporulation were constructed. In these, the phenotype that is exhibited is that of the earlier sporulation block. The same procedure was applied to sporulation mutants damaged in the same stage of development. The results provide a basis for placing in a temporal order different mutations concerned in stage II and stage IV of spore development. In general, the order indicated by the phenotypes of the double mutants is in agreement with the order derived on biochemical grounds. Double oligosporogenous mutants have also been constructed. Their sporulation incidence is roughly equal to the product of the incidences of the parent strains, idicating that separate factors are involved in overcoming each oligosporogenous block. The number of dependent sequential steps in sporulation is estimated as not less than about 12.