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.     

Spore Germination of Bacillus megaterium QM B1551 Mutants

Auxotrophic or antibiotic-resistant mutants of Bacillus megaterium QM B1551 are capable of initiation of germination similar to the parental (QM B1551) prototrophic strain.     

Genetic Analysis of Prototrophic Natural Variants of Candida Albicans

To facilitate genetic analysis of Candida albicans natural variants, we have isolated a dominant mycophenolic acid-resistant mutant. Mycophenolic acid-resistant auxotrophs were used to analyze prototrophic natural variants by spheroplast fusion. The fusion products were shown to be heterozygous for many of the parental chromosomes by molecular and genetic criteria. Using this approach, we have found that one type of morphologic variation is due to a recessive change and identified three dominant 5-fluorocytosine-resistant mutants. Rare fusion products express recessive parental markers. These exceptional progeny should be useful for linkage analysis and strain construction.     

Auxotrophic mutants in the selection of the rubomycin producer, Actinomyces coeruleorubidus

A total of 351 auxotrophic mutants with different antibiotic activity, including several mutants with activity higher than that of the parent prototrophic strains were obtained under the effect of gamma-rays from 3 prototrophic strains of Act. coeruleorubidus. It was shown that most of the auxotrophic mutants did not preserve the property of biochemical insufficiency on passages on complete media. A mutant strain 1059-32 with activity 2 times higher than that of the prototrophic strain 2-39 and the parent auxotrophic culture was obtained from the revertants. Requirements in 29 growth factors including 17 amino acids, 4 nitrous bases, 8 vitamins and coenzymes were determined in 46 stable auxotrophic mutants isolated. The effect of the specific and non-specific growth factors on the culture antibiotic production was studied.     

Acetic acid inhibits nutrient uptake in Saccharomyces cerevisiae: auxotrophy confounds the use of yeast deletion libraries for strain improvement

Acetic acid inhibition of yeast fermentation has a negative impact in several industrial processes. As an initial step in the construction of a Saccharomyces cerevisiae strain with increased tolerance for acetic acid, mutations conferring resistance were identified by screening a library of deletion mutants in a multiply auxotrophic genetic background. Of the 23 identified mutations, 11 were then introduced into a prototrophic laboratory strain for further evaluation. Because none of the 11 mutations was found to increase resistance in the prototrophic strain, potential interference by the auxotrophic mutations themselves was investigated. Mutants carrying single auxotrophic mutations were constructed and found to be more sensitive to growth inhibition by acetic acid than an otherwise isogenic prototrophic strain. At a concentration of 80 mM acetic acid at pH 4.8, the initial uptake of uracil, leucine, lysine, histidine, tryptophan, phosphate, and glucose was lower in the prototrophic strain than in a non-acetic acid-treated control. These findings are consistent with two mechanisms by which nutrient uptake may be inhibited. Intracellular adenosine triphosphate (ATP) levels were severely decreased upon acetic acid treatment, which likely slowed ATP-dependent proton symport, the major form of transport in yeast for nutrients other than glucose. In addition, the expression of genes encoding some nutrient transporters was repressed by acetic acid, including HXT1 and HXT3 that encode glucose transporters that operate by facilitated diffusion. These results illustrate how commonly used genetic markers in yeast deletion libraries complicate the effort to isolate strains with increased acetic acid resistance.     

Improvement in citric acid production by haploidization of Aspergillus niger diploid strains

Segregation of diploid strains by a haploidizing agent was used to improve citric acid producing strains of Aspergillus niger. Stable diploid strains were obtained via protoplast fusion between two citric acid-producing strains from different genealogies, one for shaking culture and the other for solid culture. Diploid strains were treated by benomyl as a haploidizing agent, and many segregants were obtained. Prototrophic segregants were selected and their haploidy was confirmed by their conidial size and DNA contents. The prototrophic segregants were very variable in their citric acid productivities, some of them better either in shaking culture or in solid culture than both the parental strains. The presence of methanol stimulated citric acid production by the parental and the diploid strains. However, all prototrophic segregants derived from one diploid strain had higher productivities in solid cultures without methanol than in those with methanol.     

Temperature-sensitive multicellular mutants of Wangiella dermatitidis.

Three temperature-sensitive morphological mutants of Wangiella dermatitidis were isolated and characterized. The mutants grew in the yeastlike morphology at the permissive temperature (25 degrees C) but expressed a multicellular (Mc) phenotype at the restrictive temperature (37 degrees C). Cultures of Mc 2 and 3 incubated at the restrictive temperature showed rapid reductions in the percentage of budded cells in the population. In contrast, budding continued for several generations in cultures of Mc 1. Incubation of cultures of Mc 2 and 3 at the restrictive temperature for 48 h resulted in nearly total conversion of yeastlike cells to the multicellular form; about 50% of the cells of Mc 1 had converted to multicellular forms after 48 h at the restrictive temperature. Studies using radiolabeled compounds documented that DNA, RNA, and protein synthesis continued at the restrictive temperature. The results suggest that multicellularity is the result of inhibition of bud emergence and cell separation without inhibition of growth nuclear division, and cytokinesis.     

Chlamydospore Production and Germ-Tube Formation by Auxotrophs of Candida albicans

A prototrophic strain and 21 auxotrophic strains of Candida albicans were assessed for their capacity to produce chlamydospores and germ tubes. All of the mutants were able to produce germ-tubes in human serum but only two mutants produced them in defined medium with L-alpha-amino-n-butyric acid as the sole source of nitrogen. Most auxotrophs were not able to produce chlamydospores on corn meal agar with 1% Tween 80, but they could be induced to do so if the medium was supplemented with their growth requirement(s). Although L-cysteine was able to support the growth of two methionine mutants, it did not support chlamydospore formation when added to corn meal agar with 1% Tween 80. Mutants of C. albicans that do not form chlamydospores could be incorrectly identified in laboratories that rely on chlamydospore formation for identification.     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

Characterization of Altered Forms of Glycyl Transfer Ribonucleic Acid Synthetase and the Effects of Such Alterations on Aminoacyl Transfer Ribonucleic Acid Synthesis In Vivo

The glycyl transfer ribonucleic acid (tRNA) synthetase (GRS) activities of several Escherichia coli glyS mutants have been partially characterized; the K(m) for glycine and the apparent V(max) of several of the altered GRS differ significantly from the parental GRS. Paradoxically, some of the altered forms exhibit more activity in vitro than the GRS from a prototrophic strain (GRS(L)); several parameters of these activities have been studied in an attempt to resolve this problem. The amount of acylated tRNA(Gly) in vivo was examined to assess the GRS activities inside the cells. During exponential growth in media containing glycine, moderate amounts of acylated tRNA(Gly) occur in the glyS mutants; glycine deprivation leads to a dramatic drop in the amount of acylated tRNA(Gly). An alternative measure of the in vivo activities of the altered enzymes is the efficiency of suppression of the trpA36 locus by su(36) (+); glyS mutants grown with added glycine exhibit one-third to one-fourth the suppression efficiency of the prototrophic glyS(H) parent, presumably because they are less efficient, even in the presence of high levels of glycine, in charging the tRNA(Gly) species which functions as the translational suppressor.     

Transmission and expression of mutations to nalidixic acid resistance among products of protoplast fusion crosses of Candida albicans

Earlier studies suggested that heritable resistance to nalidixic acid (Nal) induced in the asexual, pathogenic yeast Candida albicans by growth on Nal results from mitochondrial mutation. To determine conclusively whether mutations to Nal resistance are cytoplasmic or nuclear, several stable Nal-resistant (Nalr) mutants exhibiting distinctive differences in degrees of Nal resistance were obtained from each of two doubly auxotrophic strains (Ade-, Thr- and Arg-, His-), both derived from the same wild-type stock. Inheritance of Nal resistance was then assessed in a series of protoplast fusion crosses between complementing auxotrophs. The initial, intact cellular products of a fusion cross are prototrophic heterokaryons which frequently assort single parental nuclei into monokaryotic blastospores containing biparental cytoplasms. Occasional karyogamy within heterokaryons also yields prototrophic hybrid monokaryons which can undergo recombinations for chromosomal markers through spontaneous or induced mitotic crossing-over. Segregation and expression of Nal resistance among non-hybrid, parental-type monokaryons from Nalr X Nals heterokaryons showed that Nalr mutations are nuclear and that their expressions are not noticeably affected by admixture of cytoplasms of sensitive and resistant parental strains. Analyses of heterokaryons and hybrid monokaryons from Nalr X Nals and Nalr X Nalr crosses demonstrated that Nal resistance is recessive to sensitivity, and that independent Nalr mutations arise at one gene in the Ade-, Thr- strain and at a separate, complementing single gene in the Arg-, His- strain. Prior work demonstrated that induction of Nalr mutations in wild-type C. albicans depends profoundly on the (i) carbon and nitrogen, (ii) growth temperature, (iii) contact with particular metabolic inhibitors and (iv) division stage of cells during exposure to Nal. The present observations indicate that the character of cellular auxotrophies can determine the genetic loci at which Nalr mutations can be recovered.     

Intraspecific heterokaryon and fruit body formation in Coprinus macrorhizus by protoplast fusion of auxotrophic mutants

Summary Fusion of protoplasts of Coprinus macrorhizus mutants with different amino acid requirements resulted in the production of prototrophic clones at frequencies of 1–4% of the protoplasts surviving the fusion treatment. The frequencies were at least 200 times higher than those of the appearance of revertants. Few prototrophic colonies appeared also when the mutant protoplasts were individually subjected to fusion treatment, or when they were mixedly cultured without fusion treatment. It was thus concluded that intraspecific heterokaryons were formed by protoplast fusion.The auxotrophic mutants did not form fruit bodies when cultured singly or mixedly with each other. In contrast, the heterokaryons produced by protoplast fusion between the mutants of compatible mating types developed into fruit bodies with intermediate morphology of those of the strains from which the mutants were derived. Heterokaryons were also formed by fusion of mutant protoplasts with identical mating genotype, but they failed to form fruit bodies.Abbreviations PEG polyethyleneglycol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid     

Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.     

Nonparental progeny resulting from protoplast fusion inTrichoderma in the absence of parasexuality

The purpose of this study was to characterize a number of progeny from intra- and interstrain protoplast fusion within the genusTrichoderma. We wished to determine whether parasexuality or other genetic mechanisms occur in these fungi. When two different auxotrophs of the same strain were fused, rapidly growing prototrophic progeny were obtained in high frequencies. When single spore isolates of these strains were prepared, equal numbers of strains indistinguishable from the two parental auxotrophic strains were obtained, even though 10⁹–10¹⁰ conidia were tested per strain. Thus, progeny from intrastrain fusions all appeared to be balanced heterokaryons, and no evidence of recombination between the two parental strains was obtained. When 16 separate interstrain fusions were conducted, very different results were obtained, regardless of whether fusions were within or between species. Following interstrain fusions, presumptive somatic hybrids developed very slowly and in low numbers as compared with hybrids from intrastrain fusions. Most were weakly prototrophic. These slow-growing progeny were unstable and sectors developed from them. Such sectors themselves were unstable and gave rise to other progeny. Usually sectors were more strongly prototrophic and more rapid growing than the original progeny strain. Sectoring gave rise to a very wide range of morphotypes. Most of these morphotype variants were stable through conidiation; thus, these types did not occur as a consequence of heterokaryosis. Isozyme analysis was conducted on over 1000 progeny strains. Nearly all progeny were identical to one or the other parental isozyme phenotypes. A few progeny, when tested as soon as possible after fusion, exhibited the isozyme phenotypes of both parents, but such biparental banding patterns were rapidly lost upon subsequent reculturing. Isozyme banding patterns of multimeric enzymes never gave band patterns indicative of heterokaryosis or heterozygosis. Banding patterns indicative of heterozygous diploids or recombinants were never detected. Despite the extreme variation in morphotype and nutritional requirements among progeny, isozyme banding patterns of derived progeny from any fusion were invariably identical to one or the other parental strains. From these results, we conclude that protoplast fusion in the genusTrichoderma gives rise to great variability, but that the classical parasexual cycle is not required for variation to occur.     

MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola

The dimorphic ascomycete pathogen Mycosphaerella graminicola switches from a yeastlike form to an infectious filamentous form that penetrates the host foliage through stomata. We examined the biological function of the mitogen-activated protein kinase-encoding gene MgHog1 in M. graminicola. Interestingly, MgHog1 mutants were unable to switch to filamentous growth on water agar that mimics the nutritionally poor conditions on the foliar surface and, hence, exclusively developed by a yeastlike budding process. Consequently, due to impaired initiation of infectious germ tubes, as revealed by detailed in planta cytological analyses, the MgHog1 mutants failed to infect wheat leaves. We, therefore, conclude that MgHog1 is a new pathogenicity factor involved in the regulation of dimorphism in M. graminicola. Furthermore, MgHog1 mutants are osmosensitive, resistant to phenylpyrrole and dicarboximide fungicides, and do not melanize.     

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion.

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crosings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.     

Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli K12

Pyruvate and ethanol were both effective electron donors for nitrite reduction by Escherichia coli K12. The pyruvate-dependent rate decreased by approximately 50% when either a cysG mutation, which results in loss of NADH-dependent nitrite reductase activity (EC 1.6.6.4), or a chl mutation, which results in loss of the formate-nitrite oxidoreductase activity, was introduced into the prototrophic parental strain CGSC4315. A double mutant deficient in both of these previously described activities retained only 2% of the rate of nitrite reduction of the parental strain after growth on glucose or 5% after growth on pyruvate. We conclude that any third pathway for nitrite reduction contributes little to the in vivo rate of nitrite reduction by wild-type strains.     

The use of mitochondrial mutants in the isolation of hybrids involving industrial yeast strains

Summary Methods for isolation of hybrids of industrial yeast strains, obtained by PEG-mediated protoplast fusion, using mitochondrial mutations to antibiotic resistance and to the petite condition as markers, are described. One of the industrial prototrophic strains, carrying a rescuable mutation to antibiotic resistance, was converted to the petite form, and protoplasts obtained from it were fused with protoplasts from antibiotic-sensitive prototrophic strains of brewing and distiller's yeasts or with an auxotrophic laboratory strain carrying several chromosomal mutations. The parent of the petite strain was also able to metabolize starch, which was used as an additional character for confirming the hybrid nature of the strains isolated on the basis of their antibiotic resistance.Presented in part at the 5th International Protoplast Symposium held at the Attila Josef University, Szeged, Hungary, July 9–14, 1979