Citric acid production by the diploid strains of Aspergillus niger obtained by protoplast fusion

Summary Diploid strains were obtained following protoplast fusion between two citric acid producers of Aspergillus niger, one for the solid culture and the other for the shaking culture. In the shaking culture, all the diploid strains exhibited lower productivities than one parental strain. However, in the solid culture, some diploid strains exhibited higher productivities than either parental strain; the best diploid strain produced 1.2 times as much citric acid as the parental strain in solid culture.     

Formation of autodiploid strains in Aspergillus niger and their application to citric acid production from starch

Autodiploid strains were induced by colchicine treatment of Aspergillus niger WU-2223L, a citric acid-producing strain. In shaking culture, a representative autodiploid strain, L-d1, yielded higher citric acid than the parental strain, WU-2223L. When glucose was used as a carbon source, L-d1 and WU-2223L produced 67.2 g/l and 62.0 g/l of citric acid, respectively, from 120 g/l of glucose in 9 d-cultivation. Furthermore, the autodiploid strain L-d1 produced 49.6 g/l of citric acid, 1.4 times as much as that produced by WU-2223L from 120 g/l of soluble starch. During the whole period of cultivation with starch, the extracellular glucoamylase activity of L-d1 was on the same level as that of WU-2223L, but the extracellular acid-protease activity of L-d1 was much higher. The addition of pepstatin, an inhibitor of acid protease, to the culture broth at 2 d greatly increased the extracellular glucoamylase activity, and citric acid production by L-d1 reached a level of 59.0 g/l. During several subcultivations on both minimal and complete agar media, the autodiploid strains were genetically stable since they formed diploid conidia in their uniform colonies without producing sectors, and maintained citric acid productivity. However, when cultivated on minimal and complete agar media containing benomyl as a haploidizing reagent, the autodiploid strains readily formed sectors of haploid segregants. The properties of the haploid strains obtained by the benomyl treatment of the autodiploid strains were similar in morphology and citric acid productivity to those of the parental strain, WU-2223L. These results indicated that the enhanced production of citric acid from soluble starch by the autodiploid strains was due to autodiploid formation but not to gene mutation caused by the colchicine treatment.     

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.     

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.     

Haploid recombinants formed as sectors in the intraspecific fusants of Aspergillus niger producing citric acid

Protoplasts of nutritionally complementary strains of Aspergillus niger producing citric acid were treated in a polyethylene glycol solution, plated onto hypertonic minimal medium and the intraspecific fusants were obtained. When transferred and cultivated on minimal medium, almost all fo the fusants continued to grow as heterokaryons. However, some fusant colonies formed sectors of the prototrophic strains (sector strains). Most of these sector strains were haploid recombinants and their properties were genetically stable when subcultivated on both minimal and supplemented media. With respect of morphological features and citric acid productivity, the recombinant strains were like either of the parental strains or an intermediate between both parental strains. The rest of the sector strains that were nonconidiation typw seemed to be haploid recombinants, although they stopped growing on the third subcultivation on both the minimal and supplemented media.     

Intergenic Complementation of Glucoamylase and Citric Acid Production in Two Species of Aspergillus

All auxotrophs of Aspergillus foetidus and all but two auxotrophs of A. niger which we isolated yield glucoamylase and citric acid, respectively, at levels below that of the prototrophic strain from which they were derived. Results of representative heterokaryon tests suggest that the nucleus was principally responsible for the inheritance of citric acid or glucoamylase production. Most somatic diploid strains of A. foetidus gave rise to higher yields of glucoamylase when compared to their haploid component strains. Both heterokaryons and somatic diploid strains of A. niger synthesized between auxotrophs which were simultaneously reduced in citric acid yields also gave rise to enhanced yields when compared with their haploid components. The yields of a heterokaryon and somatic diploid synthesized between two high producers of citric acid were not higher than those of respective haploid components. We concluded from these results that gene dosage (or ploidy) does not increase the yield of citric acid. The apparent enhancement in yields observed in diploids or heterokaryons synthesized between auxotrophs with reduced yields in both species can be interpreted as resulting from intergenic complementation.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

Chromosome Fragments in DICTYOSTELIUM DISCOIDEUM Obtained from Parasexual Crosses between Strains of Different Genetic Background

The first aneuploid strains of Dictyostelium discoideum have been unambiguously characterized, using cytological and genetic analysis. Three independently isolated, but genetically similar, fragment chromosomes have been observed in segregants from diploids formed between haploid strains derived from the NC4 and V12 isolates of D. discoideum. Once generated, the fragment chromosomes, all of which have V12-derived centromeres, can be maintained in a NC4 genetic background. Genetic evidence is consistent with the view that all three fragment chromosomes studied encompass the region from the centromere to the whiA locus of linkage group II and terminate in the interval between whiA and acrA. From cytological studies, one of the fragment chromosomes consists of approximately half of linkage group II.—We observed no deleterious effect on viability or asexual fruiting-body formation in either haploid or diploid strains carrying an additional incomplete chromosome and hence are disomic or trisomic, respectively, for part of linkage group II. The incomplete chromosome is lost at a frequency of 2 to 3% from disomic and trisomic strains, but surprisingly this loss is not increased in the presence of the haploidizing agent, benlate. A new locus (clyA), whose phenotype is altered colony morphology, is assigned to the region of linkage group II encompassed by the fragment chromosome.     

Recombinogenic activity of nalidixic acid for artificial hybrids but not for natural strains of Candida albicans: evidence for the monoploidy of natural strains

Nalidixic acid induces segregation of auxotrophs from prototrophic hybrids of Candida albicans artifically produced by fusing complementing auxotrophic protoplasts. The auxotrophies recovered are limited to those introduced through the fusion process, and patterns of segregations for linked auxotrophic markers demonstrate the segregants are products mitotic crossing-over. Nalidixic acid does not induce auxotrophies of any sort in clinical isolates of C. albicans. These findings are contrary to a current hypothesis that natural strains of C. albicans are diploid and heterozygous for a variety of auxotrophic mutations.     

Selection of mutants of Aspergillus niger showing enhanced productivity of citric acid from starch in shaking culture

Mutants with enhanced citric acid production from soluble starch were induced from Aspergillus niger WU-2223L. After UV-irradiation of a conidial suspension of strain WU-2223L, mutants were selected on modified starch-methyl red agar plates on the basis of higher amylolytic activity and acid productivity. The 8 mutants selected showed enhanced citric acid production from soluble starch in shaking culture. Among them, a representative mutant strain, 2M-43, produced 48.0gg/l of citric acid from 120 g/l of soluble starch in 9 d of cultivation in shaking culture, whereas strain WU-2223L produced 35.1 g/l. Glucoamylase activities in the culture filtrates of strains 2M-43 and WU-2223L reached maximum levels of 3.62 U/ml and 2.11 U/ml, respectively, both at 3 d of cultivation, and thereafter decreased.     

Effect of forced aeration on citric acid production by Aspergillus sp. mutants in SSF

Citric acid (CA) is one of the most important products of fermentation in the world. A great variety of agro-industrial residues can be used in solid state fermentation. Aspergillus niger parental strain (CCT 7716) and two strains obtained by mutagenesis (CCT 7717 and CCT 7718) were evaluated in Erlenmeyer flasks and glass columns using citric pulp (CP) as substrate/support, sugarcane molasses and methanol. Best results using glass columns (forced aeration) were found in the fourth day of fermentation: 278.4, 294.9 and 261.1 g CA/kg of dry CP with CCT 7716, CCT 7718 and CCT 7717, respectively. In Erlenmeyer flasks (aeration by diffusion) CA reached 410.7, 446.8 and 492.7 g CA/kg of dry CP with CCT 7716, CCT 7718 and CCT 7717, respectively. The aeration by diffusion improved CA production by the three strains. A data acquisition system specially developed for biotechnological processes analysis was used to perform the respirometric parameters measurement.     

Intraspecific protoplast fusion of citric acid-producing strains of Aspergillus niger

Intraspecific protoplast fusion was carried out between different citric acid-producing strains of Aspergillus niger. Protoplasts prepared from the mycelia of auxotrophic mutants were treated in 30% (w/v) polyethylene glycol solution containing 0.01 M CaCl₂ and 0.5 M KCl, and fusion frequencies were 2.0–6.3%. Fusants were heterokaryons and in some cases formed sectors of the prototroph. Heterodiploids were induced from a heterokaryon by d-camphor treatment. Citric acid productivity of one heterodiploid was intermediate between those of parent strains in both shaking and solid cultures.     

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.     

Recombinogenic and mutagenic effects of the antitumor antibiotic bleomycin in Aspergillus nidulans

Bleomycin, an antibiotic and antineoplastic drug that inhibits DNA synthesis and causes several types of chromosomal aberration, was found to increase mitotic recombination in Aspergillus nidulans. Heterozygous prototrophic diploid strains grown on media containing bleomycin produced significant increases of yellow and white sectors compared with controls. Further, the increased colour segregants were due to mitotic crossing-over, whereas the non-dis junctional segregants remained at the control level. Bleomycin also induced point mutations in the methionine-suppressor system of the methGl biAl strain of Aspergillus nidulans. Conidia treated in suspension with various concentrations of bleomycin increased the methionine-independent mutants 30-fold and more.     

Interspecific hybridization between Penicillium chrysogenum and Penicillium cyaneo-fulvum following protoplast fusion

Summary Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.     

Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii.     

曲霉和青霉属内杂种和亲株的蛋白质电泳图谱比较

曲霉属内黑曲霉(Aspergitlus niger)与米曲霉(A.oryzae)具有特征明显不同的可溶性蛋白质电泳图谱,其种间杂种具有双亲的部分或全部电泳带并与黑曲霉相近。来自杂种Ⅰ的多数分离子电泳带与黑曲霉相近,只有一个分离子产生米曲霉的电泳带并具有米曲霉的遗传特性。青霉属内产黄青霉(Penicillium chrysogenum)与展青霉(P.patulum)种间及种内不同菌株间的电泳图谱基本相同,种内或种间杂种具有双亲的电泳带。结果讨论了蛋白质图谱分析的意义。     

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.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

The inhibitory effects of 20 compounds including purine analogues on the growth of Micrococcus glutamicus No. 534–348, an inosinic acid-producing adenine-auxotroph, and No. 534, a prototrophic strain, were investigated.

A number of strains resistant to each of 6-merca ptoguanine, 6-thioguanine, 8-azaguanine, mitomycin C and sulfanilamide were induced from strain No. 534–348, and their inosinic acid productivities were compared with their parent strain. Among them, MGR-25, α 6-mercaptoguanine-resistant strain, accumulated more inosinic acid than its parent strain. Furthermore, the strain MGR-25 was differentiated in its morphology, frequency of spontaneous reversion to prototrophic type in adenine-deficient medium, and the effectiveness of hypoxanthine to increase the inosinic acid accumulation.     

Levels of fos, ets2, and myb proto-oncogene RNAs correlate with segregation of chromosome 11 of normal cells and with suppression of tumorigenicity in human cell hybrids.

The tumorigenicity in nude mice of human carcinoma-derived D98AH2 (D98) cells is suppressed when cell hybrids are made by fusing these cells with normal human diploid cells. Selection for hybrids that have segregated chromosomes results in the recovery of tumorigenic segregants. These segregants have all lost at least one copy of chromosome 11 of the diploid cell parent. Earlier we found that the parental D98 cells had detectable levels of mRNA specific for 13 of 21 proto-oncogenes examined. To determine if transregulation of proto-oncogenes by genes of the normal cell occurs in such hybrids, the steady-state levels of mRNA specific to 22 proto-oncogenes in the parental cells were compared with those of nontumorigenic D98 X human diploid hybrids as well as with those of their tumorigenic segregants and with the cells of the resulting tumors. The only chromosome consistently segregated in the latter was chromosome 11 of the diploid cell. fos and ets2 RNA levels and the amount of fos protein were consistently elevated in the segregants compared with amounts in the original hybrids. An unexpected finding was the inverse relationship for myb RNA that was barely detected in the parental D98 cells but was at least 10-fold elevated in hybrids that did not have segregated chromosomes compared with those that did. These patterns were evident in RNAs prepared from both subconfluent and confluent cell cultures. The findings suggest that genes of the normal cell parent can affect proto-oncogene expression. Whether the genes affecting fos, ets2, and myb RNA levels are on chromosome 11 and whether these alterations are causally related to the tumorigenic phenotype of the hybrid remain to be determined.