Selection of fitter type nuclei in heterocaryons ofAspergillus oryzae

Summary To examine the consequences of spontaneous mutation in multinucleateAspergillus oryzae, the shift of nuclear ratio in heterocaryons produced artificially between a fast grower and a slow grower on casein-containing medium was studied throughout the course of growth. It was found that the cultivation of the heterocaryon on casein-containing medium always favored the selection of the faster grower. The faster grower had a better selective advantage when cultivated in a special tube, designed to give larger amounts of oxygen to the growing cells.With 4 Figures in the Text     

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.     

Biochemical Properties of Haploid and Diploid Strains of Penicillium chrysogenum

An intensive parasexual genetics program in which industrial strains of Penicillium chrysogenum were used culminated in the isolation of a number of heterozygous diploid strains. The diploid clones were selected from heterokaryons formed from matings between mutant strains having complementary biochemical and conidial color markers. Several diploid cultures were compared with their haploid wild-type parents and other distantly related production strains on the basis of a variety of cultural and physiological criteria. The diploid strains characteristically produced conidia of larger volume and higher deoxyribonucleic acid content. Some were vigorous with respect to growth rate and onset and degree of conidiation. One diploid strain (WC-9) had a 46% greater oxygen uptake rate and oxidized glucose at a 57% greater rate than its haploid parent (M-2). It also produced 33% higher concentrations of β-galactosidase, 66% more alkaline protease, and 53% more glucose oxidase than the M-2 haploid parent. The selection of rare stable diploid mold cultures through the use of parasexual genetics offers a unique approach to the direct selection of mutants with potential for increased enzyme formation.     

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.     

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

K undu , P.N. & D as , A. 1985. A note on crossing experiments with Aspergillus niger for the production of calcium gluconate. Journal of Applied Bacteriology 59 , 1–5.
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-nitrosoguanidne (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.     

Genetic and Biochemical Studies on the Formation of Protease in Aspergillus sojae

The mutant strain X816 synthesizing protease and amylase at high rates was crossed with original strain K.S. which is characteristic of abundant sporulation and stable growth, for the purpose of breeding a new koji mold endowed with both parental merits. Diploid strains obtained from these crosses show the intermeadiate character in general both in the formation of these enzymes and in sporulation.

In this paper, the genetic characters of these diploid strains as well as the mode of changes of these enzyme activities during hybrid formation are described.     

Cathepsin B, D and H activities in muscles of chicks of fast and slow growing strains: effect of age and diet

1. Activities of cathepsins B, D and H were measured in leg and breast muscles of fast growing (broiler) and slow growing (layer) chicks at eight time intervals between 1 and 29 days of age. 2. These enzyme activities were also measured in muscles from fast and slow growing chicks given a low protein (125 g/kg crude protein) diet between the ages of 17 and 24 days. 3. Activities of none of these cathepsins differed greatly between muscle type or strain of chick. However in both strains of chick cathepsin D and H in muscles significantly decreased with increasing age (muscle size) of the chick. Cathepsin D activity also increased when muscle proteolytic rates were increased by feeding a low protein diet. This latter effect was significant only in the muscles of fast growing chicks. 4. The results suggest that lysosomal proteases are not responsible for the differences in muscle protein degradation and growth between fast and slow growing strains of chicks, or between muscle types in the chick.     

Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain

Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.     

Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms]

Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac. subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied. Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions. It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms. Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000. Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000. Apparently there is only one structural gene for subtilisin in Bac. subtilis genome. The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e. pre-subtilisin. Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications.     

GENETICS OF PHYTOPATHOGENIC FUNGI. XI. A GENETIC STUDY OF AVIRULENCE DUE TO AUXOTROPHY IN PENICILLIUM EXPANSUM BY MEANS OF THE PARASEXUAL CYCLE

Nine color and 35 auxotrophic mutants were tested for their virulence, using the ‘Jonathan’ variety of apple; only mutants requiring methionine were avirulent. One tested arginine-requiring and the methionine-requiring mutants were avirulent for the ‘Red Delicious’ variety. In vitro and in vivo, supplementation studies indicated that the avirulence was probably related to the relatively low concentration of the required amino acids at the site of inoculation. One heterocaryon and the corresponding diploid, involving 2 avirulent methionine-requiring mutants, were virulent. Six mutant loci in 3 diploid strains could be assigned to 2 linkage groups by means of the parasexual cycle.     

Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the Fl portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.     

Effect of water activity on invertase production in solid state fermentation by improved diploid strains of Aspergillus niger

Invertase production under solid state fermentation (SSF) was determined using two overproducing mutants (Aw96-3 and Aw96-4) isolated previously from the wild type strain Aspergillus niger C28B25, as well as one diploid (DAR1) and two autodiploid strains (AD96-3 and AD96-4) constructed by parasexual crossings among these mutants. Using polyurethane foam (PUF) as an inert carrier, two initial water activity (Aw) values were evaluated (0.99 and 0.96). At Aw=0.99, maximal activity was reached by diploid AD96-4 (48.91 IU/ml) representing 30- and 13-fold increases with respect to maximal values achieved by the wild type and the haploid parental mutant (Aw96-4), respectively. Similar levels were achieved by this strain at Aw=0.96. However, diploid DAR1 only produced high levels of invertase at Aw=0.96 (43.90 IU/ml), whereas strain AD96-3 reached its highest production (31.10 IU/ml) at Aw=0.99. Both productivity and yields were also analysed for every strain at each Aw value.     

Effect of genetic origin of the fish on in vitro proliferation of muscle myosatellite cells of rainbow trout

In two rainbow trout Oncorhynchus mykiss strains fed 5% of body mass for 1 week, no significant differences were observed between the two strains in the in vitro proliferation rate of myosatellite cells isolated from white axial muscle and studied for 8 days following the incorporation of 5-bromo-2'-deoxyuridine. Nonetheless, the differentiation process and formation of myotubes started first in the strain displaying the fastest growth and the myotubes attained a more developed stage in these fish: more numerous and bigger myotubes. When the two strains were submitted to starvation for 1 week, the fast growing strain showed a faster response to the culture medium and a significantly higher proliferation rate than the slow growing one.     

Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains

The coexistence of different yeasts in a single vineyard raises the question on how they communicate and why slow growers are not competed out. Genetically modified laboratory strains of Saccharomyces cerevisiae are extensively used to investigate ecological interactions, but little is known about the genes regulating cooperation and competition in ecologically relevant settings. Here, we present evidences of Hsp12p‐dependent altruistic and contact‐dependent competitive interactions between two natural yeast isolates. Hsp12p is released during cell death for public benefit by a fast‐growing strain that also produces a killer toxin to inhibit growth of a slow grower that can enjoy the benefits of released Hsp12p. We also show that the protein Pau5p is essential in the defense against the killer effect. Our results demonstrate that the combined action of Hsp12p, Pau5p and a killer toxin is sufficient to steer a yeast community.     

TRANSPLANTATION OF NUCLEI IN NEUROSPORA CRASSA

Wilson , James F. (Hartnell Coll., Salinas, Calif.) Transplantation of nuclei in Neurospora crassa. Amer. Jour. Bot. 50(8): 780–786. Illus. 1963.—Heterocaryons of N. crassa were produced artificially by cell-to-cell transfer of protoplasm of genetically marked strains. The injected hyphal sections were excised and cultured separately. The criteria of prototrophic growth and recovery of conidia of the donor genotype were used to prove that functional nuclei had been transplanted. Reciprocal injections were made with 2 pairs of macroconidial and 3 pairs of microconidial strains. Growth curves and conidial ratios of these artificial heterocaryons were compared to the corresponding natural heterocaryons. In each pair of heterocaryons produced by reciprocal injection, the growth curve of only one was equivalent to the naturally formed heterocaryon of that pair. The growth curve of the other artificial heterocaryon differed either in rate or in the lag period before growth occurred. There was no apparent relation between conidial ratios of the natural heterocaryons and the artificial heterocaryons with comparable growth curves. Injection of normal nuclei into a morphological temperature-sensitive mutant produced an apparent heterocaryon which grew with normal morphology at 34 C. The apparent stability of Neurospora nuclei may make in vitro studies possible.     

Detection of protease specifically splitting actin in revertants of Shigella flexneri L-forms

Proteolytic activity of cell extracts from revertants of Shigella flexneri L-forms as well as biochemical properties of these strains and their sensitivity to antibiotics were studied. The protease found earlier in cells of strain E. coli A2 was shown to be synthesized by one of 8 revertants under study. This protease split actin and did not split some other proteins, its activity was inhibited by inhibitors of metalloproteases. Strain 5a2c which produced the protease was similar to the strain E. coli A2 and differed from other revertants in some biochemical properties, resistance to ampicillin and sensitivity to furazolidone. Thus the protease activity can be a marker of structural and functional transformation of Sh. flexneri under the influence of furazolidone.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains. II. Effect of cellular genotype: A heterozygous strain

Human diploid cell strains develop progressively higher levels of specific catalase activity as they grow. Following subculture activity falls again. A diploid cell strain heterozygous for the gene for acatalasia I (acatalasemia) was found to develop specific catalase activity at proportionately the same rate as normal cell strains. Yet the mutant gene reduced the absolute level of specific catalase activity which the culture attained at any given point in time. In this respect the heterozygous acatalasia I strain resembles the homozygous acatalasia II strain previously reported.     

Extracellular enzyme production by haploids,heterocaryons and diploids of Aspergillus nidulans

Summary Extracellular amylase, lipase and protease produced by haploids, diploids and heterocaryons of Aspergillus nidulans were analysed. Three morphologically normal strains and 8 morphologic mutants as well as various genetic combinations of the 11 strains were examined in solid culture medium containing specific substrates. The enzyme production of each strain was determined by measuring the halo around the colony. It was observed that the colonies showing less growth also showed more alterations in enzyme production. The compact strains (BVIII and B6) and the slow-growing heterocaryons (pp+M32 and pp+M35) showed the highest enzymatic index for the three enzymes simultaneously. If colony growth is not considered, then for amylase and protease the highest values were reached by some diploid and heterocaryons and for lipase by one morphological strain. The results showed that morphological mutants and some combinations could be used for higher production of amylase, lipase and protease.     

A mutant strain of Polysphondylium with defects in many genes

PN6024 is a mutant strain of P. pallidum which appeared on selection for resistance to MDMP, an inhibitor of translation. It was found to be mutant in four other traits, being resistant to tubercidin, incapable of growth at 31.5 degrees C, abnormal in development, and slow growing at 25 degrees C. Genetic crosses using the macrocyst cycle showed that these five traits are controlled by five unlinked genes. The hypothesis is that movement of a transposon to multiple new locations caused these mutations. A difference in restriction fragment pattern between PN6024 and its parent PN600 support the hypothesis. Attempts were made to find conditions generating other strains like PN6024. Selection for growth in the presence of tubercidin yielded clones which resemble PN6024 in being developmentally abnormal as well as tubercidin resistant. Tubercidin treatment also increased the frequency of clones resistant to canavanine. It is suggested that tubercidin is mutagenic because it causes movement of the putative transposon, not because it generates point mutations. Growth under conditions of stress (at 31.5 degrees C, at 8 degrees C, in the presence of 2% ethanol) had at most an erratic effect in generating strains like PN6024. Three substrains appeared spontaneously in cultures of PN6024. These differed in developmental characteristics from each other and from the parent strain. It is suggested that they carry mutations in genes which control the choices between growth and aggregation, and between aggregation and encystment.     

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.