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.     

耐高温酿酒酵母单倍体的RAPD分析

采用随机孢子分析的方法 ,从酿酒酵母的耐热菌株HU TY 1中分离了单倍体HZ系列 ,对其进行生长曲线和发酵力的测定。选取HZ-21、HZ-84菌株进行随机扩增多态性DNA片段的分析 ,显示单倍体菌株与它们的二倍体亲株以及原始出发菌株LK的基因组之间确实存在着多态性DNA片段 ,其中有些可能与菌株的耐热性相关     

Effect of the medium composition and cultivation conditions on sporulation in chemolithotrophic bacteria

The possibility of controlling endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as "S. thermosulfidooxidans subsp. thermotolerans". Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40-50 degrees C and pH 1.8-2.2 for strain 1269 and 35-40 degrees C and pH 2.5-2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth of optimum ranges.     

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.     

Sporulation of Penicillium camemberti in submerged batch culture.

Sporulation of Penicillium camemberti was studied in submerged batch fermentation. A defined medium was used with glucose and ammonium as C- and N-sources. Temperature was set to 25 degrees C at pH 5.6. Essential for submerged sporulation was the presence of calcium (14 mM) which was adsorbed to the cell walls in all sporulating strains and inhibited mycelial growth. Acetate led to highly branched conidiophores and was the second main factor for efficient sporulation. The chelating properties of citrate were necessary for keeping calcium and phosphate in solution. Fermentation conditions allowed high spore yields after 96 h (1.6 x 10(8) spores/ml). Cyclopiazonic acid, the mycotoxin common for P. camemberti was produced during fermentation. The levels observed (0.5-4 ppm at 96 h) were strain specific and not related to spore yield.     

Efficient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated α-amylase and glucoamylase genes

The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae, which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3′-half of the α-agglutinin gene as the anchor protein, and the α-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67, 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process.     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

莱氏野村菌紫外线诱变抗敌敌畏菌株的生理特性

[背景]夜蛾科害虫易对化学杀虫剂产生高抗性,但一些化学农药可以对部分虫生真菌的毒力作用效果起增幅作用,目前缺乏对莱氏野村菌(Nomuraea rileyi)的该方面研究.[目的]探究对常用有机磷杀虫剂敌敌畏具有较强耐药性的紫外线诱变莱氏野村菌突变菌株的生理特性,包括菌丝生长、产孢情况和产几丁质酶活性.[方法]在紫外线诱...     

Inheritance of the 2μm DNA Plasmid from Saccharomyces

A variety of Saccharomyces strains were examined for the presence of 2micro DNA and, if present, for the pattern of fragments produced by its digestion with site-specific (restriction) endonucleases. Two strains were found that did not contain detectable levels of 2micro DNA, and two strains contained 2micro DNA molecules having only one EcoRI restriction endonuclease recognition site rather than the usual two.-A haploid containing 2micro DNA with one EcoRI restriction site was mated with a haploid containing 2micro DNA with two EcoRI restriction sites and the resulting diploid maintained both types during vegetative growth. Sporulation of the diploid produced four spores, and the clones from these spores contained both types.-A haploid lacking 2micro DNA was mated with a haploid containing 2micro DNA and the resulting diploid contained 2micro DNA. The four clones derived from the haploid spores after sporulation of this diploid all contained 2micro DNA. A rho(-) strain without 2micro DNA was mated to a rho(+) strain with 2micro DNA, and heteroplasmons were selected that had received the nucleus from the strain without 2micro DNA and the mitochondria from the strain with 2micro DNA. Twelve of twenty-four such clones contained 2micro DNA.-I conclude that: (1) the different types of 2micro DNA identified in these strains do not restrict one another, (2) the different types are inherited extrachromosomally, (3) lack of 2micro DNA in two strains is not due to the absence of genes needed for maintenance and (4) the approximately 100 copies of 2micro DNA contained within a single cell are probably clustered within one or a few cytoplasmic organelles.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Genetic instability of sporulation-associated characters in a Bacillus subtilis mutant: relationship between sporulation, segregation and the synthesis of extracellular enzymes (kinetic studies).

In the genetically unstable, protease-overproducing 'medusa (M) strains of Bacillus subtilis, segregation of stable, wild-type-like B cells occurred mainly during sporulation. After the end of the exponential growth phase, a small fraction of M cells sporulated quickly and formed M spores, while the majority of the cells, after a 'critical period', gave rise to B segregants which sporulated after a delay. Segregation occurred without cell division. Delayed sporulation, segregation and protease overproduction are related. Similar but more complex results were obtained with the highly unstable TD strains. Sporulation and the kinetics of protease overproduction were also followed in several stable segregants. Depending on the strain, either the rate of protease production or both the rate and time course were affected. The results are interpreted in terms of sequential activation and de-activation of sporulation genes. The production of the alkaline and the neutral proteases was, in general, under common genetic control. In some strains alpha-amylase was also overproduced.     

Pulsed-field gel electrophoresis indicates genotypic heterogeneity among Campylobacter upsaliensis strains

Abstract A non-flocculent strain of Saccharomyces cerevisiae was selected after EMS mutation of a flocculent and heterozygous FLO1 locus diploid. The analysis of 25 asci from this diploid showed in all cases segregation 0F:4NF, thus confirming that it was probably affected in the desired gene. After sporulation and dissection of asci, three haploid strains were chosen, which were altered in the locus FLO1 . Crossing these three strains with two other ones having markers for ADE1 and pho11::LEU2 , we could map the mutation at ca. 4.3 cM and ca. 37.7 cM from the PHO11 and ADE1 loci respectively.     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: I. EFFECT OF MAGNESIUM AND CALCIUM IONS

Sporulation in a strain of the wild yeast, Hansenula saturnus,was investigated. The yeast was found to form spores even indistilled water. The sporulation rate (percentage of ascus-bearingindividuals) in this case was found to be markedly affectedby the cell concentration adopted in the test. The addition of inorganic nutrients to the sporulation mediumstimulates sporulation. The yeast requires either magnesiumor calcium for growth and sporulation. Higher concentrationsof these ions are required for sporulation than for growth.In both cases magnesium is effective at more dilute concentrationsthan calcium. Under the conditions of the experiments, in which the yeastforms a pellicle, the sporulation rate in the pellicle far exceedsthat in the sediment. The effects of environmental factors on the sporulation wasconsidered in relation to growth. It was found that, under theconditions of poor growth in the sporulation culture, no exogenousmagnesium and calcium are required for sporulation. In suchcases, the yeast cells are inferred to have an endogenous stockof magnesium and calcium enough for the sporulation. ¹ Present address: Laboratory of Microbiology, Department ofAgriculture, Tôhoku University, Sendai. (Received May 4, 1961; )     

Synthesis of Ribosomal Ribonucleic Acid During Sporulation of Bacillus subtilis

The incorporation of radioactive uracil into 50s and 30s ribosomal subunits and ribosomal ribonucleic acid (rRNA) was studied during the growth cycle of different sporogenic and asporogenic strains of Bacillus subtilis. It was found that partially synchronized cultures of the strains examined incorporated labeled uracil into the two ribosomal subunit species and rRNA during sporulation and during the stationary phase of the asporogenic strains. Kinetic studies have shown that, compared to vegetative cells, the percentage of uracil incorporated into the ribosomal subunits of cells taken 30 min after the end of exponential growth was decreased by about 25 to 35%. This decrease, however, appeared to be a general characteristic of stationary-phase cells and seems to depend on the nature of the sporulation medium and to some extent on the nature of the strain but not on the sp(+) or sp(-) phenotype of the strain. Moreover, by use of actinomycin D it was shown that the labeled uracil incorporated, in the presence of the drug, during the sporulation period was located in the ribosomal subunits (stable RNA). Based on these results, we concluded that during sporulation ribosomal genes are transcribed and consequently rRNA continues to be synthesized, although to a lesser extent than during vegetative growth. These results are discussed in the light of those obtained by Hussey et al.     

Improved Medium for Sporulation of Clostridium perfringens

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.     

Outgrowth and sporulation studies on Clostridium botulinum type E: influence of isoleucine.

A defined medium (CDM) is described which supported growth and sporulation of type E strains of Clostridium botulinum, but not sporulation of other serotypes of C. botulinum or C. sporogenes. As compared to growth in complex medium, spore outgrowth was delayed and both the growth rate and the cell yield was reduced. However, efficiency of sporulation of the type E MSpt strain in a chemically defined medium (CDM) was the same as that in complex medium and, in fact, sporulation was nearly synchronous and completed within 3 h of the first appearance of phase-bright endospores, compared with completion in 9 h in TPGY. Growth studies with CDM, from which single amino acids were omitted, showed that isoleucine was essential for outgrowth of heat-activated spores of the MSp+ strain, whereas valine was required for that of the Ts-25 mutant. Radioactive isoleucine was incorporated by germinating MSp+ spores at an earlier stage and at a more rapid rate than labelled methionine or mixed amino acids. Uptake studies showed that isoleucine accumulated in a prominent acid-soluble pool during outgrowth, a period when its incorporation into protein was not evident. The results suggest that the isoleucine may be required for a purpose other than protein synthesis during outgrowth.     

Biological indicators for low temperature steam and formaldehyde sterilization: the effect of defined media on sporulation, growth index and formaldehyde resistance of spores of Bacillus stearothermophilus strains

Preliminary screening was carried out on spores of 29 strains of Bacillus stearothermophilus to determine their potential as biological indicator organisms for low temperature steam and formaldehyde sterilization. Each strain was sporulated on four chemically defined media. Fourteen strains produced satisfactory sporulation on one or more of the media but there was considerable variation in the extent of sporulation. The growth index of the spores, which was dependent on both the strain of organism and the sporulation medium, ranged from 1% to 90%. The spores were appraised on the basis of their resistance to inactivation by 0.5% w/v formaldehyde in aqueous solution at 70°C. The survivor curves obtained could be characterized into five types on the basis of the shape of the curve. Only five strains of Bacillus stearothermophilus produced spores with the characteristics of high resistance, linear semi-logarithmic survivor curve and high growth index that would be required of a potential biological indicator organism.     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.