Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK).

Steady-state chemostat cultures of Azotobacter vinelandii strain CA11, carrying a deletion of genes encoding the structural polypeptides of nitrogenase nifHDK, were established in a simple defined medium chemically purified to minimize contamination by Mo. The medium contained no utilizable N source. Growth was dependent on N2 (1.1 X 10(8) viable cells X ml-1 at D = 0.176 h-1), and was inhibited by Mo (20 nM). DNA hybridization showed the deletion to be stable during prolonged (55 days) growth in the chemostat (132 doublings). Since batch cultures, using unsupplemented 'spent' chemostat medium, showed good growth (1.9 X 10(8) cells X ml-1), no requirement for subnanomolar concentrations of Mo was found. The biomass yield, as the dilution rate (D) was varied, showed that the N content of the culture, protein and dry wt. increased as D was decreased, indicating that neither N2 nor O2 were limiting growth. The limiting nutrient was not identified. Substantial amounts of H2 were evolved by the chemostat cultures, probably as the result of inhibition of O2-dependent hydrogenase activity by nitrilotriacetic acid present in the medium. Over a range of D values approx. 50% of the electron flux through the alternative system was allocated to H+ reduction. C2H2 was a poor substrate, being reduced at 0.14-0.1 times the rate of N2 fixation, calculated from the N content of the cells. SO4(2-)-limited steady-state continuous cultures of strain UW136 (wild-type for nifHDK) had a 2-fold greater biomass in the presence of MoO4(2-) (1 microM). The significance of this finding for 'Mo-limited' continuous cultures [Eady & Robson (1984) Biochem. J. 224, 853-862] is discussed.     

Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa.

In batch cultures of Pseudomonas aeruginosa, the maximum rate of exopolysaccharide synthesis occurred during exponential growth. In nitrogen-limited continuous culture, the specific rate of exopolysaccharide synthesis increased from 0.27 g g of cell-1 h-1 at a dilution rate (D) of 0.05 h-1 to 0.44 g g of cells h-1 at D=0.1 H-1. The yield of exopolysaccharide on the basis of glucose used was in the range of 56 to 64%. Exopolysaccharide was also synthesized in carbon-limited cultures at 0.19 g g of cell-1 h-1 at D=0.05 h-1 in a 33% yield. Nonmucoid variants appeared after seven generations in continuous culture and rapidly increased in proportion to the total number of organisms present.     

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.     

酵母糖化酶基因高效表达的剂量效应研究

通过原生质体融合和秋水仙素加倍技术,将糖化酵母的３个等效异位基因分别构建成交配型位点等痊基因纯合和糖化酶基因各自纯各加倍的纯合二倍体（ＳＴＡ１／ＳＴＡ１、ＳＴＡ２／ＳＴＡ２和ＳＴＡ３ＳＴＡ３）以及糖化酶基因相互组织加倍的组合二倍体（ＳＴＡ１／ＳＴＡ２、ＳＴＡ２／ＳＴＡ３和ＳＴＡ１／ＳＴＡ３）。研究了这３个糖化酶基因表达的剂量效及其相互关系。糖化酶酶活测定的结果表明,在交配型位点等位基因纯合状态下,     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

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.     

Defined conditions for synthesis of Bacillus cereus enterotoxin by fermenter-grown cultures.

A strain of Bacillus cereus produced high levels of enterotoxin when grown in a semidefined medium in a laboratory scale fermenter. The optimum conditions for enterotoxin synthesis by cultures grown in this medium, which contained Casamino Acids and yeast extract, were found to be: inoculation of vigorously gorwing culture at the 1% level, addition of glucose at a concentration of 1%, control of culture pH at 8.0, incubation at 32 degrees C, use of a moderate stirring rate, and addition of air at low flow rates to minimize foaming. The enterotoxin yield in fermenter-grown cultures was approximately 20 to 50 times higher than the yield obtained in shake flask cultures.     

Selection of strain and optimization of mutanase production in submerged cultures of Trichoderma harzianum

Nineteen fungal strains belonging to different genera were tested for extracellular mutanase production in shaken flasks. The optimal enzymatic activity was achieved by Trichoderma harzianum F-470, a strain for which the mutanase productivity has not yet been published. Some of factors affecting the enzyme production in shaken flasks and aerated fermenter cultures have been standardized. Mandels mineral medium with initial pH 5.3, containing 0.25% mutan and inoculated with 10% of the 48-h mycelium, was the best for enzyme production. A slight mutanolytic activity was also found when sucrose, raffinose, lactose and melibiose were carbon sources. Application of optimized medium and cultural conditions, as well as use of a fermenter with automatic pH control set at pH 6.0 enabled to obtain a high mutanase yield (0.33 U/ml, 2.5 U/mg protein) in a short time (2-3 days). The enzyme in crude state was stable over a pH range of 4.5-6.0, and at temperatures up to 35 degrees C; its maximum activity was at 40 degrees C and at pH 5.5.     

Defined conditions for synthesis of Bacillus cereus enterotoxin by fermenter-grown cultures.

A strain of Bacillus cereus produced high levels of enterotoxin when grown in a semidefined medium in a laboratory scale fermenter. The optimum conditions for enterotoxin synthesis by cultures grown in this medium, which contained Casamino Acids and yeast extract, were found to be: inoculation of vigorously gorwing culture at the 1% level, addition of glucose at a concentration of 1%, control of culture pH at 8.0, incubation at 32 degrees C, use of a moderate stirring rate, and addition of air at low flow rates to minimize foaming. The enterotoxin yield in fermenter-grown cultures was approximately 20 to 50 times higher than the yield obtained in shake flask cultures.     

HAPLOID PARTHENOGENETIC SPOROPHYTES OF LAMINARIA JAPONICA (PHAEOPHYCEAE)1

Parthenogenetic sporophytes were obtained from three strains of Laminaria japonica Areschoug. These sporophytes grew to maturity in the sea, producine spores that all grew into female gametophytes. These female gametophytes gave rise to another generation of parthenogenetic sporophytes during the next year, so that by the year 1990 parthenogenetic sporophytes had been cultivated for 12, 9, and 7 generations, respectively, for the three strains. When female gametophytes from parthenogenetic sporophytes were combined with normal male gametophytes, normal sporophytes that reproduced and gave rise to both female and male gametophytes were obtained. The parthenogenetic sporophytes were shorter and narrower than the normal sporophytes of the same strain. Chromosome counts on mature sporophytes showed that normal sporophytes (from fertilized eggs) were diploid (2n = approximately 40) and that the spores they produced were haploid (n = approximately 20), while nuclei from both somatic and sporangial cells in parthenogenetic sporophytes were haploid. All gametophytes were haploid. Young sporophytes derived from cultures with both female and male gametophytes were diploid, while young, sporophytes obtained from female gametophytes from parthenogenetic sporophytes had haploid, diploid, or polyploidy chromosome numbers. Polyploidy was associated with abnormal cell shapes. The presence of haploid parthenogenetic sporophytes should be use in breeding kelp strains with useful characteristics, since the sporophyte phenotype is expressed from a haploid genotype which can be more readily selected.     

Origin of plantlets and callus obtained from chile pepper anther cultures

Summary Androgenesis occurred from chile pepper (Capsicum annuum L.) anthers incubated in a continuous warm environment (29° C) with continuous light. Forty plantes and embryoids were retrieved from anther cultures and anllyzed for isozyme markers. Of these, 35 exhibited a single allele for markers suggesting microspore origin, while 5 were heterozygous indicating somatic tissue origin. Chromosome numbers were confirmed for 21 plantlets, of which 16 were haploid and 5 were diploid. However, two plants exhibited a single allele for an isozyme marker but possessed the diploid chromosome number, suggesting spontaneous doubling. Anther cultures also produced callus. Nearly 92% of the slow-growing calli sampled were heterozygous for the isozyme marker, suggesting somatic tissue origin. More than 46% of the fast-growing calli exhibited only one allele for the marker, indicating microspore origin. Callus did not regenerate plantlets. The occurrence of both heterozygous and homozygous diploid plantlets from pepper anther cultures has important implications for applied breeding programs.     

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.     

Ploidy of small individual embryo-like structures from maize anther cultures treated with chromosome doubling agents and calli derived from them

Summary In order to determine the ploidy of individual embryo-like structures (ELSs) following chromosome doubling treatments, a method was developed to determine the DNA content (ploidy level) of nuclei from single ELSs weighing as little as 1 mg using flow cytometry. About half (53%) of the ELSs which formed during anther culture of the maize inbred line used in control medium were haploid, 27% mixoploid and 20% diploid. Gibberellic acid (GA₃) increased the diploid percentage to 52% without affecting the mixoploid frequency (26%). A four day treatment with the chromosome doubling agent colchicine (50M) increased chromosome doubling while oryzalin eliminated the diploidy and mixoploidy. When regenerable callus cultures were initiated from the ELSs none were found to be mixoploid but the haploid and diploid proportions were similar to that of the ELSs analyzed. Regenerable cultures could not be initiated from the colchicine treated ELSs, however. These studies show that with the genotype used here, GA₃ and colchicine increased the amount of chromosome doubling of the ELSs while oryzalin and pronamide did not. The mixoploidy which existed in about 25% of the ELSs was never observed in calli apparently because these structures do not initiate callus or cells of only one ploidy level grew.Abbreviations ELS embryo-like structure - GA₃ gibberellin A₃     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Anther Culture of Naked Oat and the Establishment of Its Haploid Suspension Cell

This paper reported the production of haploid plants through anther culture in naked oat (Arena nuda). Calluses were induced from anthers of naked oat placed on various culture media. MS medium with 4% sucrose, 1% activated charcoal and no hormones gave the highest initiation frequencies (14.7%) of anther callus among media tested. Twelve green plants and one albino plant have been regenerated from anther calluses. Cytological examination of mitotic rooot tip ceils from three green anther plants showed that two of the plants were haploid (2n=3x=21) and one was diploid (2n=6x=42). The cell suspension cultures were established from pollen friable calluses in liquid medium. The suspension cells were cytologically stable during one year subcultures. Most of the ceils examined were haploid.     

Parasexual Genetic Analysis of the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM A3

Haploid strain A3 of the cellular slime mold Dictyostelium discoideum is valuable for biochemical studies because it is capable of axenic growth. Mutants of A3 temperature-sensitive for growth and resistant to the drugs cycloheximide, acriflavin, or methanol were isolated.--Heterozygous diploid recombinants, formed at low frequency by cell and nuclear fusion, were isolated by selecting temperature-resistant progeny of mixed cultures of two nonallelic temperature-sensitive haploids (LOOMIS 1969). Each drug-resistant mutation was found to be recessive. Two independently isolated methanol-resistant mutants were in one complementation group.--Diploids of A3 heterozygous for drug resistance formed drug-resistant segregants with a frequency of approximately 10(-4). Segregants selected for resistance to a single drug were either haploid or diploid; the fraction which was haploid varied from 0.11 to 0.86, depending on the selected marker. Segregants selected for resistance to two or three drugs were almost all haploid.--Using this parasexual cycle of diploid formation and haploidization, linkage of these temperature-sensitive and drug-resistance mutations to each other and to mutations studied by KATZ and SUSSMAN (1972) and by WILLIAMS, KESSIN and Newell (1974b) was analyzed. The methanol-resistant mutants were found to be partially resistant to acriflavin, and unlinked to the mutant selected for acriflavin resistance, which was methanol-sensitive. Of the expected seven linkage groups in D. discoideum, five, and a possible sixth, have been marked.--Linkage analysis of a mutant abnormal in morphogenesis showed that its phenotype results from two unlinked chromosomal mutations.