Growth of Escherichia coli in phosphate limited cultures : Measurements and modelling

The batch cultivation ofEscherichia coli limited by inorganic phosphate shows an exponential growth phase followed by a linear phase when the phosphate in the medium is exhausted. Data analysis and modelling of batch cultivation with different initial concentrations of the limiting factor give information about an exchangeable phosphate store, with RNA as the main component, and a non-exchangeable store.     

Comparison of the fermentation kinetics of cephalosporium acremonium in shake flasks and fermenters by means of ribonucleic acid measurements

The measurement of the RNA content was used as a convenient method for monitoring the growth ofCephalosporium acremonium in complex media containing solids. It was applied to fermentations of two mutant strains in fermenters and flasks. Pronounced differences in growth rate and cell yield were observed in the two cultivation systems.     

A comparison of growth yields obtained fromSchwanniomyces castellii and an alcohol dehydrogenase mutant

Summary The growth yields and the rates of production of -amylase and amyloglucosidase have been compared for the wild strain ofSchwanniomyces castellii and an alcohol dehydrogenase mutant. The loss of fermentative ability in the mutant strain leads to limited but significant increases in biomass yields, and in amylase production.     

Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia

Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca²⁺ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca²⁺ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.     

Yeast mutant defective in synthesis of phosphatidylinositol. Isolation and characterization of a CDPdiacylglycerol--inositol 3-phosphatidyltransferase Km mutant

1. A Km mutant of Saccharomyces cerevisiae with a lesion in CDPdiacylglycerol-inositol 3-phosphatidyltransferase was isolated. The mutant required a high concentration of myo-inositol for growth. 2. The CDPdiacylglycerol-inositol 3-phosphatidyltransferase in the mutant cells showed an apparent Km for myo-inositol over 200-times higher than that of the enzyme in wild-type cells. The maximum velocity of the mutant enzyme was comparable to that of the wild-type enzyme. 3. In mutant cells, labelled myo-inositol, phosphate and acetate were incorporated into phosphatidylinositol at much slower rates than in wild-type cells. The phosphatidylinositol content of mutant cells was markedly lower than that observed in wild-type cells. 4. Genetic analysis showed that the growth phenotype of the mutant arose from a single nuclear gene mutation in a gene coding for CDPdiacylglycerol-inositol 3-phosphatidyltransferase. 5. The mutant showed a normal level of phosphatidylserine synthase activity. The phosphatidylserine synthase gene was located between ura3 and hom3 on chromosome V, whereas the CDPdiacylglycerol-inositol 3-phosphatidyltransferase gene showed no linkage with ura3. 6. Labelled acetate was incorporated into various lipids including triacylglycerols, diacylglycerols, sterol esters and phospholipids other than phosphatidylinositol at faster rates in mutant cells than in wild-type cells. Incorporation into both the fatty acid and the sterol moieties was facilitated in the mutant. 7. A striking change in the cell-division process was observed when phosphatidylinositol synthesis was limited. The results showed that phosphatidylinositol synthesis is involved in the cell-division cycle of yeast.     

Mutation in a homolog of yeast Vps53p accounts for the heat and osmotic hypersensitive phenotypes in Arabidopsis hit1-1 mutant

Previously, the growth of Arabidopsis hit1-1 (heat-intolerant) mutant was found to be inhibited by both heat and water stress (Wu et al. in J Plant Physiol 157:543–547, 2000). In order to determine the genetic mutation underlying the hit1-1 phenotype, map-based cloning of HIT1 gene was conducted. Transformation of the hit1-1 mutant with a HIT1 cDNA clone reverts the mutant to the heat tolerance phenotype, confirming the identity of HIT1. Sequence analysis revealed the HIT1 gene encodes a protein of 829 amino acid residues and is homologous to yeast (Saccharomyces cerevisiae) Vps53p protein. The yeast Vps53p protein has been shown to be a tethering factor that associates with Vps52p and Vps54p in a complex formation involved in the retrograde trafficking of vesicles to the late Golgi. An Arabidopsis homolog of yeast Vps52p has previously been identified and mutation of either the homolog or HIT1 by T-DNA insertion resulted in a male-specific transmission defect. The growth of yeast vps53Δ null mutant also shows reduced thermotolerance, and expression of HIT1 in this mutant can partially complement the defect, supporting the possibility of a conserved biological function for Vps53p and HIT1. Collectively, the hit1-1 is the first mutant in higher plant linking a homolog of the vesicle tethering factor to both heat and osmotic stress tolerance.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Solid state fermentation of dried citrus peel byAspergillus niger

Summary Solid state fermentation of dried citrus peel was studied in a packed bed reactor withAspergillus niger QH-2. When no nitrogen source was added the growth was limited. When urea and/or ammonium sulphate were added in the proportion of 0.63 g N/100 g solid dry substrate a diauxie was observed and the final content of crude protein was higher than 10%. The results of a factorial design show that ammonium sulphate has a significant effect on the specific growth rate.     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis

The isolation and properties of a single site temperature sensitive protease mutant of Bacillus subtilis are described. Numerous criteria suggest that the mutation resides in the structural gene coding for a basic serine protease. The mutation has been mapped between aroD and lys-1 on the Bacillus subtilis chromosome. This protease exists as an intracellular and extracellular enzyme. The mutant cells are temperature sensitive for sporulation, antibiotic production, and the sporulation-specific alteration in DNA-dependent RNA polymerase β subunit. Several types of evidence indicate a direct involvement of this enzyme in a limited proteolytic cleavage of vegetative RNA polymerase β subunit, which produces the lower molecular weight β subunit found in sporulating cells. The derangement in this process is sufficient to account for the stoppage of sporulation at stage 0 when the mutant cells are grown at the non-permissive temperature.     

Inhibition of cultured cell growth by tungstate and molybdate

The growth of suspension cultured cells of Nicotiana tabacum (tobacco) was inhibited completely by 100 M tungstate. Even though molybdate reversed the tungstate inactivation of nitrate reductase activity, the growth inhibition was not reversed. The growth inhibition of N. tabacum, Daucus carota, Glycine max and Solanum tuberosum suspension cultured cells by tungstate was similar in media with or without amino acids as a source of reduced nitrogen. Only in the case of G. max was a slight reversal caused by the amino acids. Tungstate was slightly less inhibitory to the growth of a nitrate reductase-lacking mutant N. tabacum line (nia-63) than to the line with nitrate reductase. These results indicate that tungstate must inhibit the cell growth of the four species used, predominantly, in some way other than by inhibiting nitrate reductase activity. Similar studies with molybdate, a sulfate analog which apparently competes with sulfate at the ATP sulfury-lase enzyme, showed that 1 mM concentrations were completely inhibitory to cell growth. The addition of sulfate or cysteine, as a source of reduced sulfur, and amino acids, as a source of reduced nitrogen, in most cases did not reverse the molybdate inhibition appreciably. Some reversal was seen only by sulfate with D. carota cells and by cysteine plus amino acids with D. carota and G. max. These results indicate that selection for tungstate or molybdate resistance will in general not select for higher levels or other alterations in the activity of nitrate reductase or ATP sulfurylase, respectively, since these ions do not inhibit growth by primarily affecting these enzymatic steps in cultured cells of the four species studied.     

Regulation of carbon and nitrogen flow by glutamate synthase in Neurospora crassa

A glycine-resistant Neurospora crassa mutant (am-132;glyr), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for NADP-dependent glutamate dehydrogenase (GDH).] This new mutation also conferred resistance to serine and methionine sulphoximine (MS), which are inhibitors of glutamine synthetase (GS). In addition, the mutant obtained grew better on ammonium than the am-132 parental strain. Resistance to glycine was not due to increased synthesis of glutamine by an altered or induced GS, nor to increased glutamate synthesis by induction of the catabolic NAD-dependent GDH, nor to NADH-dependent glutamate synthase (GOGAT), which was as sensitive to inhibitors as the GOGAT from the parental strain. The glycine-resistance mutation lowered but did not abolish the carbon flow; this resulted in a lower content of tricarboxylic acid cycle intermediates. GOGAT activity was inhibited in vitro by several organic acids and methionine sulphone (MSF). The higher growth rate of the glycine-resistant mutant on ammonium or on ammonium plus glycine, serine or MS was explained by an increased capacity of GOGAT to synthesize glutamate in vivo due to a lower content of inhibitory tricarboxylic acid cycle intermediates; the higher glutamate content overcomes the effect of the GS inhibitors and explains the MSF resistance of the mutant.     

Regulation of mycotoxin production by Fusarium graminearum: Complementation effects between two mutant types

Summary Starting from anileu auxotroph ofFusarium graminearum producing high levels of the mycotoxin zearalenone, selection after UV irradiation gave low-producing mutants of essentially normal morphology,zea,ileu. Heterokaryons betweenzea,ileu strains and an auxotrophic strainlz,inos derived from the lazy morphological mutant ofFusarium graminearum, which has abnormal morphology and also produces little or n zearalenone, produced significant levels (over 50% of the wild-type level) of mycotoxin. The observation confirms views as to the regulatory nature of thelazy mutation.     

Characterization of a cytochrome oxidase-deficient yeast mutant which accumulates porphyrins

A cytochrome oxidase-deficient mutant (pop4) of Saccharomycescerevisiae which accumulates porphyrins has been characterized. The pop4 mutation is recessive and affects a single nuclear gene. The bulk of the accumulated porphyrins consists of uroporphyrin and its partial decar?ylation products, suggesting that the mutation causes a block at the level of uroporphyrinogen decar?ylase. However, pop4 is not allelic to hem6 or pop3, putative decar?ylase structural-gene mutants. These results suggest that there may be a uroporphyrinogen decar?ylase isoenzyme specifically involved in heme a production.     

Evaluation of UV-Induced Mutants of Chlorella vulgaris for Single Cell Protein I. The Screening Effect of Cycloheximide and 6-Methylpurine

Chlorella vulgaris was irradiated with UV doses allowing a 0.1 per cent survival. Dark recovery and photoreactivation were carried out either in the presence or in the absence of an inhibitor of protein synthesis. Faster growing and faster greening colonies were selected, and the amino acid composition of the mutant strains were evaluated. Higher growth and photosynthetic rates, higher chlorophyll content and lower respiration rate were shown by all mutants irrespective of the selection procedure. Selection against cycloheximide led to higher protein and RNA, but lower carotenoid content, whereas mutants selected against 6-methylpurine showed, in addition to a protein and carotenoid increase, a more favourable protein: RNA ratio.     

Characterization of an Arabidopsis cadmium-resistant mutant <Emphasis Type="Italic">cdr3</Emphasis>-<Emphasis Type="Italic">1D</Emphasis> reveals a link between heavy metal resistance as well as seed development and flowering

A lot of studies have identified many key genes involved in heavy metal detoxification and tolerance in plants; however, our understanding of its molecular mechanisms is far from complete. To gain insight into the regulatory mechanisms for heavy metal detoxification and tolerance, we performed a mutant screen for identifying Arabidopsis (Arabidopsis thaliana) cadmium (Cd)-resistant mutants. A Cd-resistant mutant cdr3-1D (c a d mium-r esistant) was isolated because of its increased root growth and fresh weight in Cd stress, and genetic analysis showed that cdr3-1D is a single dominant nuclear mutation. Compared with the wild type, the cdr3-1D mutant was more resistant to heavy metals Cd, Pb, and copper as well as hydrogen peroxide. Moreover, we also observed that seeds of the cdr3-1D mutant were larger than those of wild type, and that cdr3-1D displayed early flowering compared with wild type. A lower Cd/Pb content was detected in cdr3-1D plants than in wild-type plants when subjected to Cd/Pb treatment, which was associated, at least in part, with increase of expression of AtPDR8/AtPDR12, a pump excluding Cd/Pb and/or Cd/Pb-containing toxic compounds from the cytoplasm, respectively. In addition, enhanced Cd/Pb resistance of the cdr3-1D mutant was partially glutathione (GSH) dependent, which was related to increase of expression of GSH1 gene involved in GSH synthesis and consequently increased GSH content. Taken together, our results provide genetic evidence indicating that CDR3 is involved in the regulation of heavy metal resistance as well as seed development and flowering.     

Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe

A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4. To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpb1 gene, which encodes RpII LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures. First, we examined the effects of the mutation on haploid yeast cells. The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed. Tetrad analysis suggested that these phenotypes were associated with the mutation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These observations demonstrate that the S. pomberpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4.     

Effect of phosphorus deficiency on levels of phosphorus compounds in spirodela

When Spirodela plants are transferred to a phosphate-deficient medium, growth slows down immediately, and ceases after 14 days. During this time, inorganic phosphate content falls from 30 to 0.7 μmoles/g fresh weight of tissue, phosphate ester content from 3.5 to 0.6 μmoles/g, phospholipid content from 3.5 to 1.2 μmoles/g, and residual phosphate (mainly RNA) content from 7.5 to 2.0 μmoles/g. Relative proportions of the various phosphate esters, and relative proportions of the various phospholipids, are not markedly affected by phosphate deficiency. Turnover rates of phosphate esters are somewhat higher in phosphate-deficient tissue. In control tissue, inorganic phosphate is present in 2 pools; a metabolic (12%) and a non-metabolic pool (88%). In phosphate-deficient tissues, most of the inorganic phosphate (>90%) is in the metabolic pool. Non-metabolic phosphate is presumably stored in the vacuole, and is not readily accessible to the tissue, so that growth normally occurs at the expense of external phosphate. During deficiency, growth is limited by the rate at which phosphate can be transported through the tonoplast and tissue to the growing point. Growth ceases when the supply of non-metabolic phosphate is exhausted. Metabolic phosphate is presumably located in the cytoplasm: it can not be used for growth. Nor can the plant respond to deficiency by making some phosphorus compounds at the expense of others. In this respect, phosphorus deficiency and nitrogen deficiency are dissimilar.