Low molecular weight sulphydryl compounds and the expression of a cell division mutant of Chlamydomonas reinhardi

Cultures of the cytokinesis-deficient mutant of Chlamydomonas reinhardi, cyt₁ normally contain a mixture of uninucleate, binucleate and multinucleate cells. Inclusion of ethanol, diamide or mercaptoethanol in the culture medium increases the proportions of binucleate and multinucleate cells. A similar effect has previously been found with both cobalt and benzimidazole. Ethanol, cobalt and benzimidazole all raise internal cysteine or cystine levels very markedly in both mutant and wild-type cells. Changes in free cysteine or cystine levels and proportions of multinucleate cells in the mutant are induced over similar concentrations of ethanol and over similar time periods in a fixed concentration of ethanol. Diamide, thought to be a specific glutathione oxidising agent, and mercaptoethanol do not raise internal cysteine/ cystine levels. Agents which induce changes in levels of sulphydryl compounds or their oxidation-reduction status therefore seem to alter the expression of the mutant. Since similar changes in sulphydryl compounds only produce much lesser effects in cell division in wild type cells, it is proposed that cells of the mutant cyt₁ are in some way hypersensitive to such changes.     

Effect of cobalt on <Emphasis Type="Italic">Escherichia coli</Emphasis> metabolism and metalloporphyrin formation

Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe–S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.     

Differential requirements of two insect cell lines for growth in serum-free medium

Summary The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 × 10⁶ TCID₅₀ extracellular virus and 4.4×10⁸ polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects.     

Transport of cystine and cysteine and cell growth in cultured human diploid fibroblasts: effect of glutamate and homocysteate

Human diploid fibroblasts take up cystine in the culture medium and the cystine is immediately reduced to cysteine in the cells. It is found that cysteine thus formed is rapidly released from the cells into the medium and accumulates there. The system transporting cysteine is convincingly similar to the ASC system described by Christensen et al. (1967). Since cysteine in the medium is sensitive to autoxidation and readily changes back to cystine, the uptake of cystine seems crucial to the cells. Inhibitors of cystine uptake, such as glutamate and homocysteate, potently reduce the intracellular and extracellular levels of cysteine. These inhibitors modify the cell growth depending upon the cystine concentration is physiological. An excessive concentration of cystine is in itself inhibitory action is antagonized by glutamate or homocysteate.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

In vitro utilization of L-cystine by keratinophilic fungi inhabiting a gelatin factory

Twenty-six different species of keratinophilic fungi were examined to determine their ability to utilize free cystine. Of the fungi tested, the majority metabolized free L-cystine in a glucose-peptone culture medium. Cystine was used as source of sulfur, and carbon and nitrogen as well. Excess sulfur was excreted into the culture fluid, as thiosulfate and sulfate, following oxidation. The rate of cystine oxidation varied with the different fungal strains, but was maximal for Graphium penicilloideus (88.5%). Low quantities of thiols were found in the medium. Cystine oxidation and inorganic thiosulfate excretion were found to correlate significantly (r = 0.94).     

Spore and crystal formation inBacillus thuringiensis var.thuringiensis during growth in cystine and cysteine

The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation inBacillus thuringiensis var.thuringiensis was studied. The effect was well pronounced when the cystine/cysteine additions were made after the stationary phase. Heat stable spores and crystals were formed when the culture was provided with a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentration of cystine or cysteine (0.15%), only heat labile spores were formed without the production of the crystal. When the cystine/cysteine concentration was high (0.25%), spore and crystal formation were completely inhibited. Partial reversal of inhibition of sporulation was brought about by sodium sulphate or Zinc sulphate and lead, copper, cadmium or cobalt acetate at 0.2 mM or at 0.2% of sodium or potassium pyruvate, citrate, cisaconitate, oxalosuccinate, ∞ -keto-glutarate, succinate, fumarate, malate, or oxalacetate. Glutamate (0.2%) overcame the inhibitory effect of cystine/cysteine completely. The structural changes observed using phase contrast microscopy were dependent upon the concentration of cystine/cysteine.     

The Relationship of Cobalt Requirement to Vitamin B12-dependent Methionine Synthesis in Rhizobium meliloti

As a growth factor, Rhizobium meliloti required cobalt ion, or vitamin B₁₂ which was found to be incorporated into the cells without decomposition to cobalt ion. Trial of replacement for cobalt ion by the addition of various compounds to the cobalt-deficient medium revealed that methionine could substitute for cobalt ion and promote the growth in response to its concentration. Furthermore, B₁₂-dependent methionine synthetase was demonstrated in the cell-free extracts of this microorganism. The morphological change of R. meliloti by the additions to the medium was observed microscopically.     

Effects of various compounds on growth of yeast cells of Histoplasma capsulatum

Summary A number of compounds were screened for their effects on growth of the yeast cells ofHistoplasma capsulatum. Included were penicillin and related compounds, sulfhydryl inhibitors, various organic sulfur compounds recently synthesized for the first time, and compounds structurally related to the required metabolites, thiamine and cystine or cysteine. Cephalothin was the only one of the penicillin related compounds which inhibited growth. This occurred only when a high concentration (8.3 × 10^–4 M) was used. Of the analogues of cystine tested, allylglycine had the greatest inhibitory effect on growth of the yeast cells in the synthetic medium, but it failed to inhibit growth in a complex medium containing peptones and plasma. Among the sulfhydryl inhibitors, the maleimides were the most effective, producing complete inhibition of growth in the peptone medium at 10µg/ml or less. At subinhibitory concentrations the cultures tended to become mycelial. The action of the maleimides was reversed by cystine over a range of concentrations. At low concentrations, some of the disulfide derivatives of thiamine stimulated growth equally as well as thiamine, but at concentrations of 100 to 150µg/ml, they completely inhibited growth. On the basis of results obtained to date, three classes of the new organic sulfur compounds being tested offer promise as sources of potentially useful chemotherapeutic agents. These classes, which differ widely in structure, are as follows: the benzyl decylaminoethyl disulfides, the acyl disulfides, and the trithiopercarbamates.This investigation was supported by Public Health Service Research Grant AI-03524 from the National Institute of Allergy and Infectious Diseases.     

Analysis of β-tubulin Gene Fragments from Benzimidazole-sensitive and -tolerant Cercospora beticola

Cercospora leaf spot of sugar beet, caused by the fungus Cercospora beticola, is a major foliar pathogen on sugar beet. Fungicide sprays have been used extensively to manage Cercospora leaf spot, including the benzimidazole fungicides. Resistance to benzimidazoles has been observed in isolates of C. beticola. The precise genetics of this resistance is not known in this fungus. We tested benzimidazole‐tolerant and ‐sensitive isolates and found a single mutation in the β‐tubulin gene of benzimidazole‐tolerant isolates that corresponds to a mutation known to confer benzimidazole tolerance in other ascomycetes. This mutation is predicted to cause a change from glutamic acid to alanine in the protein product. Isolates containing this mutation further show an increased sensitivity to an N‐phenylcarbamate, as would be predicted based on the mutant phenotype found in other filamentous fungi. Only a single mutation was found in isolates from different regions of the United States, isolated in different growing seasons.     

Distribution of amino acids in the cellular fractions ofStaphylococcus aureus

WhenStaphylococcus aureus cells were labeled with a single radioactive amino acid for 20 minutes, the highest activity, except for alanine, leucine, and glycine, was found in the free pool. Significant amounts of the above amino acids and also valine and methionine were incorporated into the protein — cell wall fraction.Cells previously labeled with a single amino acid underwent a net loss of radioactivity when transferred to buffer, glucose, or complete medium. An exception was glycine. The greatest loss in activity occurred in the free pool.While some amino acids (alanine, cystine) were transferred from the free pool to the protein — cell wall fraction under all conditions tested, others (glutamic acid, proline) were transferred only under conditions of growth.Cells labeled with certain single amino acids and then transferred to a complete medium lost a significant portion of the label. The most extreme case noted was proline, but other amino acids also effluxed from the cell under these conditions.     

Bacterial breakdown of benomyl. I. Pure cultures

With different soil and water samples as inoculum and the benzimidazole fungicides benomyl (either as Benlate or as the pure compound) and thiabendazole as selective agents, a large number of, mainly fluorescent, Pseudomonas strains were isolated which nearly all were able to grow in a mineral medium with benomyl as the sole source of carbon. However, no growth occurred with any of a series of other benzimidazole compounds, viz. benzimidazole, 2-aminobenzimidazole (2-AB), thiabendazole and fuberidazole. Although benomyl—or rather its non-enzymatic breakdown product methyl benzimidazol-2-yl carbamate (MBC)—was partially degraded to 2-AB, most probably n-butylamine, which arises after splitting off of the butylcarbamoyl side chain, was the actual carbon source for the Pseudomonas isolates.When incorporated in a lactate medium, 2-AB markedly inhibited the growth of Pseudomonas spp. at a concentration of 250 g/ml, with complete inhibition being attained at 500 g/ml. For Bacillus spp. grown in liquid peptone media benzimidazole compounds were inhibitory at concentrations of 500–1000 g/ml, with a toxicity increasing in the order: benzimidazole 相似文献   

Thioredoxin or glutaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucleotide synthesis.

We have shown previously that Escherichia coli cells constructed to lack both thioredoxin and glutaredoxin are not viable unless they also acquire an additional mutation, which we called X. Here we show that X is a cysA mutation. Our data suggest that the inviability of a trxA grx double mutant is due to the accumulation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in the sulfate assimilation pathway. The presence of excess cystine at a concentration sufficient to repress the sulfate assimilation pathway obviates the need for an X mutation and prevents the lethality of a novel cys+ trxA grx double mutant designated strain A522. Mutations in genes required for PAPS synthesis (cysA or cysC) protect cells from the otherwise lethal effect of elimination of both thioredoxin and glutaredoxin even in the absence of excess cystine. Both thioredoxin and glutaredoxin have been shown to be hydrogen donors for PAPS reductase (cysH) in vitro (M. L.-S. Tsang, J. Bacteriol. 146:1059-1066, 1981), and one or the other of these compounds is presumably essential in vivo for growth on minimal medium containing sulfate as the sulfur source. The cells which lack both thioredoxin and glutaredoxin require cystine or glutathione for growth on minimal medium but maintain an active ribonucleotide reduction system. Thus, E. coli must contain a third hydrogen donor active with ribonucleotide reductase.     

The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide

When sulfur compounds are scarce or difficult to process, Escherichia coli adapts by inducing the high‐level expression of sulfur‐compound importers. If cystine then becomes available, the cystine is rapidly overimported and reduced, leading to a burgeoning pool of intracellular cysteine. Most of the excess cysteine is exported, but some is adventitiously degraded, with the consequent release of sulfide. Sulfide is a potent ligand of copper and heme moieties, raising the prospect that it interferes with enzymes. We observed that when cystine was provided and sulfide levels rose, E. coli became strictly dependent upon cytochrome bd oxidase for continued respiration. Inspection revealed that low‐micromolar levels of sulfide inhibited the proton‐pumping cytochrome bo oxidase that is regarded as the primary respiratory oxidase. In the absence of the back‐up cytochrome bd oxidase, growth failed. Exogenous sulfide elicited the same effect. The potency of sulfide was enhanced when oxygen concentrations were low. Natural oxic‐anoxic interfaces are often sulfidic, including the intestinal environment where E. coli dwells. We propose that the sulfide resistance of the cytochrome bd oxidase is a key trait that permits respiration in such habitats.     

CRISPR/Cas9 engineering of albino cystinuria Type A mice

Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co‐injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off‐target editing. No off‐target indels were observed for the top 10 predicted off‐targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.     

Soy Peptides Enhance Heterologous Membrane Protein Productivity during the Exponential Growth Phase of Saccharomyces cerevisiae

In this study, the production of eight G protein-coupled receptors by Saccharomyces cerevisiae was compared using two types of media, one of which contained soy peptides and the other free amino acids. Yeast cell growth improved in the medium with soy peptides, and the expression levels of six of the receptors increased during the exponential phase by an average of 2.3-fold as against the free amino acid-based medium. The enhancement of protein expression by soy peptides can be explained by alleviation of metabolite stress due to amino acid source depletion caused by heterologous protein expression.     

The inhibitory effect of glutamate on the growth of a murine hybridoma is caused by competitive inhibition of the x- C transport system required for cystine utilization

Glutamic acid was found to be growth inhibitory to a murinelymphocyte hybridoma in a concentration-dependent manner from 3to 12 mM glutamate. At 12 mM glutamate there was a 70% decreasein the specific growth rate of the cells. Attempts to alleviateinhibition or adapt cells to growth in glutamate-based mediawere unsuccessful. It is proposed that elevated glutamate levelsimpair adequate uptake of cystine, a critical amino acid for thesynthesis of glutathione. Glutathione is required by cells toprevent intracellular oxidative stress. The measured rate ofuptake of U-¹⁴C L-cystine into the cells was found to havethe following parameters: K_m = 0.87 mM, V_max = 0.9nmole/mg cell protein per min. The uptake was sodiumindependent and resembled the previously described x^- _ctransport system, with elevated glutamate levels causingextensive inhibition. Glutamate at a concentration of 1.4 mMcaused a 50% decrease in cystine uptake from the serum-freegrowth medium. Glutamate was taken up from the external medium(K_m = 20 mM and V_max = 12.5 nmole/mg cell protein permin) by the same transport system in a stereo specific, sodiumindependent manner. Of the amino acids examined, it was foundthat cystine and homocysteic acid were the most extensiveinhibitors of glutamate uptake and that inhibition was competitive. Metabolic profiles of the cells grown in culturescontaining enhanced glutamate levels revealed an overallincrease in net production of alanine, serine, asparagine andaspartate. A substantially increased specific consumption ofglutamate was accompanied by a decreased consumption of cystine,valine and phenylalanine.The combined kinetic and metabolic results indicate thatglutamate and cystine are taken up by the anionic transportsystem x^- _c. The increasing levels of glutamate in themedium result in a decreased transport of cystine by this systemdue to competitive inhibition by glutamate.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

The Production of Phenazine Antibiotics by the Pseudomonas aureofaciens Strain with Plasmid-Controlled Resistance to Cobalt and Nickel

Plasmid pBS501, responsible for the resistance of the wild-type Pseudomonas sp. BS501(pBS501) to cobalt and nickel ions, was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393(pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501(pBS501) (1.2 versus 0.2 g Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393(pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393(pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici1818 in a medium containing 0.5 mM cobalt or nickel.     

Proteomic responses of the coccolithophore Emiliania huxleyi to zinc limitation and trace metal substitution

Zinc concentrations in pelagic surface waters are within the range that limits growth in marine phytoplankton cultures. However, the influence of zinc on marine primary production and phytoplankton communities is not straightforward due to largely uncharacterized abilities for some phytoplankton to access zinc species that may not be universally bioavailable and substitute zinc with cobalt or cadmium. We used a quantitative proteomic approach to investigate these strategies and other responses to zinc limitation in the coccolithophore Emiliania huxleyi, a dominant species in low zinc waters. Zinc limitation resulted in the upregulation of metal transport proteins (ZIP, TroA-like) and COG0523 metallochaperones. Some proteins were uniquely sensitive to growth under replete zinc, substitution of zinc with cobalt, or enhancement of growth with cadmium, and may be useful as biomarkers of zinc stress or substitution in situ. Several proteins specifically upregulated under cobalt-supported or cadmium-enhanced growth appear to reflect stress responses, despite titration of these metals to optimal nutritive levels. Relief from zinc limitation by zinc or cadmium resulted in increased expression of a δ-carbonic anhydrase. Our inability to detect metal binding enzymes that are specifically induced under cobalt- or cadmium-supported growth suggests cambialism is important for zinc substitution in E. huxleyi.