ISOLATION AND INVESTIGATION OF INDUCED ASPOROGENIC MUTANTS

1960 ◽  
Vol 6 (2) ◽  
pp. 135-151 ◽  
Author(s):  
D. G. Lundgren ◽  
G. Beskid
Keyword(s):  
Minimal Medium ◽  
Synthetic Medium ◽  
Normal Growth ◽  
Building Blocks ◽  
Culture Technique ◽  
Temperature Sensitive ◽  
Good Growth ◽  
Active Growth ◽  
Casein Hydrolyzate ◽  
Weight Protein

A simple chemically defined (minimal) medium was developed which supported good growth and sporulation of B. cereus var. lacticola. Normal growth and sporulation habits in the medium were determined employing an active growth-culture technique. Induced asporogenic mutants were isolated employing ultraviolet light as the mutagen. Mutants differed in their sporulation habits from the normal culture when grown at 37 °C under the standardized conditions in a minimal synthetic medium. Three mutant cultures appeared to be temperature-sensitive mutants as regards sporulation, that is, in the minimal medium mutant cultures were able to sporulate when grown at 28 °C but were non-sporulating at 37 °C. Sporulation blocks were partially reversed when casein hydrolyzate and some low molecular weight protein building blocks were added to the synthetic medium. The pH changes of the medium during growth and sporulation of normal and mutant cultures were compared.

fusB is an allele of nadD, encoding nicotinate mononucleotide adenylyltransferase in Escherichia coli

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2427-2433 ◽  
Author(s):  
Martin Stancek ◽  
Leif A. Isaksson ◽  
Monica Rydén-Aulin
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Temperature Sensitivity ◽  
Minimal Medium ◽  
Synthetic Medium ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Atp Binding Site ◽  
Pleiotropic Phenotype

Isolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD+ and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+ synthesis is decreased at 30 °C but the level is still sufficient to support normal growth. At 42 °C, NAD+ synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD+ levels affects ability to grow on minimal medium at 42 °C, while a large decrease leads to a more pleiotropic phenotype.

scholarly journals Distinct Amino Acid Availability-Dependent Regulatory Mechanisms of MepS and MepM Levels in Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Yung Jae Kim ◽  
Byoung Jun Choi ◽  
Si Hyoung Park ◽  
Han Byeol Lee ◽  
Ji Eun Son ◽  
...  
Keyword(s):  
Amino Acid ◽  
Minimal Medium ◽  
Molecular Mechanisms ◽  
Aromatic Amino Acids ◽  
Normal Growth ◽  
Regulatory Mechanisms ◽  
Dependent Manner ◽  
Bacterial Physiology ◽  
Protein Levels ◽  
Amino Acid Availability

Peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and antibiotic resistance. However, the regulatory mechanisms of their expression are poorly understood. In this study, we have uncovered novel regulatory mechanisms of the protein levels of the synthetically lethal PG endopeptidases MepS and MepM, which are involved in PG synthesis. A mutant defective for both MepS and MepM was lethal in an amino acid-rich medium, whereas it exhibited almost normal growth in a minimal medium, suggesting the expendability of MepS and MepM in a minimal medium. Protein levels of MepS and MepM dramatically decreased in the minimal medium. Although MepM was revealed as a substrate of Prc, a periplasmic protease involved in the proteolysis of MepS, only the decrease in the MepS level in the minimal medium was affected by the prc depletion. Phenotypic and biochemical analyses showed that the presence of aromatic amino acids in the medium induced the accumulation of MepS, but not MepM, while the presence of glutamate increased the level of MepM, but not MepS. Together, these results demonstrate that the protein levels of the two major PG endopeptidases are regulated in an amino acid availability-dependent manner, but their molecular mechanisms and signaling are significantly distinct.

scholarly journals The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

2000 ◽  
Vol 11 (7) ◽  
pp. 2335-2347 ◽  
Author(s):  
Desmond C. Raitt ◽  
Anthony L. Johnson ◽  
Alexander M. Erkine ◽  
Kozo Makino ◽  
Brian Morgan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Response Regulator ◽  
Coiled Coil ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

scholarly journals Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Daniel Christoph Volke ◽  
Johann Rohwer ◽  
Rainer Fischer ◽  
Stefan Jennewein
Keyword(s):  
Metabolic Engineering ◽  
Metabolic Control ◽  
Quantitative Description ◽  
Normal Growth ◽  
Building Blocks ◽  
Metabolic Control Analysis ◽  
Mep Pathway ◽  
Control Analysis ◽  
Significant Control ◽  
Theoretical Yield

Abstract Background Terpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpenoids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. Results We applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and isopentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a flux control coefficient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway flux) in the isoprene-producing strain and therefore exerted significant control over the flux though the MEP pathway. At higher dxs expression levels, the intracellular concentration of 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the flux towards isoprene. The higher intracellular concentrations of MEcPP led to the efflux of this intermediate into the growth medium. Discussion These findings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineering of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids.

XVII.—Utilization of Dietary Purines and Pyrimidines by Drosophila melanogaster

1957 ◽  
Vol 66 (4) ◽  
pp. 339-359 ◽  
Author(s):  
James H. Sang
Keyword(s):  
Drosophila Melanogaster ◽  
Orotic Acid ◽  
De Novo ◽  
Normal Growth ◽  
Energy Utilization ◽  
Good Growth ◽  
Response Curves ◽  
Adenylic Acid ◽  
Multicellular Organisms

SynopsisDrosophila melanogaster larvæ when cultured aseptically on a synthetic diet require exogenous ribose nucleic acid (RNA) for normal growth even though they can synthesize their own endogenous RNA from simple precursors. The optimum dietary supply lies between 0.4 and 0.7 per cent RNA. Individual bases, nucleosides and nucleotides which make up RNA cannot substitute for the whole polynucleotide, but adenine, adenosine, adenylic acid, guanosine and guanylic acid are used and stimulate growth to varying degrees. The pyrimidines and their nucleosides and nucleotides are not used when fed singly.It is shown that the de novo synthesis of purines may be more difficult than that of pyrimidines, and that if a source of purines is supplied (as adenylic acid), then the nucleosides and nucleotides of both cytosine and uracil are utilized by the larvæ, whereas the free bases are not. Cytidylic and uridylic acids seem to be interchangeable, and together with an adequate supply of adenylic acid give as good growth as RNA. Orotic acid and 2—6-diaminopurine are not used by the larvæ under the conditions described, but hypoxanthine and inosine are: xanthine and xanthosine can also be shown to have an effect on growth.Dose-response curves were determined for adenylic, guanylic, cytidylic and uridylic acids under conditions which allow the determination of the optimal supplies of each. These are found to be about 0.110, 0.080, 0.025 and 0.025 per cent, respectively. The requirement of RNA is therefore primarily a requirement of adenylic acid, since more than enough of the other nucleotides should be available when the supply of RNA is optimal. The optimal supply of adenine corresponds almost exactly with the optimal supply of adenylic acid, though a somewhat delayed larval development may be a result of energy utilization in the base-nucleoside-nucleotide conversion.These results are discussed in the light of our knowledge of purine and pyrimidine utilization in other multicellular organisms, particularly the rat, and possible applications of the findings are considered.

scholarly journals Molecular Basis for Rab Prenylation

2000 ◽  
Vol 150 (1) ◽  
pp. 89-104 ◽  
Author(s):  
Christelle Alory ◽  
William E. Balch
Keyword(s):  
Mammalian Cells ◽  
Normal Growth ◽  
Kinetic Properties ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Inherited Disease ◽  
Vesicular Traffic ◽  
Null Strain

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

scholarly journals t-SNARE Phosphorylation Regulates Endocytosis in Yeast

2002 ◽  
Vol 13 (5) ◽  
pp. 1594-1607 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Michael Marash ◽  
Adina Weinberger ◽  
Jeffrey E. Gerst
Keyword(s):  
Protein Phosphatase ◽  
Normal Growth ◽  
Gene Inactivation ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutation ◽  
Phosphatase Activation ◽  
Mating Factor ◽  
In Cells

Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lacking the v- and t-SNAREs involved in endocytosis are also rescued by CAPP activation. Yeast lacking the Tlg1 or Tlg2 t-SNAREs, the Snc v-SNAREs, or both, undergo endocytosis after phosphatase activation. CAPP activation correlated with restored uptake of FM4-64 to the vacuole, the uptake and degradation of the Ste2 receptor after mating factor treatment, and the dephosphorylation and assembly of Tlg1,2 into SNARE complexes. Activation of the phosphatase by treatment with C2-ceramide,VBM/ELO gene inactivation, or by the overexpression of SIT4 was sufficient to confer rescue. Finally, we found that mutation of single PKA sites in Tlg1 (Ser31 to Ala31) or Tlg2 (Ser90 to Ala90) was sufficient to restore endocytosis, but not exocytosis, to snc cells. These results suggest that endocytosis is also modulated by t-SNARE phosphorylation in vivo.

BIOCHEMICAL CHANGES OCCURRING IN A MINIMAL MEDIUM DURING GROWTH AND SPORULATION: COMPARISON OF SPOROGENIC BACILLUS CEREUS WITH ASPOROGENIC MUTANTS

1961 ◽  
Vol 7 (4) ◽  
pp. 543-551 ◽  
Author(s):  
G. Beskid ◽  
D. G. Lundgren
Keyword(s):  
Dipicolinic Acid ◽  
Synthetic Medium ◽  
Diaminopimelic Acid ◽  
Temperature Sensitive ◽  
Organic P ◽  
Sensitive Mutant ◽  
Inorganic P ◽  
Similar Chemical ◽  
Metabolic Products ◽  
Chemical Studies

Chemical changes in a minimal synthetic medium were studied during growth and sporulation of normal and temperature-sensitive mutant cultures of B. cereus at 28 °C. Similar chemical studies were made when the cultures were grown at 37 °C; the mutants were asporogenic (nonsporulating) at this temperature.Concurrent events, essentially the relationships between spore formation, the consumption of zinc, and consumption of manganese, were noted in sporulating and nonsporulating cultures. Other components of the medium also investigated included: glucose, amino-N, inorganic-P, potassium, iron, and magnesium. Metabolic products detected in the medium included: diaminopimelic acid, dipicolinic acid, ammonia-N, amide-N, peptide-N, and organic-P.

STUDIES OF ASPOROGENIC MUTANTS OF BACILLUS CEREUS: PRELIMINARY INVESTIGATIONS WITH CALCIUM AND ZINC

1962 ◽  
Vol 8 (6) ◽  
pp. 823-833 ◽  
Author(s):  
J. J. Cooney ◽  
D. G. Lundgren
Keyword(s):  
Heat Resistance ◽  
Bacillus Cereus ◽  
Trichloroacetic Acid ◽  
Minimal Medium ◽  
Soluble Fraction ◽  
Dipicolinic Acid ◽  
Insoluble Fraction ◽  
Temperature Sensitive ◽  
Vegetative Cells ◽  
Culture Cycle

The physiology of spore formation was studied in Bacillus cereus and a temperature-sensitive asporogenic mutant. The parent organism sporulates when cultured in a minimal medium at either 28 °C or 37 °C while the mutant sporulates only at 28 °C. The blocking of sporulation at 37 °C has been referred to as "abortive" sporulation. Uptake of calcium and zinc was followed during growth and sporulation or "abortive" sporulation. Calcium and dipicolinic acid (DPA) levels in sporogenic cultures increased as the medium calcium was increased. The asporogenic mutant took up less calcium and synthesized little DPA. Heat resistance of spores increased as the calcium and DPA increased. Over 99% of Ca45or Zn65were released from labelled spores when autoclaved to release DPA. Chemical fractionations were made of cells labelled with Zn65and Ca45and harvested at different times during the culture cycle. Smaller percentages of calcium than of zinc were located in the cold trichloroacetic acid soluble fraction. The alcohol-soluble, ether-insoluble fraction of spores contained a greater percentage of calcium than was found in vegetative cells. Cells which had undergone "abortive" sporulation contained the same percentage of calcium in this fraction as homologous vegetative cells.

COMPARATIVE RESPONSE OF TWO SAPROPHYTIC AND TWO PLANT PARASITIC SOIL FUNGI TO TEMPERATURE, HYDROGEN-ION CONCENTRATION, AND NUTRITIONAL FACTORS

1961 ◽  
Vol 39 (1) ◽  
pp. 65-79 ◽  
Author(s):  
E. W. B. Ward ◽  
A. W. Henry
Keyword(s):  
Soil Fungi ◽  
Inorganic Nitrogen ◽  
Synthetic Medium ◽  
Trichoderma Viride ◽  
Nitrogen Sources ◽  
Ion Concentration ◽  
Good Growth ◽  
Hydrogen Ion Concentration ◽  
Nutritional Factors ◽  
Ph Values

The behavior of two soil saprophytes, Trichoderma viride and Trichocladium asperum, and two root-infecting fungi, Ophiobolus graminis and Fomes annosus, was compared under various conditions in laboratory culture.On an agar-solidified organic medium optimum temperatures for growth were approximately: T. viride 25–30 °C, T. asperum 20–25 °C, O. graminis 20–25 °C, F. annosus 25 °C. T. viride rapidly outgrew the other fungi in the optimum range but this relationship changed at lower temperatures, its growth rate being equalled by that of O. graminis at 10 °C. T. viride was the only fungus to grow at 35 °C. In a synthetic liquid medium adjusted to pH values from 3.0–7.0 with a citrate–phosphate buffer, growth of O. graminis and F. annosus was sharply reduced at pH values below 5.0. T. viride made good growth at pH 3.1 and reduction in growth of T. asperum occurred only below pH 4.0. Both parasites required thiamine for growth in a synthetic medium and O. graminis also required biotin; in addition they showed a preference for organic as opposed to inorganic nitrogen sources. T. viride and T. asperum grew well with KNO3 as nitrogen source and neither required vitamins. D-Glucose, D-fructose, and D-mannose were readily utilized, and D-arabinose poorly utilized, by all four fungi. Utilization of other hexoses, pentoses, disaccharides, and polysaccharides varied considerably between the fungi.The relationship of the results obtained to the observations of others on the ecology of soil fungi is discussed and the possibility that combinations of physical and nutritional factors may favor specific fungi in the soil is considered.

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