scholarly journals Regulation of fitness in yeast overexpressing glycolytic enzymes: parameters of growth and viability

1992 ◽  
Vol 59 (1) ◽  
pp. 35-48 ◽  
Author(s):  
R. F. Rosenzweig
Keyword(s):  
Growth Rate ◽  
Stationary Phase ◽  
Specific Activity ◽  
Current Model ◽  
Carbon Sources ◽  
Fold Increase ◽  
Culture Cell ◽  
Phase Density ◽  
Enzyme Specific Activity ◽  
High Copy Plasmid

SummaryCurrent models predict that large increases over wild-type in the activity of one enzyme will not alter an organism's fitness. This prediction is tested in Saccharomyces cerevisiae through the use of a high copy plasmid that bears one of the following: hexokinase B (HEXB), phosphoglucose isomerase (PGI), phosphofructokinase (PFKAandPFKB), or pyruvate kinase (PYK). Transformants containing these plasmids demonstrate a four to ten-fold increase in enzyme specific activity over either the parent strain or transformants containing the plasmid alone. Haploid and diploid transformants derived from independent backgrounds were grown on both fermentable and non-fermentable carbon sources and evaluated for several components of fitness. These include growth rate under non-limiting conditions, maximum stationary phase density, and viability in extended batch culture. Cell viability is not affected by overproduction of these enzymes. Growth rate and stationary phase density do not differ significantly among strains that overexpressHEXB, PGIor contain the vector alone.PFKA, Btransformants show reduced growth rate on glucose in one background only. For these loci the current model is confirmed. By contrast, when grown on glucose, yeast overexpressingPYKdemonstrate reduced growth rate and increased stationary phase density in both backgrounds. These effects are abolished in cells containing plasmids with a Tn5 disrupted copy of thePYKgene. Our results are consistent with reports that the PYK locus may exert control over the yeast cell cycle and suggest that it will be challenging to model relations between fitness and activity for multifunctional proteins.

scholarly journals Transcription Initiation of the Yeast IMD2 Gene Is Abolished in Response to Nutrient Limitation through a Sequence in Its Coding Region

2003 ◽  
Vol 23 (17) ◽  
pp. 6279-6290 ◽  
Author(s):  
Mafalda Escobar-Henriques ◽  
Martine A. Collart ◽  
Bertrand Daignan-Fornier
Keyword(s):  
Growth Rate ◽  
Nucleotide Sequence ◽  
Stationary Phase ◽  
Nutrient Limitation ◽  
Transcription Initiation ◽  
Carbon Sources ◽  
Regulatory Sequence ◽  
Coding Region ◽  
Active Mechanism ◽  
Low Levels

ABSTRACT The yeast IMD2 to IMD4 and GUA1 genes, involved in GMP synthesis, are highly expressed in exponentially growing cells but are shut off when cells cease to grow upon nutrient limitation. We show for the IMD2 gene that this effect is not specific to certain carbon sources or to growth rate. Strikingly, the cis elements responsible for this nutritional response are contained within a 23-nucleotide sequence in the coding region of the IMD2 gene. Despite its very unusual location, this regulatory sequence mediates the repression of transcription initiation. From our data, we conclude that GMP synthesis is downregulated upon nutrient limitation through an active mechanism. We show that this transcriptional shutoff abolishes any possibility of the induction of IMD2, even under drastic conditions of guanylic nucleotide limitation. Taken together, these results indicate that low levels of guanylic nucleotides could be required for proper entry into stationary phase.

scholarly journals Complex Regulation of Urease Formation from the Two Promoters of the ure Operon of Klebsiella pneumoniae

2007 ◽  
Vol 189 (21) ◽  
pp. 7593-7599 ◽  
Author(s):  
Qiong Liu ◽  
Robert A. Bender
Keyword(s):  
Growth Rate ◽  
Klebsiella Pneumoniae ◽  
Specific Activity ◽  
Regulatory System ◽  
Fold Increase ◽  
Sole Source ◽  
Proximal Promoter ◽  
Absolute Requirement ◽  
Activity Difference ◽  
Operon Expression

ABSTRACT Klebsiella pneumoniae can use urea as the sole source of nitrogen, thanks to a urease encoded by the ureDABCEFG operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen. Much of this nitrogen regulation of urease formation depended on the nitrogen regulatory system acting through the nitrogen assimilation control protein, NAC. In a strain deleted for the nac gene, nitrogen limitation resulted in only a 7-fold increase in the specific activity of urease, in contrast to the 70-fold increase seen in that of the wild type. The ure operon was transcribed from two promoters. The proximal promoter (P1) had an absolute requirement for NAC; little or no transcription was seen in the absence of NAC. The distal promoter (P2) was independent of NAC, but its activity increased about threefold when the growth rate of the cells was limited by the nitrogen source. Transcriptional regulation of P1 and P2 accounted for most of the changes in urease activity seen under various nitrogen conditions. However, when transcription of ureDABCEFG was less than 20% of its maximum, the amount of active urease formed per transcript of ure decreased almost linearly with decreasing transcription. This may reflect a defect in the assembly of active urease and accounted for as much as a threefold activity difference under the conditions tested here. Thus, the ure operon was transcribed from a NAC-independent promoter (P2) and the most strongly NAC-dependent promoter known (P1). Most of the regulation of urease formation was transcriptional, but when ure transcription was low, assembly of active urease also was defective.

scholarly journals Agroresidues enhanced peroxidase activity expression by Bacillus sp. MABINYA-1 under submerged fermentation

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Ayodeji O. Falade ◽  
Leonard V. Mabinya ◽  
Anthony I. Okoh ◽  
Uchechukwu U. Nwodo
Keyword(s):  
Wheat Straw ◽  
Peroxidase Activity ◽  
Submerged Fermentation ◽  
Specific Activity ◽  
Inorganic Nitrogen ◽  
Carbon Sources ◽  
Fold Increase ◽  
Kraft Lignin ◽  
Inorganic Nitrogen Source ◽  
Maize Stover

Abstract Agroresidues have continued to gain preference over conventional carbon sources for microbial enzyme production due to the low price and abundance in the environment. Therefore, this study aimed at improving peroxidase yield by Bacillus sp. MABINYA-1 (BMAB-1) using agroresidues under submerged fermentation. The culture parameters that support maximum peroxidase yield by BMAB-1 was initially determined and the results showed that peroxidase activity expression was optimum at pH 5, 30 °C and 150 rpm while veratryl alcohol and ammonium sulphate served as the best peroxidase-inducer and inorganic nitrogen source, respectively. BMAB-1 exhibited maximum peroxidase expression (17.50 ± 0.10 U/mg) at 72 h using kraft lignin liquid medium (KLLM) under the optimized culture conditions. Upon utilization of selected agroresidues (sawdust, wheat straw and maize stover) as sole carbon sources by BMAB-1 in the fermentation process, peroxidase activity was significantly enhanced when compared with glucose (14.91 ± 0.31 U/mg) and kraft lignin (17.50 ± 0.10 U/mg). Sawdust produced the highest peroxidase yield (47.14 ± 0.41 U/mg), followed by maize stover (37.09 ± 0.00 U/mg) while wheat straw yielded the lowest peroxidase specific activity (21.65 ± 0.35 U/mg). This indicates that utilization of sawdust by BMAB-1 resulted in 3.2- and 2.7-fold increase in peroxidase activity expression as compared to glucose and kraft lignin, respectively. The aptitude of BMAB-1 to utilize agroresidues would reduce the cost of peroxidase production by the bacteria since the substrates are cheaper than the conventional carbon sources and are, as well, more readily available.

scholarly journals Maintenance of Mitochondrial Morphology Is Linked to Maintenance of the Mitochondrial Genome in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1147-1156 ◽  
Author(s):  
Theodor Hanekamp ◽  
Mary K Thorsness ◽  
Indrani Rebbapragada ◽  
Elizabeth M Fisher ◽  
Corrine Seebart ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Mitochondrial Dna ◽  
Carbon Sources ◽  
Mitochondrial Morphology ◽  
Yeast Saccharomyces Cerevisiae ◽  
Slow Growth Rate ◽  
Dna Migration ◽  
Mitochondrial Dna Stability ◽  
Extragenic Suppressors

Abstract In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology. Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA. Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability. A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10.

scholarly journals LED Illumination Spectrum Manipulation for Increasing the Yield of Sweet Basil (Ocimum basilicum L.)

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 344
Author(s):  
Md Momtazur Rahman ◽  
Mikhail Vasiliev ◽  
Kamal Alameh
Keyword(s):  
Growth Rate ◽  
Fold Increase ◽  
Photosynthetic Photon Flux Density ◽  
Ocimum Basilicum ◽  
Photon Flux ◽  
Photon Flux Density ◽  
White Led ◽  
Experimental Conditions ◽  
Sweet Basil ◽  
Optimal Illumination

Manipulation of the LED illumination spectrum can enhance plant growth rate and development in grow tents. We report on the identification of the illumination spectrum required to significantly enhance the growth rate of sweet basil (Ocimum basilicum L.) plants in grow tent environments by controlling the LED wavebands illuminating the plants. Since the optimal illumination spectrum depends on the plant type, this work focuses on identifying the illumination spectrum that achieves significant basil biomass improvement compared to improvements reported in prior studies. To be able to optimize the illumination spectrum, several steps must be achieved, namely, understanding plant biology, conducting several trial-and-error experiments, iteratively refining experimental conditions, and undertaking accurate statistical analyses. In this study, basil plants are grown in three grow tents with three LED illumination treatments, namely, only white LED illumination (denoted W*), the combination of red (R) and blue (B) LED illumination (denoted BR*) (relative red (R) and blue (B) intensities are 84% and 16%, respectively) and a combination of red (R), blue (B) and far-red (F) LED illumination (denoted BRF*) (relative red (R), blue (B) and far-red (F) intensities are 79%, 11%, and 10%, respectively). The photosynthetic photon flux density (PPFD) was set at 155 µmol m−2 s−1 for all illumination treatments, and the photoperiod was 20 h per day. Experimental results show that a combination of blue (B), red (R), and far-red (F) LED illumination leads to a one-fold increase in the yield of a sweet basil plant in comparison with only white LED illumination (W*). On the other hand, the use of blue (B) and red (R) LED illumination results in a half-fold increase in plant yield. Understanding the effects of LED illumination spectrum on the growth of plant sweet basil plants through basic horticulture research enables farmers to significantly improve their production yield, thus food security and profitability.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

scholarly journals TOR and RAS pathways regulate desiccation tolerance inSaccharomyces cerevisiae

2013 ◽  
Vol 24 (2) ◽  
pp. 115-128 ◽  
Author(s):  
Aaron Z. Welch ◽  
Patrick A. Gibney ◽  
David Botstein ◽  
Douglas E. Koshland
Keyword(s):  
Growth Rate ◽  
Stress Response ◽  
Heat Shock ◽  
Desiccation Tolerance ◽  
Ribosome Biogenesis ◽  
Inverse Correlation ◽  
Fold Increase ◽  
Nutrient Deprivation ◽  
Camp Pathway ◽  
Dividing Cells

Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance.

scholarly journals Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacteriumSynechococcus elongatusUTEX 2973

2018 ◽  
Vol 115 (50) ◽  
pp. E11761-E11770 ◽  
Author(s):  
Justin Ungerer ◽  
Kristen E. Wendt ◽  
John I. Hendry ◽  
Costas D. Maranas ◽  
Himadri B. Pakrasi
Keyword(s):  
Growth Rate ◽  
Photosynthetic Rate ◽  
Rapid Growth ◽  
Mutational Analysis ◽  
Specific Activity ◽  
Synechococcus Elongatus ◽  
Fast Growth ◽  
Close Relative ◽  
Genetic Changes ◽  
Photosynthetic Productivity

Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously describedSynechococcus elongatusUTEX 2973 as the fastest growing cyanobacterium known.Synechococcus2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strainSynechococcus elongatus7942. The two strains differ at 55 genetic loci, andsome of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis ofSynechococcus2973 and identified three specific genes,atpA,ppnK, andrpaA, with SNPs that confer rapid growth. The fast-growth–associated allele of each gene was then used to replace the wild-type alleles inSynechococcus7942. Upon incorporation, each allele successively increased the growth rate ofSynechococcus7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity ofSynechococcus7942. We also determined that the fast-growth–associated allele ofatpAyielded an ATP synthase with higher specific activity, while that ofppnKencoded a NAD+kinase with significantly improved kinetics. TherpaASNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.

scholarly journals Introducing a Thermo-Alkali-Stable, Metallic Ion-Tolerant Laccase Purified From White Rot Fungus Trametes hirsuta

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Si ◽  
Hongfei Ma ◽  
Yongjia Cao ◽  
Baokai Cui ◽  
Yucheng Dai
Keyword(s):  
Specific Activity ◽  
Endocrine Disrupting Chemicals ◽  
Environmental Pollutants ◽  
Fold Increase ◽  
Industrial Applications ◽  
White Rot ◽  
White Rot Fungus ◽  
Metallic Ions ◽  
Trametes Hirsuta ◽  
Ph Range

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.

scholarly journals Evaluation of Changes Induced in the Probiotic Escherichia coli M17 Following Recurrent Exposure to Antimicrobials

2021 ◽  
pp. 158-167
Author(s):  
M. J. A. Mbarga ◽  
I. V. Podoprigora ◽  
E. G. Volina ◽  
A. V. Ermolaev ◽  
L. A. Smolyakova
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Silver Nanoparticles ◽  
Biofilm Formation ◽  
Fold Increase ◽  
Bacterial Attachment ◽  
Diffusion Method ◽  
Cross Resistance ◽  
Microdilution Method ◽  
Similar Work

Introduction: It is already well known that the exposure of certain bacteria, pathogenic or not, to antimicrobials is likely to increase their virulence and induce the development of direct or cross resistance to antimicrobials, but there is almost no information available regarding probiotics. Aim: To assess the changes induced in susceptibility to antibiotics, biofilm formation, growth rate and relative pathogenicity in the probiotic Escherichia coli M17 (EC-M17) after long exposure to antimicrobials namely ampicillin, kanamycin, cefazolin and silver nanoparticles (AgNPs). Methods: After determining the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the 4 antimicrobials above-mentioned by the microdilution method, EC-M17 was exposed to increasing subinhibitory doses ranging from MIC/8 to MIC for 8 days. The susceptibility to antibiotics of the mutants obtained was assessed by the Kirby Bauer disc diffusion method, biofilm formation by the Congo red agar method and with crystal violet bacterial attachment assay, and relative pathogenicity was assessed using a Galleria melonella waxworm model. Results: Exposure to antimicrobials induces noticeable changes in EC-M17. The highest adaptation to antimicrobials was observed on AgNPs with 8-fold increase in MIC and 16-fold increase in MBC of AgNPs. EC-M17 exposed to ampicillin, kanamycin and silver nanoparticles became resistant to ampicillin, ceftazidime, ceftazidime/clavulanate and tetracycline while exposure to cefazolin induced a significant decrease in sensitivity to tetracycline and ampicillin and resistance to ceftazidime/clavulanate and ceftazidime. The strain exposed to ampicillin was the only one to produce more biofilm than the control strain and except the EC-M17 exposed to cefazolin, all other EC-M17 strains were more pathogenic on G. melonella model than the control. Conclusion: Data in this investigation suggest that repeated exposure of the probiotic EC-M17 to antimicrobials may induce changes in antimicrobials susceptibility, biofilm formation, growth rate, and relative pathogenicity. Therefore, as far as possible, the probiotic E. coli M17 should not be used in combination with antibiotics and further investigations are required to expand similar work on more probiotics in order to avoid resistance build-up which might be transmitted by horizontal transfer.

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