scholarly journals Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3945 ◽  
Author(s):  
Alexey Gilman ◽  
Yanfen Fu ◽  
Melissa Hendershott ◽  
Frances Chu ◽  
Aaron W. Puri ◽  
...  
Keyword(s):  
Growth Rates ◽  
Natural Environment ◽  
Culture Condition ◽  
Metabolic Flux ◽  
Metabolic Switch ◽  
Growth Defects ◽  
Fermentative Metabolism ◽  
Starvation Condition ◽  
Bioreactor Cultures ◽  
Starvation Conditions

The bacteria that grow on methane aerobically (methanotrophs) support populations of non-methanotrophs in the natural environment by excreting methane-derived carbon. One group of excreted compounds are short-chain organic acids, generated in highest abundance when cultures are grown under O2-starvation. We examined this O2-starvation condition in the methanotroph Methylomicrobium buryatense 5GB1. The M. buryatense 5GB1 genome contains homologs for all enzymes necessary for a fermentative metabolism, and we hypothesize that a metabolic switch to fermentation can be induced by low-O2 conditions. Under prolonged O2-starvation in a closed vial, this methanotroph increases the amount of acetate excreted about 10-fold, but the formate, lactate, and succinate excreted do not respond to this culture condition. In bioreactor cultures, the amount of each excreted product is similar across a range of growth rates and limiting substrates, including O2-limitation. A set of mutants were generated in genes predicted to be involved in generating or regulating excretion of these compounds and tested for growth defects, and changes in excretion products. The phenotypes and associated metabolic flux modeling suggested that in M. buryatense 5GB1, formate and acetate are excreted in response to redox imbalance. Our results indicate that even under O2-starvation conditions, M. buryatense 5GB1 maintains a metabolic state representing a combination of fermentation and respiration metabolism.

scholarly journals Generation of an Isogenic Collection of Yeast Actin Mutants and Identification of Three Interrelated Phenotypes

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 533-543
Author(s):  
Johanna L Whitacre ◽  
Dana A Davis ◽  
Kurt A Toenjes ◽  
Sharon M Brower ◽  
Alison E M Adams
Keyword(s):  
Crystal Structure ◽  
Growth Rates ◽  
Small Region ◽  
Wild Type ◽  
Large Collection ◽  
Growth Defects ◽  
Cytoskeletal Function ◽  
Highly Correlated ◽  
The Relationship ◽  
Mutant Cells

Abstract A large collection of yeast actin mutations has been previously isolated and used in numerous studies of actin cytoskeletal function. However, the various mutations have been in congenic, rather than isogenic, backgrounds, making it difficult to compare the subtle phenotypes that are characteristic of these mutants. We have therefore placed 27 mutations in an isogenic background. We used a subset of these mutants to compare the degree to which different actin alleles are defective in sporulation, endocytosis, and growth on NaCl-containing media. We found that the three phenotypes are highly correlated. The correlations are specific and not merely a reflection of general growth defects, because the phenotypes are not correlated with growth rates under normal conditions. Significantly, those actin mutants exhibiting the most severe phenotypes in all three processes have altered residues that cluster to a small region of the actin crystal structure previously defined as the fimbrin (Sac6p)-binding site. We examined the relationship between endocytosis and growth on salt and found that shifting wild-type or actin mutant cells to high salt reduces the rate of α-factor internalization. These results suggest that actin mutants may be unable to grow on salt because of additive endocytic defects (due to mutation and salt).

Preliminary Studies on the Growth and Movements of the Yellow-bellied Toad, Bombina variegata (Anura: Discoglossidae)

1984 ◽  
Vol 5 (2) ◽  
pp. 81-86 ◽  
Author(s):  
Barbara Płytycz ◽  
Janusz Bigaj
Keyword(s):  
Body Length ◽  
Growth Rates ◽  
Natural Environment ◽  
Small Area ◽  
Older Individuals ◽  
South Eastern ◽  
Bombina Variegata ◽  
Small Growth

Yellow-bellied toads were studied in their natural environment in a mountain locality in south-eastern Poland. In June 1981, 353 specimens were captured in a small area, marked by yellow skin autografts, and released. 49 individuals were recaptured in July 1982 in the same area, and 17 animals were recaptured at other places 200-1200 m away. Movements were carried out mainly by juveniles. Large increases in body length of young individuals, probably yearlings, and small growth rates in older individuals, were generally found.

scholarly journals Inclusion of maintenance energy improves the intracellular flux predictions of CHO

2020 ◽  
Author(s):  
Diana Széliová ◽  
Jerneja Štor ◽  
Isabella Thiel ◽  
Marcus Weinguny ◽  
Michael Hanscho ◽  
...  
Keyword(s):  
Growth Rates ◽  
High Throughput Screening ◽  
Energy Demand ◽  
Metabolic Flux ◽  
Chinese Hamster ◽  
Tca Cycle ◽  
Metabolic Model ◽  
Maintenance Energy ◽  
Uptake Rates ◽  
Wide Range

AbstractChinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by pFBA using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.

scholarly journals Deciphering the Principles of Bacterial Nitrogen Dietary Preferences: a Strategy for Nutrient Containment

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Jilong Wang ◽  
Dalai Yan ◽  
Ray Dixon ◽  
Yi-Ping Wang
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Growth Rates ◽  
Regulatory Networks ◽  
Metabolic Flux ◽  
Nitrogen Sources ◽  
Fast Growth ◽  
Ammonia Assimilation ◽  
Wild Type ◽  
Dietary Preferences

ABSTRACT A fundamental question in microbial physiology concerns why organisms prefer certain nutrients to others. For example, among different nitrogen sources, ammonium is the preferred nitrogen source, supporting fast growth, whereas alternative nitrogen sources, such as certain amino acids, are considered to be poor nitrogen sources, supporting much slower exponential growth. However, the physiological/regulatory logic behind such nitrogen dietary choices remains elusive. In this study, by engineering Escherichia coli , we switched the dietary preferences toward amino acids, with growth rates equivalent to that of the wild-type strain grown on ammonia. However, when the engineered strain was cultured together with wild-type E. coli , this growth advantage was diminished as a consequence of ammonium leakage from the transport-and-catabolism (TC)-enhanced (TCE) cells, which are preferentially utilized by wild-type bacteria. Our results reveal that the nitrogen regulatory (Ntr) system fine tunes the expression of amino acid transport and catabolism components to match the flux through the ammonia assimilation pathway such that essential nutrients are retained, but, as a consequence, the fast growth rate on amino acids is sacrificed. IMPORTANCE Bacteria exhibit different growth rates under various nutrient conditions. These environmentally related behaviors reflect the coordination between metabolism and the underlying regulatory networks. In the present study, we investigated the intertwined nitrogen metabolic and nitrogen regulatory systems to understand the growth differences between rich and poor nitrogen sources. Although maximal growth rate is considered to be evolutionarily advantageous for bacteria (as remarked by François Jacob, who said that the “dream” of every cell is to become two cells), we showed that negative-feedback loops in the regulatory system inhibit growth rates on amino acids. We demonstrated that in the absence of regulatory feedback, amino acids are capable of supporting fast growth rates, but this results in ammonia leaking out from cells as “waste,” benefiting the growth of competitors. These findings provide important insights into the regulatory logic that controls metabolic flux and ensures nutrient containment but consequently sacrifices growth rate.

scholarly journals In Silico-guided Metabolic Engineering of Bacillus Subtilis for Efficient Biosynthesis of Purine Nucleosides by Blocking the Key Backflow Nodes

2021 ◽  
Author(s):  
Aihua Deng ◽  
Qidi Qiu ◽  
Qinyun Sun ◽  
Zhenxiang Chen ◽  
Junyue Wang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Engineering ◽  
In Silico ◽  
Metabolic Flux ◽  
De Novo ◽  
Feedback Inhibition ◽  
Purine Nucleotides ◽  
Metabolic Switch ◽  
Purine Synthesis ◽  
Wide Range

Abstract Background: Purine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproductions of purine nucleosides by de novo metabolic engineering have been a great challenge due to their strict and complex regulatory machinery involved in the biosynthetic pathways. Results: In this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on the genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes Drm (Purine nucleotides toward PPP) and YwjH (PPP-EMP) for resolving the competitive relationship between biomass and purine nucleotides synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides and greatly increased the inosine production to 13.98–14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of purine operon by promoter replacement further enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, deleting the second backflow node YwjH and overexpressing Zwf were combined to increase the inosine production to 22.01±1.18 g/L by enhancing the metabolic flow of PPP. Through switching on the flux node of the glucose-6- phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81±1.23 g/L of inosine by a pgi-based metabolic switch in shake-flask cultivation, suggesting the highest yield in de novo engineered inosine bacteria. Under the guidance of the in silico-designed strategy, a general chassis bacterium was generated for the first time to efficiently synthesize inosine, adenosine, guanosine, IMP, and GMP, providing the sufficient precursor for the synthesis of various purine intermediates. Conclusions: Overall, in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring the efficient targets, representing a superior strategy for efficient biosynthesis of the biotechnological products.

scholarly journals Diversion of the Metabolic Flux from Pyruvate Dehydrogenase to Pyruvate Oxidase Decreases Oxidative Stress during Glucose Metabolism in Nongrowing Escherichia coli Cells Incubated under Aerobic, Phosphate Starvation Conditions

2004 ◽  
Vol 186 (21) ◽  
pp. 7364-7368 ◽  
Author(s):  
Patrice L. Moreau
Keyword(s):  
Oxidative Stress ◽  
Pyruvate Dehydrogenase ◽  
Metabolic Flux ◽  
Thiobarbituric Acid ◽  
Pyruvate Oxidase ◽  
Prolonged Incubation ◽  
Alkyl Hydroperoxide ◽  
Escherichia Coli Cells ◽  
Low Levels ◽  
Starvation Conditions

ABSTRACT Ongoing aerobic metabolism in nongrowing cells may generate oxidative stress. It is shown here that the levels of thiobarbituric acid-reactive substances (TBARSs), which measure fragmentation products of oxidized molecules, increased strongly at the onset of starvation for phosphate (Pi). This increase in TBARS levels required the activity of the histone-like nucleoid-structuring (H-NS) protein. TBARS levels weakly increased further in ΔahpCF mutants deficient in alkyl hydroperoxide reductase (AHP) activity during prolonged metabolism of glucose to acetate. Inactivation of pyruvate oxidase (PoxB) activity decreased the production of acetate by half and significantly increased the production of TBARS. Overall, these data suggest that during incubation under aerobic, Pi starvation conditions, metabolic flux is diverted from the pyruvate dehydrogenase (PDH) complex (NAD dependent) to PoxB (NAD independent). This shift may decrease the production of NADH and in turn the adventitious production of H2O2 by NADH dehydrogenase in the respiratory chain. The residual low levels of H2O2 produced during prolonged incubation can be scavenged efficiently by AHP. However, high levels of H2O2 may be reached transiently at the onset of stationary phase, primarily because H-NS may delay the metabolic shift from PDH to PoxB.

scholarly journals Organization of the Pre-autophagosomal Structure Responsible for Autophagosome Formation

2008 ◽  
Vol 19 (5) ◽  
pp. 2039-2050 ◽  
Author(s):  
Tomoko Kawamata ◽  
Yoshiaki Kamada ◽  
Yukiko Kabeya ◽  
Takayuki Sekito ◽  
Yoshinori Ohsumi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ternary Complex ◽  
Nutrient Depletion ◽  
Autophagosome Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Atg Proteins ◽  
Starvation Condition ◽  
Putative Site ◽  
Starvation Conditions

Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11Δ cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1–Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.

scholarly journals Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1860-1872 ◽  
Author(s):  
Abhishek Murarka ◽  
James M. Clomburg ◽  
Ramon Gonzalez
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Reducing Power ◽  
Building Blocks ◽  
Exponential Phase ◽  
Flux Analysis ◽  
Wild Type ◽  
Fermentative Metabolism

The fermentative metabolism of d-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh−) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh− were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD+ ratio supported the proposed role of PDH. Moreover, the NADH : NAD+ ratio in a strain deficient in both PDH and the ox-PPP (Pdh−Zwf−) was even lower than that observed for Pdh−. Strain Pdh−Zwf− also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh−. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh− mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD+ ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.

scholarly journals Let-7 family of microRNA is required for maturation and adult-like metabolism in stem cell-derived cardiomyocytes

2015 ◽  
Vol 112 (21) ◽  
pp. E2785-E2794 ◽  
Author(s):  
Kavitha T. Kuppusamy ◽  
Daniel C. Jones ◽  
Henrik Sperber ◽  
Anup Madan ◽  
Karin A. Fischer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Fatty Acid ◽  
Large Scale ◽  
Metabolic Flux ◽  
Embryonic Stem ◽  
Acid Oxidation ◽  
Loss Of Function ◽  
Metabolic Switch ◽  
Cardiac Maturation

In metazoans, transition from fetal to adult heart is accompanied by a switch in energy metabolism-glycolysis to fatty acid oxidation. The molecular factors regulating this metabolic switch remain largely unexplored. We first demonstrate that the molecular signatures in 1-year (y) matured human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are similar to those seen in in vivo-derived mature cardiac tissues, thus making them an excellent model to study human cardiac maturation. We further show that let-7 is the most highly up-regulated microRNA (miRNA) family during in vitro human cardiac maturation. Gain- and loss-of-function analyses of let-7g in hESC-CMs demonstrate it is both required and sufficient for maturation, but not for early differentiation of CMs. Overexpression of let-7 family members in hESC-CMs enhances cell size, sarcomere length, force of contraction, and respiratory capacity. Interestingly, large-scale expression data, target analysis, and metabolic flux assays suggest this let-7–driven CM maturation could be a result of down-regulation of the phosphoinositide 3 kinase (PI3K)/AKT protein kinase/insulin pathway and an up-regulation of fatty acid metabolism. These results indicate let-7 is an important mediator in augmenting metabolic energetics in maturing CMs. Promoting maturation of hESC-CMs with let-7 overexpression will be highly significant for basic and applied research.

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