Engineering of Carbon Distribution between Glycolysis and Sugar Nucleotide Biosynthesis in Lactococcus lactis

ABSTRACT We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an ∼100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.

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The control of synthesis of bacterial cell walls. Interaction in the synthesis of nucleotide precursors

Biochemical Journal ◽

10.1042/bj1360871 ◽

1973 ◽

Vol 136 (4) ◽

pp. 871-876 ◽

Cited By ~ 13

Author(s):

Raymond G. Anderson ◽

L. Julia Douglas ◽

Helen Hussey ◽

James Baddiley

Keyword(s):

Cell Wall ◽

Bacillus Licheniformis ◽

Bacterial Cell ◽

Cell Walls ◽

Energy Charge ◽

The Other ◽

Cell Wall Synthesis ◽

Sugar Nucleotides ◽

Sugar Phosphates

Phosphoenolpyruvate–UDP-N-acetylglucosamine enolpyruvyltransferase, UDP-N-acetylglucosamine pyrophosphorylase and CDP-glycerol pyrophosphorylase activities were demonstrated in soluble extracts from Bacillus licheniformis A.T.C.C. 9945. The effect of various nucleotides, sugar nucleotides and sugar phosphates on the nucleotide pyrophosphorylases was investigated. UDP-N-acetylglucosamine pyrophosphorylase was inhibited by UDP-MurAc-pentapeptide (UDP-N-acetylmuramyl-l-alanyl-d-glutamyl- meso-diaminopimelyl-d-alanyl -d-alanine) and CDP-glycerol. CDP-glycerol pyrophosphorylase was inhibited by UDP-MurAc-pentapeptide and stimulated by UDP-N-acetylglucosamine. Interaction between a precursor of one cell-wall polymer and an enzyme involved in the synthesis of a precursor of a second polymer has therefore been demonstrated. The possible role of such interaction in the control of bacterial cell-wall synthesis is discussed. Of the other compounds investigated mono- and di-nucleotides were shown to be inhibitory, indicating that nucleotide pyrophosphorylase activities may be influenced by the energy charge of the cell.

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Adaptations in the Muscle Cell to Training: Role of the Na+-K+-Atpase

Canadian Journal of Applied Physiology ◽

10.1139/h00-016 ◽

2000 ◽

Vol 25 (3) ◽

pp. 204-216 ◽

Cited By ~ 15

Author(s):

Howard J. Green

Keyword(s):

Metabolic Flux ◽

Contractile Activity ◽

Integral Membrane Protein ◽

Adaptive Responses ◽

Muscle Strain ◽

Muscle Contractility ◽

Exercise Adaptation ◽

Altered Regulation ◽

Key Words Muscle

The plasticity of skeletal muscle is evident following the onset of regular contractile activity where extensive adaptations can be observed at all levels of organization. Among the properties subject to altered regulation is the Na+-K+-ATPase, an integral membrane protein distributed throughout the sarcolemma and t-tubule, which functions to maintain high Na+ and K+ transmembrane gradients. This protein is uniquely positioned to control muscle excitation and contraction processes, metabolic flux rates, and contractility. Pronounced and rapid upregulation in the Na+-K+-ATPase content can be observed within the first days of exercise and well before the other major ATPase proteins involved in Ca2+ and actomyosin cycling. Moreover, the Na+-K+-ATPase is subject to complex messenger regulation, involved both in the accommodation and the adaptive responses to contractile activity. This emphasizes that adaptive responses can be mediated soon after the onset of training and may have profound affects on muscle contractility and other cellular adaptations. Key Words: muscle, strain, exercise, adaptation, accommodation

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Induction of mitochondrial heat shock proteins and mitochondrial biogenesis in endothelial cells upon acute methylglyoxal stress: Evidence for hormetic autofeedback

10.1101/2021.11.30.470545 ◽

2021 ◽

Author(s):

Ruben Bulkescher ◽

Thomas Fleming ◽

Claus Rodemer ◽

Rebekka Medert ◽

Marc Freichel ◽

...

Keyword(s):

Endothelial Cells ◽

Heat Shock ◽

Heat Shock Proteins ◽

Mitochondrial Biogenesis ◽

Metabolic Flux ◽

Metabolic Stress ◽

Oxidative Capacity ◽

Mitochondrial Proteins ◽

Shock Proteins

Increased metabolic flux produces potentially harmful side-products, such as reactive dicarbonyl and oxygen species. The reactive dicarbonly methylglyoxal (MG) can impair oxidative capacity, which is downregulated in type 2 diabetes. Heat shock proteins (HSPs) of subfamily A (Hsp70s) promote ATP-dependent processing of damaged proteins during MG exposure which also involve mitochondrial proteins. Since the protection of mitochondrial proteins could promote higher production of reactive metabolites due to increased substrate flux, tight regulation of HspA-mediated protein handling is important. We hypothesized that stress-inducible HspAs (HspA1A/HspA1B) are pivotal for maintaining mitochondrial biogenesis during acute MG-stress. To analyze the role of stress-inducible HspA1A/HspA1B for maintenance of mitochondrial homeostasis during acute MG exposure, we knocked out HSPA1A/HSPA1B in mouse endothelial cells. HSPA1A/HSPA1B KO cells showed upregulation of the mitochondrial chaperones HspA9 (mitochondrial Hsp70/mortalin) and HspD1 (Hsp60) as well as induction of mitochondrial biogenesis upon MG exposure. Increased mitochondrial biogenesis was reflected by elevated mitochondrial branching, total count and area as well as by upregulation of mitochondrial proteins and corresponding transcription factors. Our findings suggest that mitochondrial HspA9 and HspD1 promote mitochondrial biogenesis during acute MG stress, which is counterregulated by HspA1A/HspA1B to prevent mitochondrial overstimulation and to maintain balanced oxidative capacity under metabolic stress conditions. These data support an important role of HSPs in MG-induced hormesis.

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The role of the flagellar transition region: inferences from the analysis of a Chlamydomonas mutant with defective transition region structures

Journal of Cell Science ◽

10.1242/jcs.99.4.731 ◽

1991 ◽

Vol 99 (4) ◽

pp. 731-740

Author(s):

JONATHAN W. JARVIK ◽

JOSEPH P. SUHAN

Keyword(s):

Transition Region ◽

Basal Body ◽

Wild Type ◽

Central Pair ◽

Normal Transition ◽

Thin Section Electron Microscopy ◽

Concomitant Reduction ◽

Section Electron ◽

Type Variable

Thin-section electron microscopy of the Chlamydomonas reinhardtii mutant vfl-2 revealed striking defects in the transition region between basal body and flagellum. In place of the highly organized transition cylinders and stellate fibers characteristic of wild type, variable quantities of poorly organized electron-dense material were present. In many cases the transition region was penetrated by central pair microtubules that passed from the axoneme into the basal body. On the basis of these observations we propose that an important function of the structures present in the normal transition region is to physically exclude the central pair microtubules from the basal body. The transition region is the site of flagellar autotomy – the process by which doublet microtubules are severed and flagella are released from the cell. It has been claimed that autotomy is caused by contraction of the centrin-containing stellate fibers, resulting in the mechanical severing of the doublet microtubules and a concomitant reduction of the diameter of the axoneme adjacent to the abscission point. Our observations do not support this claim in that vfl-2 cells, which lack organized stellate fibers, display effective autotomy unaccompanied by detectable narrowing of the axoneme.

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REGULATION OF PROTEIN SYNTHESIS: ROLE OF NAD+ AND SUGAR PHOSPHATES IN LYSED RABBIT RETICULOCYTES Supported by USPHS grant #AI12066.

From Gene to Protein: Information Transfer in Normal and Abnormal Cells ◽

10.1016/b978-0-12-604450-8.50098-5 ◽

1979 ◽

pp. 631

Author(s):

R.J. Suhadolnik ◽

J.M. Wu ◽

C.P. Cheung ◽

A. Bruzel ◽

J. Mosca

Keyword(s):

Protein Synthesis ◽

Sugar Phosphates ◽

Rabbit Reticulocytes

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Reproductive Potential of Yeast Cells Depends on Overall Action of Interconnected Changes in Central Carbon Metabolism, Cellular Biosynthetic Capacity, and Proteostasis

International Journal of Molecular Sciences ◽

10.3390/ijms21197313 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7313

Author(s):

Roman Maslanka ◽

Renata Zadrag-Tecza

Keyword(s):

Calorie Restriction ◽

Carbon Metabolism ◽

Metabolic Flux ◽

Reproductive Potential ◽

Central Carbon Metabolism ◽

Yeast Cells ◽

Reproductive Capacity ◽

Hexokinase 2 ◽

Central Carbon

Carbon metabolism is a crucial aspect of cell life. Glucose, as the primary source of energy and carbon skeleton, determines the type of cell metabolism and biosynthetic capabilities, which, through the regulation of cell size, may affect the reproductive capacity of the yeast cell. Calorie restriction is considered as the most effective way to improve cellular physiological capacity, and its molecular mechanisms are complex and include several nutrient signaling pathways. It is widely assumed that the metabolic shift from fermentation to respiration is treated as a substantial driving force for the mechanism of calorie restriction and its influence on reproductive capabilities of cells. In this paper, we propose another approach to this issue based on analysis the connection between energy-producing and biomass formation pathways which are closed in the metabolic triangle, i.e., the respiration-glycolysis-pentose phosphate pathway. The analyses were based on the use of cells lacking hexokinase 2 (∆hxk2) and conditions of different glucose concentration corresponding to the calorie restriction and the calorie excess. Hexokinase 2 is the key enzyme involved in central carbon metabolism and is also treated as a calorie restriction mimetic. The experimental model used allows us to explain both the role of increased respiration as an effect of calorie restriction but also other aspects of carbon metabolism and the related metabolic flux in regulation of reproductive potential of the cells. The obtained results reveal that increased respiration is not a prerequisite for reproductive potential extension but rather an accompanying effect of the positive role of calorie restriction. More important seems to be the changes connected with fluxes in central carbon metabolic pathways resulting in low biosynthetic capabilities and improved proteostasis.

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YtsJ Has the Major Physiological Role of the Four Paralogous Malic Enzyme Isoforms in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00167-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4727-4736 ◽

Cited By ~ 38

Author(s):

Guillaume Lerondel ◽

Thierry Doan ◽

Nicola Zamboni ◽

Uwe Sauer ◽

Stéphane Aymerich

Keyword(s):

Enzyme Activity ◽

Bacillus Subtilis ◽

Malic Enzyme ◽

Metabolic Flux ◽

Physiological Role ◽

Extreme Case ◽

Growth Defect ◽

Flux Analysis ◽

Malic Enzyme Activity

ABSTRACT The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD+ than with NADP+. YtsJ activity, in contrast, was 70-fold higher with NADP+ than with NAD+, with Km values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.

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