Escherichia coli thymine auxotrophs suppressible by carbon dioxide

Two independently-isolated thymine-requiring mutant strains of Escherichia coli were found to possess an unusual phenotype: in the presence of CO2, growth was independent of thymine. The two strains showed different profiles of temperature sensitivity. Glycine, serine and methionine were unable to relieve the thymine-dependence. Both strains were susceptible to trimethoprim under conditions where they were thymine-independent. The results are consistent with the occurrence of partial defects in thymidylate synthase.     

Control of the rate of photosynthetic carbon dioxide fixation

A simplified model of the reductive pentose phosphate pathway of photosynthesis is analysed in order to quantify the degree to which each of the constituent reactions controls the rate of CO₂ fixation (given by the control coefficient). The analysis focuses on the four largely irreversible reactions of the cycle together with the first irreversible reaction in the sucrose and starch synthetic pathways. The model assumes that the other reactions are at equilibrium. The photorespiratory and electron transport systems are not included in the model. The analysis demonstrates that: (1) an analytical approach can be used to investigate the distribution of flux control in autocatalytic and moiety-conserved cycles; (2) measurements of enzyme kinetic parameters and certain fluxes and substrate concentrations can be used to solve the equations defining the enzyme control coefficients; (3) the conservation of total stromal phosphate and the intricate regulatory mechanisms of the photosynthetic system result in a relationship between the control coefficients that is complex and may defy any intuitive assessment of ‘rate limitation’; (4) ribulose-1,5-bisphosphate carboxylase / oxygenase may, under certain conditions, be a major controller of the rate of CO₂ fixation and, by regulating the concentration of ribulose 1,5-bisphosphate, may be important in governing the ratio of organic to inorganic phosphate in the stroma; (5) the other enzymes may also serve an important role in determining the distribution of phosphate between organic and inorganic species because they catalyze reactions at the branch points between starch and sucrose synthesis and ribulose 1,5-bisphosphate regeneration; (6) these enzymes that catalyze ‘branch-pint’ reactions may have negative control coefficients because of their ability to reduce the total concentration of cycle intermediates; (7) an approach combining the use of the equations presented in this paper and flux and substrate concentration measurements may be adequate for determining the control coefficients of several enzymes of the reductive pentose phosphate pathway.     

The small world in biophysical systems structural properties of glycolysis and the TCA cycle in Escherichia coli

It has been shown that the central metabolic network of Escherichia coli is of the small-world type. In this paper, we present that the metabolic network of glycolysis and TCA cycle as a part of the E. coli metabolism is also a small-world network. We found that the hubs of the studied network are consistent with those found in the complete metabolic network. The evolutionary meaning of this finding is discussed.     

Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli

Microbial cells possess numerous sensing/regulator systems in order to respond rapidly to environmental changes. Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. A group of global regulators, which include the one component Fnr protein and the two-component Arc system, coordinate the adaptive responses. To quantitate the contribution of Arc and FNR-dependent regulation under microaerobic conditions, the gene expression pattern of the electron transfer chain genes and the TCA cycle genes in wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA, fnr mutant, in glucose limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state using QRT-PCR. It was found that the TCA cycle genes, icd, gltA, sucC, and sdhC are repressed by ArcA while Fnr has a minor or no effect on the expression of these genes under microaerobic conditions. The expression levels of the electron transfer chain genes, nuoA, ndh, and ubiE, were not significantly affected by either ArcA or Fnr regulation proteins, while a lower expression of cydA (up to 9-fold lower) and a higher expression of cyoA (up to 31-fold higher) were observed in cultures of the arcA mutant strain compared to those of the wild type. Since significantly higher NADH/NAD+ ratios were previously observed in cultures of the arcA mutant strain compared to the wild type it seems that the cytochrome o oxidase (the product of cyoABCDE) cannot efficiently support aerobic respiration when the cells are grown under microaerobic conditions.     

Effects of carbon dioxide concentration on anaerobic fermentations of Escherichia coli

Summary The effect of dissolved carbon dioxide concentration in the anaerobic growth of Escherichia coli was investigated. E. coli was grown anaerobically with the dissolved CO₂ concentration controlled over the range from 8x10^-6 M to 3.7x10^-2 M in the liquid phase. The maximum specific growth rate was 0.75h^-1 at 1.3x10^-3 M CO₂ and the maximum yield of cells on glucose was 0.32 at 1.75x10^-4 M CO₂. The maximum specific growth rate occurs close to the concentration of CO₂ prevalent in the mammalian gut where E. coli naturally resides.Alberta Research Council contribution, paper 1364     

Escherichia coli cad operon functions as a supplier of carbon dioxide

We examined the gene expression of the Escherichia coli cad operon, which consisted of the genes cadB and cadA (lysine decarboxylase), using cells possessing cadB–lacZ fusion gene. The cad operon was expressed when O₂ was limited, and the expression was optimal at pH6.3. The β-galactosidase activity was lowered by the addition of sodium carbonate to the medium. The expression of the cad operon was reduced in cells containing the plasmid-encoding ornithine decarboxylase, which produced carbon dioxide, indicating that the gene expression of the cad operon was regulated by carbon dioxide (or its derivatives). It is known that the Krebs cycle is a major pathway for producing carbon dioxide, and that its activity is repressed when O₂ is limited. Thus, our present results suggested that the physiological role of the cad operon is to supply carbon dioxide when its internal level is lowered under O₂-limiting conditions at a low pH.     

Temperature‐dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli

Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two‐stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr^?1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (p_R) and its expression was controlled by the temperature‐sensitive repressor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity, we performed one‐stage and two‐stage bioreactor cultivations. The two‐stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/hr (+48%) and volumetric productivity of 0.39 g/L/hr (+22%) in comparison to the one‐stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L. The temperature‐dependent activation of gene expression by the Lambda promoters (p_R/p_L) has been frequently used to improve protein or, in a few cases, metabolite production in two‐stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock‐down an essential gene (icd) in E. coli to design a two‐stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the potential to be generally used to improve cell factory performance.

     

Malate synthesis by dark carbon dioxide fixation in leaves

The rates of dark CO₂ fixation and the label distribution in malate following dark ¹⁴CO₂ fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark ¹⁴CO₂ fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO₂ at rates of 1.4, 3.4, 0.23, and 1.0 μmoles of CO₂/mg of chlorophyll· hour, respectively. Net CO₂ fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO₂ for the duration of the 23-hour experiment.

A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the β-carboxyl (C₄) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C₄ as much as 15 to 20%.

The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO₂ fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix ¹⁴CO₂ more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls.