Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for beta-galactosidase in the presence of glucose, although repression of beta-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.     

Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12

Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K(i) for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.     

Aspartase-hyperproducing mutants of Escherichia coli B

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspartase-hyperproducing mutants of Escherichia coli B.

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

The respiratory chain in a ubiquinone-deficient mutant of Saccharomyces cerevisiae.

1. Two allelic mutants of Saccharomyces cerevisiae with a deficiency in the biosynthesis of ubiquinone have been isolated. The properties of one particular mutant strain were investigated. Submitochondrial particles of this strain contain maximally 3% of the amount of ubiquinone in wild-type particles; the amounts of other components of the respiratory chain are essentially normal. 2. The respiratory rates of mutant cells, mitochondria and submitochondrial particles are low with ubiquinone-dependent substrates, but are restored to normal levels by addition of Q-1; the restored respiration is antimycin sensitive. Intact cells and mitochondria show respiratory control both in the absence and presence of Q-1. 3. The NADH:Q-1 oxidoreductase of submitochondrial particles of the mutant followspseudo first-order kinetics in [Q-1]. QH2-1 inhibits competitively with respect to Q-1, the Ki for QH2-1 being equal to the Km for Q-1. 4. Succinate dehydrogenase in both wild-type and mutant submitochondrial particles can be activated by NADH. 5. The turnover number of succinate dehydrogenase in the mutant, measured with phenazine methosulphate as primary electron acceptor, is about one-half that of wild-type particles. The turnover numbers measured with Q-1 as electron acceptor are about the same in the two types of particles. 6. The kinetics of redox changes in cytochrome b, in the presence of antimycin and oxygen, are distinctly different in the mutant and wild-type particles. They indicate that ubiquinone plays an important role in the phenomenon of the increased reducibility of cytochrome b induced by antimycin plus oxygen.     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Functions of Two Types of NADH Oxidases in Energy Metabolism and Oxidative Stress of Streptococcus mutans

We have previously identified two distinct NADH oxidases corresponding to H(2)O(2)-forming oxidase (Nox-1) and H(2)O-forming oxidase (Nox-2) induced in Streptococcus mutans. Sequence analyses indicated a strong similarity between Nox-1 and AhpF, the flavoprotein component of Salmonella typhimurium alkyl hydroperoxide reductase; an open reading frame upstream of nox-1 also showed homology to AhpC, the direct peroxide-reducing component of S. typhimurium alkyl hydroperoxide reductase. To determine their physiological functions in S. mutans, we constructed knockout mutants of Nox-1, Nox-2, and/or the AhpC homologue; we verified that Nox-2 plays an important role in energy metabolism through the regeneration of NAD(+) but Nox-1 contributes negligibly. The Nox-2 mutant exhibited greatly reduced aerobic growth on mannitol, whereas there was no significant effect of aerobiosis on the growth on mannitol of the other strains or growth on glucose of any of the strains. Although the Nox-2 mutants grew well on glucose aerobically, the end products of glucose fermentation by the Nox-2 mutant were substantially shifted to higher ratios of lactic acid to acetic acid compared with wild-type cells. The resistance to cumene hydroperoxide of Escherichia coli TA4315 (ahpCF-defective mutant) transformed with pAN119 containing both nox-1 and ahpC genes was not only restored but enhanced relative to that of E. coli K-12 (parent strain), indicating a clear function for Nox-1 as part of an alkyl hydroperoxide reductase system in vivo in combination with AhpC. Surprisingly, the Nox-1 and/or AhpC deficiency had no effect on the sensitivity of S. mutans to cumene hydroperoxide and H(2)O(2), implying that the existence of some other antioxidant system(s) independent of Nox-1 in S. mutans compensates for the deficiency.     

Glucose metabolism in the yeast Schwanniomyces castellii: role of phosphorylation site I and an alternative respiratory pathway.

Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture. The wild-type and mutant strains exhibited the same behavior. Oxidative metabolism was observed when the substrate uptake rate (qS) was low. Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1. When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway. The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain).     

Increased production of succinic acid in Escherichia coli by overexpression of malate dehydrogenase

Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1).     

Protein production by<Emphasis Type="Italic"> Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> wild-type and <Emphasis Type="Italic">ΔptsG</Emphasis> mutant strains with IPTG induction at the onset

During Escherichia coli growth on glucose, uptake exceeds the requirement of flux to precursors and the surplus is excreted as acetate. Beside the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. The deletion of ptsG, the gene coding for one of the components (IICB(Glc)) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced glucose consumption and acetate excretion. Induction of protein production at the onset of cultivation decreased growth rate and glucose consumption rate for both the WT and the mutant strains. The mutant strain produced beta-galactosidase at higher rates than the wild-type strain while directing more carbon into biomass and CO(2) and less into acetate.     

The Cu(II)-reductase NADH dehydrogenase-2 of Escherichia coli improves the bacterial growth in extreme copper concentrations and increases the resistance to the damage caused by copper and hydroperoxide

NADH dehydrogenase-2 (NDH-2) from Escherichia coli respiratory chain is a membrane-bound cupric-reductase encoded by ndh gene. Here, we report that the respiratory system of a ndh deficient strain suffered a faster inactivation than that of the parental strain in the presence of tert-butyl hydroperoxide due to endogenous copper. The inactivation was similar for both strains when copper concentration increased in the culture media. Furthermore, several ndh deficient mutants grew less well than the corresponding parental strains in media containing either high or low copper concentrations. A mutant strain complemented with ndh gene almost recovered the parental phenotype for growing in copper limitation or excess. Then, NDH-2 gives the bacteria advantages to diminish the susceptibility of the respiratory chain to damaging effects produced by copper and hydroperoxides and to survive in extreme copper conditions. These results suggest that NDH-2 contributes in the bacterial oxidative protection and in the copper homeostasis.     

Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.     

Method for isolating mutants overproducing nicotinamide adenine dinucleotide and its precursors

A procedure has been developed for isolating mutants which are defective with respect to nicotinamide adenine dinucleotide (NAD) metabolism. It is based on the well known V-factor requirement of Haemophilus parainfluenzae. This procedure was used to isolate a series of mutants from Escherichia coli. The pyridine metabolism of wild-type and mutant E. coli cells falls in one of four distinct classes. Class A includes wild-type E. coli and represents strains that are normal with respect to pyridine metabolism. Class B mutants have altered internal pools of NAD. The intracellular NAD concentration of different class B mutants varies over a 10-fold range. Class C mutants excrete pyridine mononucleotides, and class D mutants excrete NAD. The production of pyridine nucleotides by class C and D mutants exceeds that of wild-type E. coli by a factor of at least ten. The mutant strains generally have normal generation times and achieve normal cell densities in minimal medium.     

Glutamate transport in wild-type and mutant strains of Escherichia coli

Halpern, Yeheskel S. (Hebrew University-Hadassah Medical School, Jerusalem, Israel), and Meir Lupo. Glutamate transport in wild-type and mutant strains of Escherichia coli. J. Bacteriol. 90:1288-1295. 1965.-Mutants of Escherichia coli able to grow on glutamate as their source of carbon showed glutamate dehydrogenase and glutamate-oxaloacetate transaminase activities similar to those possessed by the parent strain. The mutants took up glutamate at a much faster rate and showed a several-fold greater capacity for concentrating the amino acid than did the corresponding parent strains. Curvilinear double reciprocal plots of velocity of uptake versus glutamate concentration were obtained with the E. coli H strains. A break in the curve of glutamate uptake was observed with the E. coli K-12 strains when incubated in a glucose medium. It is suggested that these findings may be due to allosteric activation of glutamate permease by its substrate.     

Pyruvate formate-lyase is not essential for nitrate respiration by Escherichia coli

Abstract Defined deletion mutants of Escherichia coli defective for the synthesis of pyruvate formate-lyase (PFL) or pyruvate dehydrogenase (PDH) were analysed in regards their growth in batch culture and their enzyme levels under fermentative and nitrate respiratory conditions. A pfl mutant proved not to be completely auxotrophic for acetate when grown anaerobically in glucose minimal medium. In contrast, a pfl aceEF double mutant exhibited an absolute requirement for acetate, indicating that PDH is the source of acetyl-CoA in the pfl mutant. Growth of both pfl and aceEF single mutants under nitrate respiratory conditions was essentially indistinguishable from the wild-type. Thus, either PFL or PDH can be used to catabolise pyruvate in nitrate-respiring cells. The activities of PFL and PDH measured after growth with nitrate are commensurate with this proposal.     

A comparative proteomic approach to understand the adaptations of an H+ -ATPase-defective mutant of Corynebacterium glutamicum ATCC14067 to energy deficiencies

F172-8, an H(+)-ATPase-defective mutant of the glutamic acid-producing bacterium Corynebacterium glutamicum ATCC 14067, exhibits enhanced rates of glucose consumption and respiration compared to the parental strain when cultured in a biotin-rich medium with glucose as the carbon source. We conducted a comparative proteomic analysis to clarify the mechanism by which the enhanced glucose metabolism in this mutant is established using a proteome reference map for strain ATCC 14067. A comparison of the proteomes of the two strains revealed the up-regulated expression of the several important enzymes such as pyruvate kinase (Pyk), malate:quinone oxidoreductase (Mqo), and malate dehydrogenase (Mdh) in the mutant. Because Pyk activates glycolysis in response to cellular energy shortages in this bacterium, its increased expression may contribute to the enhanced glucose metabolism of the mutant. A unique reoxidation system has been suggested for NADH in C. glutamicum consisting of coupled reactions between Mqo and Mdh, together with the respiratory chain; therefore, the enhanced expression of both enzymes might contribute to the reoxidation of NADH during increased respiration. The proteomic analysis allowed the identification of unique physiological changes associated with the H(+)-ATPase defect in F172-8 and contributed to the understanding of the adaptations of C. glutamicum to energy deficiencies.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

Oxidative phosphorylation in intact chl-r mutants of Escherichia coli K 12.

The efficiency of oxidative phosphorylation was estimated in intact resting cells of Escherichia coli K 12, strain PA 601 (chl-s) and its chl-r mutants, all of them grown anaerobically in the presence of nitrate. The oxidation of endogenous NADH in intact chl-s cells was accompanied by the formation of ATP whatever the terminal electron acceptor, oxygen or nitrate, so that it was possible to conclude that the energy conservation sites are operating with either of the two acceptors in cells grown anaerobically in the presence of nitrate. For chl-r mutants oxidation of endogenous NADH correlated with ATP-production was found only with oxygen as electron acceptor. It is concluded that the energy-conservation sites are preserved in these mutants, the nitrate respiratory chain of which is altered. This assumption is corroborated by the effects of uncouplers of oxidative phosphorylation on ATP-synthesis.