Induction by Oxygen of Respiration and Phosphorylation of Anaerobically Grown Escherichia coli

The changes occurring in the respiratory enzymes of anaerobically grown Escherichia coli strain B and E. coli 15 T⁻A⁻U⁻bar during exposure to oxygen were studied. Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity reached its peak soon after O₂ exposure; cytochrome content and succinate oxidase activity increased more slowly, and these increases paralleled each other. The activities of isocitrate and malate dehydrogenases also increased, but the increase was less than that of the succinate and NADH oxidases; exposure to O₂ had no effect on the succinate and NADH dehydrogenase activities. On the other hand, the glycolytic activity decreased slowly after O₂ exposure. The incorporation of ³²P into acid-soluble organic phosphate esters paralleled the respiratory rate during the first 60 min after O₂ exposure, but continued to increase after the respiration reached a plateau. The sensitivity of ³²P incorporation to the uncoupler carbonyl cyanide m-chlorophenylhydrazone also increased with time. The observed relationship between the development of the respiratory chain and the energy-conserving mechanism during O₂ exposure is discussed. Synthesis of the respiratory enzymes upon exposure to oxygen was dependent on concomitant protein and ribonucleic acid synthesis but not on deoxyribonucleic acid synthesis.     

The Effect of Cadmium on Electron and Energy Transfer Reactions in Corn Mitochondria

The effects of cadmium on isolated corn shoot mitochondria were determined. In the absence of phosphate cadmium stimulated the oxidation of exogenous NADH optimally at 0.025 mM, but was inhibitory at 0.1 mM and above. The presence of phosphate negated the cadmium stimulation of exogenous NADH oxidation and permitted inhibitions only at higher cadmium concentrations. Succinate or malate + pyruvate oxidation in the absence of phosphate was inhibited to a greater extent by cadmium than when phosphate was present. ADP/O and respiratory control ratios were reduced by cadmium but generally were less sensitive to cadmium than state 4 or minus phosphate respiration. The data suggest that the site of cadmium effect is likely to be early in electron transport. Cadmium had a pronounced effect on mitochondrial swelling under either passive or active conditions. When succinate or exogenous NADH were being oxidized swelling occurred at 0.05 mM cadmium, but with malate + pyruvate the cadmium concentration had to exceed 1.0 mM. Phosphate (2 mM) prevented the swelling. Dithiothreitol, a SH group protector, prevented any effect of cadmium on swelling or respiration which suggests that sulfhydryl groups are likely involved in the cadmium-membrane interaction.     

Biomass relationship to growth and phosphate uptake ofPseudomonas fluorescens,Escherichia coli andAcinetobacter radioresistens in mixed liquor medium

The ability ofPseudomonas fluorescens, Escherichia coli andAcinetobacter radioresistenns to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10²–10⁵ cells ml^–1) were used. At a high initial biomass concentration (10⁸ cells ml^–1), phosphate was removed during the lag phase and during logarthmic growth byP. fluorescens. Escherichia coli. at high initial biomass concentrations (10⁷ cells ml^–1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantily of phosphate during the stationary growth phase.Acinetobacter radioresistens, at high initial cell densities (10⁶, 10⁷ cells ml^–1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase.Pseudomonas fluorescens removed phosphate more thanA. radioresistens andE. coli with specific average ranges from 3.00–28.50 mg L^–1 compared to average ranges of 4.92–17.14 mg L^–1 forA. radioresistens and to average ranges of 0.50–8.50 mg L^–1 forE. coli.     

A continuous culture study of an ATPase-negative mutant of Escherichia coli

For anaerobic glucose-limited chemostat cultures of Escherichia coli a value of 8.5 was found for Y _ATP ^max . For anaerobic glucose- or ammoniumlimited chemostat cultures of the ATPase-negative mutant M2-6 of E. coli Y _ATP ^max values of 17.6 and 20.0 were found, respectively. From these data it can be concluded that in the wild type during anaerobic growth 51–58% of the total ATP production is used for energetization of the membrane. Using the Y _ATP values obtained in the anaerobic experiments a P/O ratio of 1.46 could be calculated for aerobic experiments with the wild type. It is concluded that from the energy obtained by respiration in wild type E. coli about 60% is used for membrane energetization and only about 40% for the actual formation of ATP. No dramatic difference in the maintenance requirement for ATP or glucose has been observed between glucose- and ammonium-limited chemostat cultures of the mutant. The large difference in maintenance requirement observed for such cultures of the wild type is therefore supposed to be made possible by ATP hydrolysis by the ATPase.     

Toxicity of 2-mercaptobenzothiazole towards bacterial growth and respiration

Active sludge systems containing benzothiazoles may be intoxicated by 2-mercaptobenzothiazole (MBT). This toxicity towards several bacteria is now confirmed and is situated at around 100 mg MBT l^–1. Octanol-water partition coefficients indicated that MBT might interact with membrane-bound systems. This was confirmed through experiments showing that bacterial cell respiration was inhibited using lactate or succinate as substrates. Using these substrates and also NADH, it was found that their oxidation was also inhibited using isolated membrane fragments of Escherichia coli and Paracoccus denitrificans. Methylene blue reduction was also found to be inhibited. The oxidation of ascorbate was not inhibited in P. denitrificans. From these results it is suggested that MBT might interact with the respiratorychain at the level of flavoproteins or quinones and Fe-S clusters.     

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.     

Preparation and Properties of Mitochondria from Naegleria gruberi*

Whole cell respiration rates were measured polarographically for Naegleria gruberi during growth in agitated cultures. Log growth phase amebae consumed 80 ng atoms O/min/mg cell protein. At stationary phase, respiration rate decreased 4–fold. Intact mitochondria were isolated from N. gruberi and their oxidative and phosphorylative capacities were studied polarographically. As with the mammalian system, the mitochondria oxidized succinate and NAD-linked substrates, but unlike rat liver mitochondria, those from the protozoan rapidly oxidized citrate and NADH. The rates of substrate oxidation were ADP-dependent, with ADP:O ratios equalling ? 2.8 for NAD-linked substrates and ? 2.2 for succinate. The respiratory control ratios. 2 to 4 for 11 substrates, were dependent on Pi, Mg²⁺, and serum albumin. Potassium cyanide, azide, malonale, and rotenone inhibited electron transport the same way as that of the mammalian system: however, amytal inhibited both glutamate and succinate respiration. Pentachlorophenol, DNP, and bilirubin uncoupled oxidation from phosphorylation. Difference spectra of oxidized and dithionite-reduced mitochondria had distinct absorption bands of flavins and of c-, b-, and α-type cytochromes.     

Oxidative phosphorylation in membrane vesicles of a gram-positive methylotroph

Membrane preparations, capable of high rates of respiration-linked ATP synthesis, have been obtained from a gram-positive methylotrophic bacterium Bacillus sp. MGA3. NADH, succinate, reduced TMPD and methanol were shown to be suitable substrates for the oxidative phosphorylation. Esterification of orthophosphate was dependent on electron transfer, as evidenced by the requirement for both substrate and oxygen. Phosphorylation was also dependent on ADP and was destroyed by boiling the membrane preparation. The phosphorylation was markedly uncoupled by carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone (CCCP) and was inhibited by N,N-dicyclohexylcarbodiimide (DCCD). KCN caused strong inhibition of substrate oxidation as well as phosphorylation for all substrates tested. Rotenone, amytal and antimycin A caused inhibition when NADH or methanol were used as substrates. Antimycin A inhibited respiration and ATP synthesis with succinate as substrate and had no effect on ascorbate —N,N,N,N-tetramethyl-p-phenylenediimide (TMPD) oxidation by membrane preparations of Bacillus sp. MGA3. P/O ratios determined were 2.4 with NADH, 1.7 with succinate and 0.8 with reduced TMPD. The measured P/O ratio with methanol-oxidizing system was similar to that with NADH (about 2.4).Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - TMPD N,N,N,N-tetramethyl-p-phenylenediimide - Q ubiquinone Q     

Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density     

Heterologous expression,purification, and characterization of xylose reductase from <Emphasis Type="Italic">Candida shehatae</Emphasis>

The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His₆-tagged fusion protein. The recombinant XR had K_m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.     

调控大肠杆菌胞内ATP和NADH水平促进琥珀酸生产

琥珀酸作为一种重要的C4平台化合物,广泛应用于食品、化学、医药等领域。利用大肠杆菌(Escherichia coli)发酵生产琥珀酸受胞内辅因子不平衡的影响,存在产率低、生产强度低、副产物多等问题。为此,对不同氧气条件下琥珀酸产量和化学计量学分析发现,微厌氧条件下E.coli FMME-N-26高效积累琥珀酸需要借助三羧酸循环(tricarboxylic acid cycle,TCA)为还原性三羧酸途径(reductive tricarboxylic acid pathway,r-TCA)提供足够的ATP和NADH。通过减少ATP消耗、强化ATP合成、阻断NADH竞争途径和构建NADH回补路径等代谢工程策略,组合调控胞内ATP与NADH含量,获得工程菌株E.coli FW-17。通过发酵条件优化,菌株E.coli FW-17在5 L发酵罐能积累139.52 g/L琥珀酸,比出发菌株提高了17.81%,乙酸浓度为1.40 g/L,降低了67.59%。进一步在1000 L发酵罐中进行放大实验,琥珀酸产量和乙酸浓度分别为140.2 g/L和1.38 g/L。     

Self-excreted mediator from <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 for electron transfer to carbon electrodes

Escherichia coli K-12 was cultured under anaerobic conditions to form biofilm on carbon fiber electrodes in glucose-containing medium. The anodic current increased with the development of the biofilm and depended on the glucose concentration. Cyclic voltammetric results support the presence of a redox compound(s) excreted from E. coli cells in the biofilm. The compound remained in the film under conditions of continuous flow and gave a couple of oxidation and reduction waves, which may be assigned to a menaquinone-like compound based on the mid-point potential (−0.22 V vs Ag|AgCl at pH 7.1) and its pH dependence. The catalytic current started to increase around the anodic peak potential of the redox compound and also increased by the permeabilization of the E. coli cell membranes with ethylenediamine tetraacetic acid-treatment. The results indicate that the E. coli-excreted redox compound works as a mediator for the electron transfer from the E. coli cells to the electrode as the final electron acceptor. The activity of the redox compound in the E. coli-biofilm as a mediator with some mobility was also verified for diaphorase-catalyzed electrochemical oxidation of NADH.     

Respiration-driven proton translocation in Thiobacillus versutus and the role of the periplasmic thiosulphate-oxidizing enzyme system

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H⁺/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H⁺/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H⁺/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa₃, does not function as a proton pump.Abbreviations DCCD dicyclohexyl carbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - IEF isoelectric focusing - HIC hydrophobic interaction chromatography - EAI ethyl acetimidate hydrochloride - IAI isethionyl acetimidate     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.     

Menaquinone is an obligatory component of the chain catalyzing succinate respiration in Bacillus subtilis

The question was investigated as to whether the bacterial menaquinone (MK) is a component of the electron transport chain catalyzing succinate respiration in Bacillus subtilis. Three different methods were applied, and the following consistent results were obtained. (i) Solvent extraction of MK from the bacterial membrane caused total inhibition of the respiratory activities with succinate and NADH, while the activity of succinate dehydrogenase remained unaffected. The respiratory activities were restored onincorporation of vitamin K₁ into the membrane preparation. (ii) The membrane fraction of a B. subtilis mutant containing 15% of the wild-type amount of MK, respired succinate and NADH at reduced activities. Wild-type activities were restored on fusion of the preparation to liposomes containing vitamin K₁. (iii) The membrane fraction of B. subtilis catalyzed succinate oxidation by various water-soluble naphtho- or benzoquinones at specific activities exceeding to that of succinate respiration. The results suggest that MK is involved in succinate respiration, although its redox potential is unfavorable.Abbreviations MK menaquinone - MKH₂ reduced menaquinone - E₀' standard redox potential at pH 7 - PMS phenazine methosulfate - DCPIP 2,6-Dichlorophenol-indophenol - Q ubiquinone - Q₀ 2,3-dimethoxy-5-methyl-1,4-bezoquinone - DMN, 2,3 dimethyl-1,4-naphthoquinone - DMK demethylmenaquinone     

Properties of Wheat Mitochondria. Study of Substrates, Cofactors and Inhibitors

Isolated mitochondria of wheat shoots oxidize α- ketoglutarate, DL-malate succinate and NADH with good relative respiration control and ADP: O ratio. They have high affinity for α-ketoglutarate and NADH as substrates and utilize malate and succinate with a respiration ratio of about one-half of α-ketoglutarate. The average ADP : O ratios approach the expected theoretical values, i.e., 3.6 ± 0.2 for α-ketoglutarate, 1.8 ± 0.2 for succinate, and 2.8 ± 0.2 for malate. The ADP: O ratio with NADH is 1.8 ± 0.2. The maximum coupling of oxidation and phosphorylation is obtained at concentrations of 10 mM, 2 mM, 10 mM and 8 mM for α-ketoglutarate, NADH, malate and succinate, respectively. — Wheat mitochondria have little or no dependence on added cofactors. Mitochondria prepared by our procedure apparently retain sufficient amounts of endogenous cofactors required for NAD-linked systems. FAD⁺ is found to improve succinate oxidation. Cytochrome c does not have any significant effect on respiratory parameters of wheat mitochondria. — Wheat mitochondria are some -what resistant to DNP at 1.7 × 10^-5M. Malonate seems to improve coupling of α-ketoglutarate oxidation. Other Krebs cycle intermediates have been tested on three major substrates of TCA cycle, i.e., α-ketoglutarate, malate and succinate.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。