Linkage of formate hydrogenlyase with anaerobic respiration in Proteus mirabilis

The linkage between the enzyme system catalysing formate hydrogenlyase and reductases involved in anaerobic respiration in intact cells of anaerobically grown Proteus mirabilis was studied. Reduction of nitrate and fumarate by molecular hydrogen or formate was possible under all growth conditions; reduction of tetrathionate and thiosulphate occurred only in cells harvested at late growth phase from a pH-regulated batch culture and not in cells harvested at early growth phase or in cells grown in pH-auxostat culture. Under all conditions, cells possessed the enzyme tetrathionate reductase. We conclude that linkage between tetrathionate reductase (catalysing also reduction of thiosulphate) and the formate hydrogenlyase chain is dependent on growth conditions. During reduction of high-potential oxidants such as fumarate, tetrathionate (when possible) or the artificial electron acceptor methylene blue by formate, there was no simultaneous H₂ evolution due to the formate hydrogenlyase reaction. H₂ production started only after complete reduction of methylene blue or fumarate, in the case of methylene blue after a lag phase without gas production. In preparations with a low fumarate reduction activity this was accompanied by an acceleration in CO₂ production. During reduction of thiosulphate (a low-potential oxidant) or of tetrathionate in the presence of benzyl viologen (a low-potential mediator) by formate, H₂ was evolved simultaneously. From this we conclude that formate hydrogenlyase is regulated by a factor that responds to the redox state of any electron acceptor couple present such that lyase activity is blocked when the acceptor couple is oxidised to too great an extent.     

Formic Hydrogenlyase and the Photoassimilation of Formate by a Strain of Rhodopseudomonas palustris

A photosynthetic bacterium isolated by enrichment on media containing formate as major source of cell carbon was identified as a strain of Rhodopseudomonas palustris. It grew on a wide range of simple organic compounds including alcohols, fatty acids, and hydroxyacids, on a chemically defined medium with biotin and p-aminobenzoic acid as essential growth factors. The organism grew on formate or photoautotrophically with molecular hydrogen or thiosulfate only in the presence of yeast extract. Ability to photoassimilate formate could be shown only in organisms grown in the presence of formate. The organism contained an inducible formic hydrogenlyase consisting of a soluble formic dehydrogenase, a particulate hydrogenase, and one or more intermediate, but as yet unidentified, electron carriers. The formic hydrogenlyase could be reconstituted from a particulate hydrogenase and a partially purified soluble formic dehydrogenase. Some properties of the formic dehydrogenase and hydrogenase have been compared with that of the formic hydrogenlyase system.     

On culturing Escherichia coli on a mineral salts medium during anaerobic conditions

The substrate and products of the hydrogenlyase complex, formic acid, carbon dioxide, and molecular hydrogen, are co-operatively implicated in maintaining growth of E. coli under anaerobic conditions. Growth is observed in the presence of a combination of carbon dioxide + molecular hydrogen, or carbon dioxide + formic acid in the medium. The study shows that it is possible to culture E. coli under anaerobic conditions while sparging with nitrogen, without supplementing exogenous carbon dioxide, formic acid or molecular hydrogen. This condition occurs when the strain is allowed an appropriate induction period and is present at a sufficiently high cell density, since the cell density affects the rate of e.g. CO₂ production. In a system sparged with nitrogen gas, the removal of CO₂ due to this sparging must be balanced with a cell density dependent production rate of CO₂. It is concluded that the formic hydrogenlyase complex should be considered as an integral part of the general maintenance of the anabolism of E. coli during anaerobic conditions on a mineral salts medium, as well as being a net producer of end products in E. coli metabolism.This work was supported by the Swedish National Board for Industrial and Technical Development. A. Askendahl is acknowledged for valuable assistance in the preparation of figures and P. Warkentin for language editing.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

Induced Biosynthesis of Formic Hydrogenlyase in Iron-Deficient Cells of Escherichia coli.

Fukuyama, T. (University of Washington, Seattle), and E. J. Ordal. Induced biosynthesis of formic hydrogenlyase in iron-deficient cells of Escherichia coli. J. Bacteriol. 90:673-680. 1965.-Escherichia coli cells were grown aerobically on a lactate-mineral salts medium from which iron had been removed by extraction with 8-hydroxyquinoline and chloroform. These cells carried out induced biosynthesis of formic hydrogenlyase in a reaction mixture containing glucose, formate, and phosphate without the addition of amino acids, providing adequate amounts of iron salts were present. In the absence of iron, glucose was fermented and acids were produced, but no formic hydrogenlyase developed. When iron-deficient E. coli cells were repeatedly washed, the property of carrying out induced biosynthesis of formic hydrogenlyase with glucose, formate, phosphate, and iron was lost, but was restored on addition of acid-hydrolyzed casein to the reaction mixture. An energy source (provided as glucose) was necessary for enzyme production. Iron-deficient cells were devoid of hydrogenase and formic hydrogenlyase but showed formic dehydrogenase activity when adequate amounts of selenium and molybdenum were present in the growth medium. Hydrogenase was consistently absent in iron-deficient cells but appeared concomitantly with formic hydrogenlyase during induced biosynthesis of the latter in iron-deficient cells of E. coli.     

Comparison of the Recovery of Escherichia coli from Frozen Foods and Nutmeats by Confirmatory Incubation in EC Medium at 44.5 and 45.5 C

The productivity of confirmatory EC broth for the isolation of fecal Escherichia coli was determined at 44.5 and 45.5 C. A variety of frozen pre-cooked foods and an assortment of nutmeats were examined after primary incubation in Lauryl Sulfate Tryptose (LST) broth. In 85.3% of the cases, the parallel tubes of EC broth incubated for 24 hr at 44.5 and 45.5 C gave rise to identical E. coli responses of positive, false positive, and negative. The remaining 14.7% of the reactions represent the qualitative difference between the two temperatures. The EC test at 45.5 C was more specific for E. coli, since two- to threefold fewer false positives were produced at this temperature than at 44.5 C. However, fecal E. coli recoveries were slightly higher (4%) at the lower temperature. Incubating the EC tubes from the interval of 24 to 48 hr gave rise to an additional 4.3% of E. coli recovery, but this was accompanied by an excessive production of false positives (75.9%), representing a 3.5-fold decrease in specificity. It is recommended that, in the confirmatory use of EC broth in the examination of frozen foods and nutmeats for the recovery of fecal E. coli, the test be made at 45.5 C in a water bath and limited to 24 hr of incubation only, to insure optimal specificity. During the study, a “fixed” productivity ratio was noted; E. coli⁺/LST⁺ equaled approximately one-fourth or 25%. The significance of this ratio is discussed.     

Enzymes Released from Escherichia coli With the Aid of a Servall Cell Fractionator

The release and stability of the enzymes S-adenosylhomocysteine nucleosidase, lysine decarboxylase, arginine decarboxylase, glutamic decarboxylase, formic hydrogenlyase, formic oxidase, and glucose oxidase from Escherichia coli during disruption of the organisms in a Servall-Ribi refrigerated cell fractionator were examined. With the possible exception of arginine decarboxylase, maximal activity was retained by all the enzymes reported here when the cell suspensions were processed at pressures necessary for rupture of all the organisms (15,000 to 25,000 psi). Considerable variation in the stability of different enzymes liberated by disruption at higher pressures (45,000 to 55,000 psi) was observed. It is reasonable to assume that mechanical forces rather than effects of temperature are responsible for inactivation of these enzymes.     

Temperature range for formic hydrogenlyase induction and activity in psychrophilic and mesophilic bacteria

The temperature range for induction of formic hydrogenlyase in cell suspensions of psychrophilic strain 82 was 0 C to 20 C compared to 15 C to 45 C for mesophilicEscherichia coli and 15 C to 40 C for mesophilicAerobacter aerogenes. The respective temperatures for maximum enzyme activity were 30 C, 45 C and 40 C, and for formation of the maximum amount of enzyme 15 C, 35 C and 30 C. The temperatures for all three enzyme functions, therefore, were considerably lower for the psychrophile than for the mesophiles.This research has been supported by a grant from the National Science Foundation.     

Escherichia coli variants for gas and indole production at elevated incubation temperatures.

Two strains of Escherichia coli were subjected to heat and cold-storage treatments to determine the stability of the fecal E. coli characteristics of gas production from lactose and indole production at elevated incubation temperatures. No variants were detected with repeated sublethal heat treatment. A high incidence of variants was observed with extended cold storage of the organisms in liquid and semisolid media, especially with poor nutrient composition, and in the absence of cryoprotective agents. The indole characteristic at elevated temperature was more stable than the production of gas from lactose. The critical temperature at which both gas production from lactose and the indole characteristic were lost was 44.5 degrees C. It appeared that the variants resulted from increased temperature sensitivity of the formic hydrogen lyase and tryptophanase enzymes, respectively.     

Escherichia coli variants for gas and indole production at elevated incubation temperatures

Two strains of Escherichia coli were subjected to heat and cold-storage treatments to determine the stability of the fecal E. coli characteristics of gas production from lactose and indole production at elevated incubation temperatures. No variants were detected with repeated sublethal heat treatment. A high incidence of variants was observed with extended cold storage of the organisms in liquid and semisolid media, especially with poor nutrient composition, and in the absence of cryoprotective agents. The indole characteristic at elevated temperature was more stable than the production of gas from lactose. The critical temperature at which both gas production from lactose and the indole characteristic were lost was 44.5 degrees C. It appeared that the variants resulted from increased temperature sensitivity of the formic hydrogen lyase and tryptophanase enzymes, respectively.     

Regulation of reductase formation in Proteus mirabilis

Summary Prteus mirabilis can form four reductases after anaerobic growth: nitrate reductase A, chlorate reductase C, thiosulfate reductase and tetrathionate reductase. The last three enzymes are formed constitutively. Nitrate reductase is formed only after growth in the presence of nitrate, which causes repression of the formation of thiosulfate reductase, chlorate reductase C, tetrathionate reductase and hydrogenase. Formic dehydrogenase assayed with methylene blue as hydrogen acceptor is formed under all conditions.Two groups of chlorate resistant mutants were obtained. One group does not form the reductases and formic dehydrogenase. The second group does not form nitrate reductase, chlorate reductase and hydrogenase, but forms formic dehydrogenase and small amounts of formic hydrogenlyase after growth without hydrogen acceptor or after growth in the presence of thiosulfate or tetrathionate. Nitrate prevents the formation of formic dehydrogenase, thiosulfate reductase and tetrathionate reductase in this group of mutants. Only after growth with thiosulfate or tetrathionate the reductases for these compounds are formed. Anaerobic growth of the wild type in complex medium without a fermentable carbon source is strongly stimulated by the presence of nitrate. Tetrathionate and thiosulfate have no effect at all or only a small effect. The results show that in the presence of tetrathionate or thiosulfate the bacterial metabolism is fully anaerobic, as these cells also contain formic hydrogenlyase.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

Genetic Diversity of Hydrogen-Producing Bacteria in an Acidophilic Ethanol-H2-Coproducing System, Analyzed Using the [Fe]-Hydrogenase Gene

Hydrogen gas (H₂) produced by bacterial fermentation of biomass can be a sustainable energy source. The ability to produce H₂ gas during anaerobic fermentation was previously thought to be restricted to a few species within the genera Clostridium and Enterobacter. This work reports genomic evidence for the presence of novel H₂-producing bacteria (HPB) in acidophilic ethanol-H₂-coproducing communities that were enriched using molasses wastewater. The majority of the enriched dominant populations in the acidophilic ethanol-H₂-coproducing system were affiliated with low-G+C-content gram-positive bacteria, Bacteroidetes, and Actinobacteria, based on the 16S rRNA gene. However, PCR primers designed to specifically target bacterial hydA yielded 17 unique hydA sequences whose amino acid sequences differed from those of known HPB. The putative ethanol-H₂-coproducing bacteria comprised 11 novel phylotypes closely related to Ethanoligenens harbinense, Clostridium thermocellum, and Clostridium saccharoperbutylacetonicum. Furthermore, analysis of the alcohol dehydrogenase isoenzyme also pointed to an E. harbinense-like organism, which is known to have a high conversion rate of carbohydrate to H₂ and ethanol. We also found six novel HPB that were associated with lactate-, propionate-, and butyrate-oxidizing bacteria in the acidophilic H₂-producing sludge. Thus, the microbial ecology of mesophilic and acidophilic H₂ fermentation involves many other bacteria in addition to Clostridium and Enterobacter.     

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor,a Genus of Plant Biomass-Degrading Thermophilic Bacteria

Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C₅ and C₆ sugars primarily to hydrogen gas, lactate, acetate, and CO₂ and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.     

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning,characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5–25 °C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t_1/2 value of 16 min for thermal inactivation during incubation at 40 °C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly²⁴⁴ was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly²⁴⁴→Pro substitution into the enzyme. Stability studies showed that the t_1/2 value for thermal inactivation of the mutant during incubation at 40 °C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k_cat/K_m value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.     

Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: Isolation and structural properties

The catalytically active form (E_a) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (E_i) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5′-deoxyadenosine. E_i and the activating enzyme were purified to homogeneity and structurally characterized. E_i has an α₂ oligomeric structure (2 × 85 kDa) and contains no cofactor. The amino acid composition has been determined. Out of a total of six cysteinyl residues per subunit, one shows an unusually fast reaction with iodoacetate (k₂ = 7 (m^? s^?) at pH 6.8, 30 °C), which is accompanied by loss of the activatability of the enzyme. The 1500-fold purified activating enzyme is a monomeric protein of 30 kDa. It contains a covalently bound, as yet unidentified chromophoric factor which has an optical absorption peak at 388 nm. Further studies of the in situ state of pyruvate formate-lyase detected a reversible backconversion of the active form E_a into E_i when anaerobic cells become nutrient-depleted.     

Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus

The kinetic properties of glucokinase (GLK) from the liver of active and hibernating ground squirrels Spermophilus undulatus have been studied. Entrance of ground squirrels into hibernation from their active state is accompanied by a sharp decrease in blood glucose (Glc) level (from 14 to 2.9 mM) and with a significant (7-fold) decrease of GLK activity in the liver cytoplasm. Preparations of native GLK practically devoid of other molecular forms of hexokinase were obtained from the liver of active and hibernating ground squirrels. The dependence of GLK activity upon Glc concentration for the enzyme from active ground squirrel liver showed a pronounced sigmoid character (Hill coefficient, h = 1.70 and S _0.5 = 6.23 mM; the experiments were conducted at 25°C in the presence of enzyme stabilizers, K⁺ and DTT). The same dependence of enzyme activity on Glc concentration was found for GLK from rat liver. However, on decreasing the temperature to 2°C (simulation of hibernation conditions), this dependency became almost hyperbolic (h = 1.16) and GLK affinity for substrate was reduced (S _0.5 = 23 mM). These parameters for hibernating ground squirrels (body temperature 5°C) at 25°C were found to be practically equal to the corresponding values obtained for GLK from the liver of active animals (h = 1.60, S _0.5 = 9.0 mM, respectively); at 2°C sigmoid character was less expressed and affinity for Glc was drastically decreased (h = 1.20, S _0.5 = 45 mM). The calculations of GLK activity in the liver of hibernating ground squirrels based on enzyme kinetic characteristics and seasonal changes in blood Glc concentrations have shown that GLK activity in the liver of hibernating ground squirrels is decreased about 5500-fold.     

Glycolate Formation and Excretion by Chlorella pyrenoidosa and Netrium digitus

Conditions are described whereby suspensions of Chlorella pyrenoidosa and Netrium digitus photosynthetically biosynthesize and excrete glycolate continuously in high yields. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate-linked enzymes, increased the excretion of glycolate approximately 4-fold in 1 hour (8 millimolar) and 20-fold in 4 hours (40 millimolar) in the presence of 0.2% CO₂ in air. The amount of glycolate excreted in the presence of aminooxyacetate and an atmosphere of 0.2% CO₂ in air equaled or exceeded the amount excreted in 0.2% CO₂ in O₂ minus aminooxyacetate. CO₂ and light were required for glycolate excretion. Aminooxyacetate also stimulated photosynthetic glycolate excretion in an atmosphere of 0.2% CO₂ in nitrogen or helium, although the stimulation was not as great as when air or O₂ was present.

The excreted glycolate was converted to H₂ and CO₂ by the combined action of glycolic oxidase and the formic hydrogenlyase complex found in Escherichia coli in total conversion yields of 80%.

     

Clonal Populations of Thermotolerant Enterobacteriaceae in Recreational Water and Their Potential Interference with Fecal Escherichia coli Counts

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of β-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. β-Glucuronidase activity was critical in distinguishing E. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.     

The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L.

The fate of the carbon-20 atom in gibberellin (GA) biosynthesis was studied in a cell-free system from Pisum sativum. This carbon atom is lost at the aldehyde stage of oxidation when C₂₀-GAs are converted to C₁₉-GAs. Gibberellin A₁₂ labeled with ¹⁴C at C-20 was prepared from [3-¹⁴C]mevalonic acid with a cell-free system from Cucurbita maxima and incubated with the pea system. Analysis of the gas and aqueous phases showed that ¹⁴CO₂ was formed at the same rate and in nearly equivalent amounts as ¹⁴C-labeled C₁₉-GAs whereas [¹⁴C]formic acid and [¹⁴C]formaldehyde were not detectable. The possibility that C-20 had been lost as formic acid which had then been converted to CO₂ was investigated by control incubations with [¹⁴C]formic acid. The rate of release of ¹⁴CO₂ from [¹⁴C]formic acid was only one fiftieth of the rate of ¹⁴CO₂ release from [¹⁴C]GA₁₂ as the substrate. We conclude that in the formation of C₁₉-GAs from C₂₀-GAs, the C-20 is removed directly as CO₂.Abbreviations GA_n Gibberellin A_n