Respiratory capacities of mitochondria of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under glucose limitation

A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.     

Ethylene oxidation by Nitrosomonas europaea

Incubation of whole cells of the nitrifying bacterium Nitrosomonas europaea with ethylene led to the formation of ethylene oxide. Ethylene oxide production was prevented by inhibitors of ammonium ion oxidation, and showed properties implying that ethylene is a substrate for the ammonia oxidising enzyme, ammonia monooxygenase. Endogenous substrates, hydroxylamine, hydrazine and ammonium ions were compared as sources of reducing power in terms of rates and stoichiometries of ethylene oxidation. The highest rates of ethylene oxide formation (15 mol h^-1 mg protein^-1) were obtained with hydrazine as donor. The data suggest that at high concentrations of ethylene the rate of oxidation is limited by the rate at which reducing power can be supplied to the monooxygenase, not by an intrinsic V _max. Ethylene had an inhibitory effect on the rate of ammonium ion utilisation; an approximate K _i of 80 M was derived, but the results deviated from simple competitive behaviour. Measurement of relative rates of ethylene oxide formation and ammonium ion utilization led to a k _cat/K _m value for ethylene of 1.1 relative to NH ₄ ⁺ , or 0.04 relative to the true natural substrate, NH₃. The effects of higher concentrations of ethylene oxide on oxygen uptake rates were also investigated. The results imply that ethylene oxide is also a substrate for the monooxygenase, but with a much lower affinity than ethylene.     

Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

Energy coupling and respiration inNitrosomonas europaea

Intact cells ofNitrosomonas europaea grown in an ammonium salts medium will oxidise ammonium ions, hydroxylamine and ascorbate-TMPD; there is no oxidation of carbon monoxide, methane or methanol. TheK _m value for ammonia oxidation is highly pH dependent with a minimum value of 0.5 mM above pH 8.0. This suggests that free ammonia is the species crossing the cytoplasmic membrane(s). The measurement of respiration driven proton translocation indicates that there is probably only one proton translocating loop (loop 3) association with hydroxylamine oxidation. The oxidation of endogenous substrates is sometimes associated with more than one proton-translocating loop. These results indicate that during growth hydroxylamine oxidation is probably associated with a maximum P/O ratio of 1.Abbreviations H⁺/O ratio g equiv. H⁺ translocated/g atom O consumed     

Role of nitrite reductase in the ammonia-oxidizing pathway of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Metabolism of ammonia (NH₃) and hydroxylamine (NH₂OH) by wild-type and a nitrite reductase (nirK) deficient mutant of Nitrosomonas europaea was investigated to clarify the role of NirK in the NH₃ oxidation pathway. NirK-deficient N. europaea grew more slowly, consumed less NH₃, had a lower rate of nitrite (NO₂ ⁻) production, and a significantly higher rate of nitrous oxide (N₂O) production than the wild-type when incubated with NH₃ under high O₂ tension. In incubations with NH₃ under low O₂ tension, NirK-deficient N. europaea grew more slowly, but had only modest differences in NH₃ oxidation and product formation rates relative to the wild-type. In contrast, the nirK mutant oxidized NH₂OH to NO₂ ⁻ at consistently slower rates than the wild-type, especially under low O₂ tension, and lost a significant pool of NH₂OH–N to products other than NO₂ ⁻ and N₂O. The rate of N₂O production by the nirK mutant was ca. three times higher than the wild-type during hydrazine-dependent NO₂ ⁻ reduction under both high and low O₂ tension. Together, the results indicate that NirK activity supports growth of N. europaea by supporting the oxidation of NH₃ to NO₂ ⁻ via NH₂OH, and stimulation of hydrazine-dependent NO₂ ⁻ reduction by NirK-deficient N. europaea indicated the presence of an alternative, enzymatic pathway for N₂O production.     

The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.     

Cell yields of Vibrio succinogenes growing with formate and fumarate as sole carbon and energy sources in chemostat culture

Vibrio succinogenes which gains all the ATP by anaerobic electron transport phosphorylation, was grown in continuous culture on a defined medium with formate and fumarate as sole energy sources. The growth yield at infinite dilution rate (Y ^max) was obtained by extrapolation from the growth yields measured at various dilution rates. With formate as the growth limiting substrate, Y ^max was found as 14 g dry cells/mol formate. Under these conditions growth was limited by the rate of energy supply, because formate is used only as a catabolic substrate (Bronder et al. 1982). The Y _ATP ^max calculated from the ATP requirement for cell synthesis was 18 g dry cells/mol ATP. This gives an ATP/2e ratio of 0.8. The ATP/2e ratio in vitro had been measured as 1 (Kröger and Winkler 1981). It is concluded that growing V. succinogenes gain at least 80% the stoichiometrically possible amount of ATP, when growth is limited by energy supply.     

Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species

Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.     

Utilization of benzylpenicillin as carbon,nitrogen and energy source by a Pseudomonas fluorescens strain

A bacterium which utilizes benzylpenicillin as carbon, nitrogen and energy source was isolated from a lake sediment. The organism was identified as a strain of Pseudomonas fluorescens with a GC content of 59.71 Mol %. After growth of the organism on a mineral salts medium containing benzylpenicillin, the derivatives benzylpenicilloic acid, benzylpenilloic acid and benzylpenicillenic acid were found in culture media. There was no indication that the phenylacetate side chain of benzylpenicillin is decomposed. In uninoculated culture media benzylpenicillin, benzylpenicilloic acid and benzylpenicillenic acid were demonstrable. The following compounds were found to be absent from inoculated or uninoculated culture fluids: d-penicillamine, l-valine, l-cysteine, benzylpenillic acid and 6-aminopenicillanic acid. The organism possesses penicillinase. Penicillin acylase was not demonstrable. The reaction product of penicillinase, benzylpenicilloic acid, supports only little growth. There is no growth on 6-aminopenicillanic acid with or without NH₄Cl. Relatively little growth occurs on 6-aminopenicillanic acid in the presence of phenylacetic acid.The data indicate that the nucleus of the benzylpenicillin molecule is utilized as carbon, nitrogen and energy source. During growth a part of the substrate is destroyed into scarcely usable benzylpenicilloic acid; hereby the antibiotic is detoxified.Abbreviations TLC thin-layer chromatography - DNPH 2,4-dinitrophenylhydrazine     

Utilization of hydrogen and formate by Campylobacter spec. under aerobic and anaerobic conditions

A free-living aspartate-fermenting Campylobacter spec. was shown to utilize hydrogen produced in mixed culture by Clostridium cochlearium from glutamate. Resting cells of Campylobacter were shown to reduce aspartate, fumarate and malate as well as nitrate, nitrite, hydroxylamine, sulphite, thiosulphate and elemental sulphur with molecular hydrogen. Growth of Campylobacter spec. was demonstrated with formate as electron donor and nitrate, thiosulphate, elemental sulphur or oxygen as electron acceptor in the presence of acetate as carbon source.     

Utilization of l-alanine as carbon and nitrogen source by Deusulfovibrio HL21

Desulfovibrio HL21 is unable to grow with amino acids as energy substrates. Alanine, serine, aspartate and to some extent glutamate were used as carbon and nitrogen sources in the presence of hydrogen as the energy substrate. Dense cell suspensions converted alanine stoichiometrically to acetate, NH ₄ ⁺ and presumably HCO ₃ ^- , but at a very low rate. Desulfovibrio HL21 cells grown with alanine as carbon and nitrogen source contained increased levels of NAD(P)-dependent l-alanine dehydrogenase as compared to cells grown with NH₄Cl as nitrogen source. Unfavourable kinetic properties of this alanine dehydrogenase, repression of the synthesis of the enzyme by NH ₄ ⁺ and a low rate of NADH oxidation all have a negative effect on the rate of degradation of alanine and may partly explain the inability of the strain to grow with alanine as an energy substrate.     

Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures

Shoot tips of Triticum aestivum L. cvs. Turbo and Nandu, both summer wheat varieties, were excised from 4 and 10 day-old seedlings, and used for induction of embryogenic callus. A modified L3 medium, supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-d) for culture initiation, and 5 M 2,4-d for subculturing, was optimal; 90% of 4 day-old Turbo seedlings formed embryogenic callus. Optimal plant regeneration was achieved from callus incubated on a modified MS medium without 2,4-d, but supplemented with 2.22 M 6-benzylaminopurine and 0.27 M naphthaleneacetic acid. Plantlets formed via embryogenesis from all embryogenic Turbo calli initiated from 4 day-old explants, with a mean number of 8 regenerants per explant. Regeneration occured via embryogenesis only. Results obtained using Nandu were within the same range.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Utilization of organic compounds as the sole source of nitrogen by Thiobacillus thiooxidans

Thiobacillus thiooxidans DSM 504 was shown to grow with adenine, hypoxanthine, xanthine and uric acid as sole sources of nitrogen. Growth with these compounds was observed after lag periods of varying lengths, unless the cells had been previously grown with the same purine base. The disappearance of adenine was accompanied by a temporary accumulation of hypoxanthine in the medium. The utilization of purines was inhibited by ammonia (1 mM). Guanine, pyrimidines and some other organic compounds were not utilized.Non-standard abbreviation U-¹⁴C uniformly labeled by ¹⁴C     

Effects of gaseous NO2 on cells of Nitrosomonas eutropha previously incapable of using ammonia as an energy source

Cells of Nitrosomonas eutropha grown under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were initially unable to oxidize ammonia (ammonium) and hydroxylamine when transferred to oxic conditions. Recovery of ammonia and hydroxylamine oxidation activity was dependent on the presence of NO₂. Under oxic conditions, without addition of NO₂, ammonia consumption started after 8 – 9 days, and small amounts of NO and NO₂ were detectable in the gas atmosphere. Removing these nitrogen oxides by intensive aeration, ammonia oxidation activity decreased and broke off after 15 days. Addition of gaseous NO₂ (25 ppm) led to a fast recovery of ammonia oxidation (3 days). Simultaneously, the arrangement of intracytoplasmic membranes (ICM) changed from circular to flattened vesicles, the protein pattern revealed an increase in the concentration of a 27 and a 30 kDa polypeptide, and the cytochrome c content increased significantly.     

Utilization of sulfonates as sole sulfur source by soil bacteria including Comamonas acidovorans

Bacteria able to use cysteate, taurine or isethionate as sole source of carbon and energy were isolated from the soil. Tests of sulfur assimilation showed that sulfonate sulfur and sulfate sulfur supported comparable cell yields. Methanesulfonate, 1-dodecanesulfonate and p-toluenesulfonate also served as sole source of sulfur for strain I91, identified as Comamonas (Pseudomonas) acidovorans. Competition studies with strain I91 showed that the presence of sulfate inhibits cysteate, isethionate or taurine incorporation. Pseudomonas aeruginosa PAO1, Comamonas acidovorans 14 and 105, and Acidovorax (Pseudomonas) facilis 332 used cysteate, isethionate, or taurine as sole source of sulfur while P. aeruginosa PAO716 and PAO718 used only taurine.     

Improved Taxol production in suspension cultures of Taxus chinensis var. mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor

Taxus chinensis var. mairei was grown in two-liquid-phase culture in a 2.5 l airlift external loop reactor for production of Taxol. A mixture of oleic acid and dibutyl phthalate (1:1, v/v) in a volume ratio of 8:92 to the culture medium gave higher production of Taxol (up to 12 mg l^–1) with an aeration rate of 1 vvm. By combination of the in situ extraction, precursor feeding and additional sugar introduction, Taxol production (16.7 mg l^–1) was 5 times higher than that in the case without any additives (3.4 mg l^–1) and 1.4 times of that in the case with only thein situ extraction (12 mg l^–1). Taxol was extracted from the organic solvents with water and methanol (1:1:1.4, by vol.) and lyophilized giving a total yield of ca. 90%.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

Utilization of cyanobacteria by Brachionus calyciflorus: Anabaena flos-aquae (NRC-44-1) as a sole or complementary food source

The rotifer Brachionus calyciflorus can utilize the cyanobacterium Anabaena flos-aquae as either a sole or supplementary food source in laboratory culture. Positive population growth rates accompany food densities of 10 or 100 µg dry weight ml^–1, but slightly negative rates are found at a lower density (1.0 µg ml^–1). These results are consistent for rotifers feeding on two strains of A. flos-aquae, UTEX-1444 and NRC-44-1, with slightly enhanced survivorship and reproduction with the latter food. A 1:1 mixture (by dry weight) of Euglena gracilis and A. flos-aquae (NRC-44-1) produces survivorship comparable to that of control rotifer cohorts fed E. gracilis alone, but elicits significantly greater fecundity and population growth rates than found with the control food suspension at the same biomass density.