Aerobic biodegradation of 4-methylpyridine and 4-ethylpyridine by newly isolated Pseudonocardia sp. strain M43

A filamentous bacterium capable of utilizing 4-methylpyridine and 4-ethylpyridine as the sole source of carbon, nitrogen and energy was isolated from sludge. The organism, designated as strain M43, clustered most closely with members of the genus Pseudonocardia by 16S rRNA gene sequence analysis. During the degradation of 4-methylpyridine and 4-ethylpyridine, c. 60% of nitrogen in the pyridine ring was released as ammonia. Metabolite analyses showed that 2-hydroxy-4-methylpyridine and 2-hydroxy-4-ethylpyridine were transiently accumulated during the degradation of 4-methylpyridine and 4-ethylpyridine, respectively. Strain M43 was also able to degrade pyridine, 3,4-dimethylpyridine, 4-carboxypyridine and 2-hydroxy-4-methylpyridine. The results indicate that degradation of 4-methylpyridine and 4-ethylpyridine by strain M43 proceeded via initial hydroxylation.     

Biodegradation of 2-methyl, 2-ethyl, and 2-hydroxypyridine by an Arthrobacter sp. isolated from subsurface sediment

A bacterium capable of degrading 2-methylpyridine was isolated by enrichment techniques from subsurface sediments collected from an aquifer located at an industrial site that had been contaminated with pyridine and pyridine derivatives. The isolate, identified as an Arthrobacter sp., was capable of utilizing 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine as primary C, N, and energy sources. The isolate was also able to utilize 2-, 3-, and 4-hydroxybenzoate, gentisic acid, protocatechuic acid and catechol, suggesting that it possesses a number of enzymatic pathways for the degradation of aromatic compounds. Degradation of 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine was accompanied by growth of the isolate and release of ammonium into the medium. Degradation of 2-methylpyridine was accompanied by overproduction of riboflavin. A soluble blue pigment was produced by the isolate during the degradation of 2-hydroxypyridine, and may be related to the diazadiphenoquinones reportedly produced by other Arthrobacter spp. when grown on 2-hydroxypyridine. When provided with 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine simultaneously, 2-hydroxypyridine was rapidly and preferentially degraded; however there was no apparent biodegradation of either 2-methylpyridine or 2-ethylpyridine until after a seven day lag. The data suggest that there are differences between the pathway for 2-hydroxypyridine degradation and the pathway(s) for 2-methylpyridine and 2-ethylpyridine.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.     

Choice between autotrophy and heterotrophy in Pseudomonas oxalaticus. Growth in mixed substrates

1. The type of metabolism adopted by Pseudomonas oxalaticus during growth on a variety of carbon sources was studied. 2. The only substrate upon which autotrophic growth was observed is formate. 3. In mixtures of formate and those substrates upon which the organism can grow faster than on formate, e.g. succinate, lactate or citrate, heterotrophic metabolism results. 4. In mixtures of formate and those substrates upon which the organism can grow at a similar rate to that on formate, e.g. glycollate or glyoxylate, the predominant mode of metabolism adopted is heterotrophic utilization of the C₂ substrate coupled with oxidation of formate as ancillary energy source. 5. P. oxalaticus grows on oxalate 30% slower than on formate. In mixtures of formate and oxalate, the predominant mode of metabolism adopted is autotrophic utilization of formate coupled with oxidation of oxalate as ancillary energy source. 6. In mixtures of formate and those substrates upon which the organism grows at a much lower rate than on formate, e.g. glycerol and malonate, the predominant mode of metabolism adopted is autotrophic utilization of formate. 7. It is concluded that synthesis of the enzymes involved in autotrophic metabolism is controlled by a combination of induction and metabolite repression.     

Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment

A methanogenic acetate enrichment was initiated by inoculation of an acetate-mineral salts medium with domestic anaerobic digestor sludge and maintained by weekly transfer for 2 years. The enrichment culture contained a Methanosarcina and several obligately anaerobic nonmethanogenic bacteria. These latter organisms formed varying degrees of association with the Methanosarcina, ranging from the nutritionally fastidious gram-negative rod called the satellite bacterium to the nutritionally nonfastidious Eubacterium limosum. The satellite bacterium had growth requirements for amino acids, a peptide, a purine base, vitamin B12, and other B vitamins. Glucose, mannitol, starch, pyruvate, cysteine, lysine, leucine, isoleucine, arginine, and asparagine stimulated growth and hydrogen production. Acetate was neither incorporated nor metabolized by the satellite organism. Since acetate was the sole organic carbon source in the enrichment culture, organism(s) which metabolize acetate (such as the Methanosarcina) must produce substrates and growth factors for associated organisms which do not metabolize acetate.     

Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment.

A methanogenic acetate enrichment was initiated by inoculation of an acetate-mineral salts medium with domestic anaerobic digestor sludge and maintained by weekly transfer for 2 years. The enrichment culture contained a Methanosarcina and several obligately anaerobic nonmethanogenic bacteria. These latter organisms formed varying degrees of association with the Methanosarcina, ranging from the nutritionally fastidious gram-negative rod called the satellite bacterium to the nutritionally nonfastidious Eubacterium limosum. The satellite bacterium had growth requirements for amino acids, a peptide, a purine base, vitamin B12, and other B vitamins. Glucose, mannitol, starch, pyruvate, cysteine, lysine, leucine, isoleucine, arginine, and asparagine stimulated growth and hydrogen production. Acetate was neither incorporated nor metabolized by the satellite organism. Since acetate was the sole organic carbon source in the enrichment culture, organism(s) which metabolize acetate (such as the Methanosarcina) must produce substrates and growth factors for associated organisms which do not metabolize acetate.     

Isolation,characterization, and metabolic activities of Bacillus brevis degrading isonicotinic acid

An organism isolated from soil by enrichment on isonicotinic acid (INA) was characterized as Bacillus brevis. It used sugars more readily than amino acids as growth substrates. The organism also used isoniazid, 2-hydorxypyridine, and benzoic and p-hydroxybenzoic acids. This bacterium did not metabolize 2-hydroxy-INA, citrazinic acid, or other mono- and dihydroxypyridine compounds as well as intermediates of the maleamate pathway. Accumulation of hydroxylated pyridine compounds was not detected during fermentation, or incubation of INA with resting cells in the presence or absence of inhibitors. Succinic semialdehyde was isolated and characterized as a key intermediate and was rapidly oxidized by INA-adapted cells. Formate was detected as a product of INA metabolism, and formate but not formamide was oxidized by INA-adapted cells; γ-aminobutyrate or γ-aminocrotonate were oxidized. A pathway for INA degradation involving oxygenative cleavage of a partially reduced pyridine ring is proposed.     

Difference in 2-thenoyltrifluoroacetone sensitivity of electron transport with and against the redox potential gradient

The effect of 2-thenoyltrifluoroacetone on electron transport with and against the redox potential gradient, with succinate or ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as electron donor, was studied in rat liver mitochondria. It was found that 2-thenoyltrifluoroacetone inhibited succinate-linked intramitochondrial pyridine nucleotide reduction at low concentrations, which neither affected succinate oxidation in the controlled state nor interfered with intramitochondrial pyridine nucleotide reduction in the ascorbate plus TMPD case. The effect of 2-thenoyltrifluoroacetone on succinate-linked intramitochondrial pyridine nucleotide reduction is not attributable either to blocking of the overall rate of electron flow in the succinate dehydrogenase branch of the respiratory chain or to interference with energy transformation. Transition from the controlled to the active state enhanced the inhibitory effect of 2-thenoyltrifluoroacetone on succinate-linked respiration, and it became as sensitive to 2-thenoyltrifluoroacetone as the succinate-linked intramitochondrial pyridine nucleotide reduction. In the light of the above findings, the possibility is discussed that electrons from succinate enter the main branch of the respiratory chain by different routes, according to whether the flow is with or against the potential gradient.     

Manifold effects of palmitoylcarnitine on endoplasmic reticulum metabolism: 11β-hydroxysteroid dehydrogenase 1, flux through hexose-6-phosphate dehydrogenase and NADPH concentration

With the exception of the oxidation of G6P (glucose 6-phosphate) by H6PDH (hexose-6-phosphate dehydrogenase), scant information is available about other endogenous substrates affecting the redox state or the regulation of key enzymes which govern the ratio of the pyridine nucleotide NADPH/NADP. In isolated rat liver microsomes, NADPH production was increased, as anticipated, by G6P; however, this was strikingly amplified by palmitoylcarnitine. Subsequent experiments revealed that the latter compound, well within its physiological concentration range, inhibited 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), the bidirectional enzyme which interconnects inactive 11-oxo steroids and their active 11-hydroxy derivatives. Notably, palmitoylcarnitine also stimulated the antithetical direction of 11β-HSD1 reductase, namely dehydrogenase. This stimulation of H6PDH may have likewise contributed to the NADPH accretion. All told, the result of these enzyme modifications is, in a conjoint fashion, a sharp amplification of microsomal NADPH production. Neither the purified 11β-HSD1 nor that obtained following microsomal sonification were sensitive to palmitoylcarnitine inhibition. This suggests that the long-chain amphipathic acylcarnitines, given their favourable partitioning into the membrane lipid bilayer, disrupt the proficient kinetic and physical interplay between 11β-HSD1 and H6PDH. Finally, although IDH (isocitrate dehydrogenase) and malic enzyme are present in microsomes and increase NADPH concentration akin to that of G6P, neither had an effect on 11β-HSD1 reductase, evidence that the NADPH pool in the endoplasmic reticulum shared by the H6PDH/11β-HSD1 alliance is uncoupled from that governed by IDH and malic enzyme.     

Purification and characterization of the amine dehydrogenase from a facultative methylotroph

Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.     

Oxidation of Inorganic Sulfur Compounds by Obligately Organotrophic Bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria which are most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Oxidation in vivo of glucose, palmitate, alanine and leucine in the pig during the neonatal period

Determination of 14CO2 content in expired air after the intravenous injection of energetic substrates marked by the radioactive carbon to the pigs showed that the oxidative intensity of these substrates decreases in the series: [6-14C]glucose greater than [1-14C] alanine greater than [1-14C]leucine greater than [1-14C]glucose. The oxidation intensity of all substrates under study except for [1-14C]palmitate in the organism of one-day satisfied pigs is considerably higher, than during the first two hours after their birth. The starvation of pigs during the first 24 hours increases the oxidation of both investigated amino acid and [1-14C]-palmitate in tissues of their organism with a decrease in the metabolic intensity of [6-14C] and [1-14C]glucose.     

Oxidation of inorganic sulfur compounds by obligatory organotrophic bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some of denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Coping with formaldehyde during C1 metabolism of Paracoccus denitrificans

Methylotrophic bacteria are capable of growth using reduced one-carbon (C1) compounds like methanol or methylamine as free energy sources. Paracoccus denitrificans, which is a facultative methylotrophic organism, switches to this type of autotrophic metabolism only when it experiences a shortage of available heterotrophic free energy sources. Since the oxidation of C1 substrates is energetically less favourable than that of the heterotrophic ones, a global regulatory circuit ensures that the enzymes involved in methylotrophic growth are repressed during heterotrophic growth. Once the decision is made to switch to methylotrophic growth, additional regulatory proteins ensure the fine-tuned expression of the participating enzymes such that the steady-state concentration of formaldehyde, the oxidation product of C1 substrates, is kept below cytotoxic levels.     

Glycerol dissimilation in Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides followed a diauxic growth curve when grown on a malate-glycerol medium, the first phase of growth being supported by malate and the second by glycerol. A soluble glycerokinase and a particulate, pyridine nucleotide-independent glycerophosphate dehydrogenase, were induced by the presence of glycerol in the medium, but neither was fully expressed nor functional until all malate had been consumed.     

Zur Kinetik der Mischsubstratutilisation bei der Citronensäure-akkumulation durch Saccharomycopsis lipolytica EH 59

The mechanism and the kinetic of the assimilation of mixed substrates during the organism growth and the product excretion by strain of Saccharomycopsis lipolytica have been studied. The assimilation of citric acid for the organism growth was prevented when glucose and/or n-paraffins are present as substrates. Citric acid concentrations higher than 30 g/l in the fermentation medium decrease the growth rate on the substrate glucose. Kinetic studies of the mixed substrate assimilation by means of a n-tetradecane-1-C-labelled paraffin fraction proved that in discontinuous as well as in continuous 1-stage-processes for microbial production of citric acid glucose as substrate is only used for the organism growth whereas the n-paraffin fraction is only used for the acid excretion.     

Inhibition of nicotinic acid and nicotinamide uptake into Bordetella pertussis by structural analogues

3-Pyridine-carboxaldehyde and 3-pyridine-aldoxime were effective and specific inhibitors of the uptake of both nicotinic acid (NA) and nicotinamide (ND) by Bordetella pertussis, although neither compound inhibited the growth of the bacteria in liquid medium or the oxidation of glutamate by washed suspensions. In contrast, the following pyridine derivatives did not inhibit uptake of NA or ND: iso-NA, iso-ND, isoniazid, 6-amino-NA and 6-amino-ND, 3-acetyl-pyridine, 3-pyridyl-acetic acid, N,N-diethyl-ND and 3-pyridine-sulphonic acid. 3- Pyridyl-carbinol was inhibitory, but less so than the first listed compounds.     

The action of lipoxygenase-1 on furan derivatives.

Several 2,5-disubstituted furans, which are known to react with peroxyacids, singlet oxygen and other active forms of oxygen were tested as potential inhibitors, co-oxidants, or substrates for soybean lipoxygenase. The furan, 10,13-epoxy-octadeca-10,12-dienoic acid, methyl ester (IV) was converted by lipoxygenase or singlet oxygen or peroxyacid to the acyclic product, methyl 10,13-dioxo-octadec-11-enoate. Apparently furan IV is able to interact with an active site of lipoxygenase (Km = 220 microM). 2,5-Dimethylfuran (I), 2,5-diphenylfuran (II) and 3-(5'-methyl-2'-furyl)propenoic acid (III) were neither substrates nor inhibitors of lipoxygenase activity. Lipoxygenase-catalyzed oxidation of furan (IV), which is inhibited by hydroquinone, is explained by a mechanism involving lipoxygenase-superoxide complex and furan-radical intermediates. Also described is the selective cleavage of furan rings by m-chloroperoxybenzoic acid to yield the 1,4-diketoethylene functional system.     

Degradation of Pyridine by Micrococcus luteus Isolated from Soil

An organism capable of growth on pyridine was isolated from soil by enrichment culture techniques and identified as Micrococcus luteus. The organism oxidized pyridine for energy and released N contained in the pyridine ring as ammonium. The organism could not grow on mono- or disubstituted pyridinecarboxylic acids or hydroxy-, chloro-, amino-, or methylpyridines. Cell extracts of M. luteus could not degrade pyridine, 2-, 3-, or 4-hydroxypyridines or 2,3-dihydroxypyridine, regardless of added cofactors or cell particulate fraction. The organism had a NAD-linked succinate-semialdehyde dehydrogenase which was induced by pyridine. Cell extracts of M. luteus had constitutive amidase activity, and washed cells degraded formate and formamide without a lag. These data are consistent with a previously reported pathway for pyridine metabolism by species of Bacillus, Brevibacterium, and Corynebacterium. Cells of M. luteus were permeable to pyridinecarboxylic acids, monohydroxypyridines, 2,3-dihydroxypyridine, and monoamino- and methylpyridines. The results provide new evidence that the metabolism of pyridine by microorganisms does not require initial hydroxylation of the ring and that permeability barriers do not account for the extremely limited range of substrate isomers used by pyridine degraders.