BACTERIAL OXIDATION OF DIPICOLINIC ACID I. : Isolation of Microorganisms, Their Culture Conditions, and End Products.

Arima, Kei (University of Tokyo, Tokyo, Japan) and Yasuo Kobayashi. Bacterial oxidation of dipicolinic acid. I. Isolation of microorganisms, their culture conditions, and end products. J. Bacteriol. 84:759-764. 1962.-In a study of the metabolic pathway(s) of dipicolinic acid (DPA) in microorganisms, 436 strains of soil microorganisms were isolated by use of an enrichment culture technique. Most of them were bacteria, and one of them, Achromobacter, which had the strongest DPA-oxidizing activity, was used for the following experiments. In DPA-free medium, the enzymes which oxidize DPA were not produced. The best culture condition for enzyme production and cell growth was: Nutrient Broth supplemented with 0.1% DPA, 30 C, and 20 hr of shake culture. End products were oxalic acid, NH(3), and CO(2). Oxalic acid was not oxidized further by this bacterium. The over-all reaction equation of DPA oxidation was determined.     

Anaerobic Biodegradation of n-Hexadecane by a Nitrate-Reducing Consortium

Nitrate-reducing enrichments, amended with n-hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H₃₄-hexadecane, fully deuterated hexadecane (d₃₄-hexadecane), or H₃₄-hexadecane and NaH¹³CO₃. Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H₂₉]pentadecanoic acid, [H₂₅]tridecanoic acid, [1-¹³C]pentadecanoic acid, [3-¹³C]heptadecanoic acid, [3-¹³C]10-methylheptadecanoic acid, and d₂₇-pentadecanoic, d₂₅-, and d₂₄-tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent β-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1-¹⁴C]hexadecane was demonstrated based on the recovery of ¹⁴CO₂ in active cultures.Linear alkanes account for a large component of crude and refined petroleum products and, therefore, are of environmental significance with respect to their fate and transport (38). The aerobic activation of alkanes is well documented and involves monooxygenase and dioxygenase enzymes in which not only is oxygen required as an electron acceptor but it also serves as a reactant in hydroxylation (2, 16, 17, 32, 34). Alkanes are also degraded under anoxic conditions via novel degradation strategies (34). To date, there are two known pathways of anaerobic n-alkane degradation: (i) alkane addition to fumarate, commonly referred to as fumarate addition, and (ii) a putative pathway, proposed by So et al. (25), involving carboxylation of the alkane. Fumarate addition proceeds via terminal or subterminal addition (C-2 position) of the alkane to the double bond of fumarate, resulting in the formation of an alkylsuccinate. The alkylsuccinate is further degraded via carbon skeleton rearrangement and β-oxidation (4, 6, 8, 12, 13, 21, 37). Alkane addition to fumarate has been documented for a denitrifying isolate (21, 37), sulfate-reducing consortia (4, 8, 12, 13), and five sulfate-reducing isolates (4, 6-8, 12). In addition to being demonstrated in these studies, fumarate addition in a sulfate-reducing enrichment growing on the alicyclic alkane 2-ethylcyclopentane has also been demonstrated (23). In contrast to fumarate addition, which has been shown for both sulfate-reducers and denitrifiers, the putative carboxylation of n-alkanes has been proposed only for the sulfate-reducing isolate strain Hxd3 (25) and for a sulfate-reducing consortium (4). Experiments using NaH¹³CO₃ demonstrated that bicarbonate serves as the source of inorganic carbon for the putative carboxylation reaction (25). Subterminal carboxylation of the alkane at the C-3 position is followed by elimination of the two terminal carbons, to yield a fatty acid that is one carbon shorter than the parent alkane (4, 25). The fatty acids are subject to β-oxidation, chain elongation, and/or C-10 methylation (25).In this study, we characterized an alkane-degrading, nitrate-reducing consortium and surveyed the metabolites of the consortium incubated with either unlabeled or labeled hexadecane in order to elucidate the pathway of n-alkane degradation. We present evidence of a pathway analogous to the proposed carboxylation pathway under nitrate-reducing conditions.     

The Oxidation of Malate by Isolated Turnip (Brassica napus L.) Mitochondria: I. ULTRASTRUCTURAL OBSERVATIONS AND THE PRODUCTS OF MALATE OXIDATION

Washed and purified turnip mitochondria oxidize malate withrespiratory control and ADP: O values approaching 3.0 and producepyruvate as the principal product. Oxaloacetate is also producedin significant amounts but is removed by an endogenous mechanism.Malate dehydrogenase appears to be important to the oxidationof malate but requires the removal of oxaloacetate. During malateoxidation the mitochondria undergo configurational changes similarto those observed in animal mitochondria. Both ‘rightside-out’ and ‘inside-out’ submitochondrialparticles have been prepared. Right side-out particles oxidizemalate in the same way as intact mitochondria, whereas the inside-outparticles have a biphasic oxidation, the first phase producingoxaloacetate and the second, NAD-requiring phase producing pyruvate.It is concluded that malate oxidation is a complex process usingseveral enzymes located in the matrix compartment with a minorcomponent possibly on the outer face of the inner membrane.     

Oxidation of p-cresol by horseradish peroxidase compound I.

Rate constants for the reaction between horseradish peroxidase compound I and p-cresol have been determined at several values of pH between 2.98 and 10.81. These rate constants were used to construct a log (rate) versus pH profile from which it is readily seen that the most reactive form of the enzyme is its most basic form within this pH range so that base catalysis is occurring. At the maximum rate a second order rate constant of (5.1 +/- 0.3) x 10(-7) M-1 s-1 at 25 degrees is obtained. The activation energy of the reaction at the maximum rate was determined from an Arrhenius plot to be 5.0 +/- 0.5 kcal/mol. Evidence for an exception to the generally accepted enzymatic cycle of horseradish peroxidase is presented. One-half molar equivalent of p-cresol can convert compound I quantitatively to compound II at high pH, whereas usually this step requires 1 molar equivalent of reductant. The stoichiometry of this reaction is pH-dependent.     

Oxidation of L-glucose by a Pseudomonad.

A new enzyme, D-threo-aldolse dehydrogenase (2S,3R-aldose dehydrogenase), found in Pseudomonas caryophylli, was capable of oxidizing L-glucose L-xylose, D-arabinose, and L-fucose in the presence of NAD+. The enzyme was synthesized constitutively and purified about 120-fold from D-glucose-grown cells. The Km values for L-glucose, L-xylose, D-arabinose, and L-fucose were 1.5 . 10(-2), 4.5 . 10(-3), 2.8 . 10(-3), and 2.1 . 10(-3), respectively. D-glucose and other aldoses inhibited the enzyme reaction; this inhibition was competitive with L-glucose as substrate and D-glucose as inhibitor. The optimum pH for the enzyme reaction was 10; the molecular weight of the enzyme was determined by gel filtration to be 7 . 10(4).     

Oxidation of melatonin by oxoferryl hemoglobin: a mechanistic study.

Reaction of melatonin with the hypervalent iron centre of oxoferryl hemoglobin, produced in aqueous solution from methemoglobin and H2O2, has been investigated at 37 degrees C and pH 7.4, by absorption spectroscopy. The reaction results in reduction of the oxoferryl moiety with formation of a heme-ferric containing hemoprotein. Stopped-flow spectrophotometric measurements provide evidence that the reduction of oxoferryl-Hb by melatonin is first-order in oxoferryl-Hb and first-order in melatonin. The bimolecular reaction constant at pH 7.4 and 37 degrees C is 112 +/- 1.0 M(-1) s(-1). Two major oxidation products from melatonin have been found by gas chromatography-mass spectroscopy: the cyclic compound 1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole (cyclic 3-hydroxy-melatonin), and N-acetyl-N'-formyl 5-methoxykynuramine (AFMK). The percentage yield of the two major products appears dependent on the ratio [oxoferryl-Hb]:[melatonin]--the higher the ratio the higher the yield of AFMK. The observed stoichiometry oxoferryl-Hb(reduced):melatonin(consumed) is 2, when the ratio [oxoferryl-Hb]:[melatonin] is 1:1, but appears >2 at higher molar ratios. The reduction of the hypervalent iron of the oxoferryl moiety may be consistent with an oxidation of melatonin by two one-electron steps.     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM: I. OXIDATION OF SULFUR

1. Thiobacillus thiooxidans, isolated in our laboratory, was foundto oxidize sulfur, but not thiosulfate. Tetrathionate is alsooxidized slightly. Its ability to oxidize sulfur is inactivatedeven by such a mild treatment as keeping the cells in a frozenstate.
2. Inhibitory action of alcohols on the sulfur oxidationincreasesas the length of carbon chain of alcohols increases.Carboxylicacids do not inhibit the sulfur oxidation at pH abovetheirpK, while they strongly inhibit the reaction at pH belowthepK.
3. The sulfur oxidation is inhibited by cyanide, azide,diethyldithiocarbamateand carbon monoxide, and the inhibitionby carbon monoxide isnot reversed by light. These results suggestthe presence ofmetal enzymes in the sulfur oxidation system.The terminal enzymeof this reaction appears to be differentfrom the usual cytochromeoxidase.

(Received May 13, 1960; )     

BASAL BODIES OF BACTERIAL FLAGELLA IN PROTEUS MIRABILIS : I. Electron Microscopy of Sectioned Material

Years ago (16, 18, 19), in a study of shadowed preparations of Proteus vulgaris that had been autolyzed in the cold, the observation was made that the flagella arose from basal bodies. However, recently (3, 7, 24, 33) doubt has been cast on the conclusion that the flagella of bacteria emerge from sizable basal bodies. This problem has, therefore, been reinvestigated with actively developing cultures of Proteus mirabilis, the cell walls of which had been expanded slightly by exposure to penicillin. Two techniques were applied: ultramicrotomy, and negative staining of whole mount preparations. This paper deals with the thin sections of bacteria after the usual fixation technique had been altered slightly: the cells were embedded in agar prior to their fixation and further processing. The flagella then remained attached to the cells and were seen to extend between the cell wall and the plasma membrane. Occasionally, the flagella appeared to be anchored in the cell by means of a hook-shaped ending. In sections of cells rich in cytoplasm, the basal bodies are particularly difficult to visualize due to their small size (25 to 45 mµ) and the lack of properties that would enable one to distinguish them from the ribonucleoprotein structures; in addition, their boundary appears to be delicate. However, when the cytoplasm is sparse in the cells, either naturally or as a result of osmotic shocking in distilled water, the flagella can be observed to emerge from rounded structures approximately 25 to 45 mµ wide. Contrary to a previous suggestion (21), the flagella do not terminate in the peripheral sites of reduced tellurite, i.e. the chondrioids. The observations in this part of the study agree with those described in the following paper (15) dealing with negatively stained preparations.     

FATTY ACID METABOLISM IN SERRATIA MARCESCENS I. : Oxidation of Saturated Fatty Acids by Whole Cells.

Bishop, D. G. (University of Sydney, Sydney, Australia), and J. L. Still. Fatty acid metabolism in Serratia marcescens. J. Bacteriol. 82:370-375. 1961.-A study has been made of the oxidation of saturated fatty acids containing between two and 18 carbon atoms by whole cells of the bacterium, Serratia marcescens. This organism was found to be capable of oxidizing all of the acids tested, but variations in the rate and total oxygen uptake were found. These variations were dependent on the length of the carbon chain in the substrate molecule and the pH of the reaction mixture. The concept that these variations are due to a cellular permeability barrier to the substrate is discussed.     

Oxidation of C1 compounds by Pseudomonas sp. MS

Pseudomonas sp. MS is capable of growth on a number of compounds containing only C₁ groups. They include trimethylsulphonium salts, methylamine, dimethylamine and trimethylamine. Although formaldehyde and formate will not support growth they are rapidly oxidized by intact cells. Methanol neither supports growth nor is oxidized. A particulate fraction of the cell oxidizes methylamine to carbon dioxide in the absence of any external electron acceptor. Formaldehyde and formate are more slowly oxidized to carbon dioxide by the particulate fraction, although they do not appear to be free intermediates in the oxidation of methylamine. Soluble NAD-linked formaldehyde dehydrogenase and formate dehydrogenase are also present. The particulate methylamine oxidase is induced by growth on methylamine, dimethylamine and trimethylamine, whereas the soluble formaldehyde dehydrogenase and formate dehydrogenase are induced by trimethylsulphonium nitrate as well as the aforementioned amines.     

Role of Alcoholic Intermediates in Formation of Isomeric Ketones From n-Hexadecane by a Soil Arthrobacter

A soil Arthrobacter species isolated from an Oregon soil was capable of transforming n-hexadecane to a series of ketonic products, the 2-,3-, and 4-hexadecanones, with evidence for accumulation of 2- and 3-hexadecanols as oxidative intermediates when yeast extract or peptone was used as a growth substrate. The accumulation and participation of internal alcohols in this type of hydrocarbon transformation has not been previously reported. In the absence of yeast extract or peptone, growth from low-level inocula was not observed when n-hexadecane or two oxidation products, 2-hexadecanol and 3-hexadecanone, were used as substrates. However, washed resting cell suspensions of the organism transformed 2-hexadecanol, or a mixture of 2-,3-, and 4-hexadecanols, to the corresponding ketones without lag, indicating the possible constitutive nature of the alcohol dehydrogenase enzyme(s) carrying out this reaction. The addition of glucose to these resting cells stimulated transformation of n-hexadecane to alcoholic and ketonic oxidation products. Formation of isomeric internal alcohols appears to be a limiting step in ketone formation by this Arthrobacter isolate.