Stickland Reaction Research Articles

Lindane degradation by cell-free extracts of Clostridium rectum.

For lindane degradation, a cell suspension of Clostridium rectum strain S-17 demands the addition of substrates such as leucine, alanine, pyruvate, a leucine-proline mixture, and molecular hydrogen. In the presence of leucine-proline mixture, lindane decomposed in parallel with isovaleric acid formation, and both lindane degradation and isovaleric acid formation were inhibited by monoiodoacetic acid, suggesting a close relation between lindane degradation and the Stickland reaction. Lindane was degraded by cell-free extracts of C. rectum in the presence of dithiothreitol (DTT). Radiogaschromatograms of n-hexane soluble metabolites from [14C] lindane showed the presence of monochlorobenzene and gamma-3,4,5,6-tetrachlorocyclohexene. Leucine, NADH, and NADPH were somewhat less active than DTT for lindane degradation in cell-free extracts. Reductive dechlorination seemed the major route of lindane degradation in cell-free extracts as well as in the intact cells of C. rectum.

Selenium requirement for the growth of Clostridium sporogenes with glycine as the oxidant in stickland reaction systems.

Clostridium sporogenes was found to have an absolute requirement for selenium to utilize glycine but not proline as oxidant in Stickland-type fermentations. No glycine reductase activity was detectable in cells from media without added selenium. The data indicate that this organism could be used for microbiological assays for very low levels of selenium in certain forms.

Studies on the bacteria isolated anaerobically from paddy field soil III. Production of fatty acids and ammonia byClostridiumspecies

Abstract The volatile fatty acids produced by the representative strains of clostridia isolated from paddy field soil were surveyed. All the strains, except Cl. tertium, utilized only amino acids as the sole source of energy for growth. All of them produced acetic and butyric acids, and some of them produced propionic acid in addition. Iso-valeric and iso-butyric acids were also produced, except by Cl. tertium. These results suggest that all the strains, except Cl. tertium, may carry out the STICKLAND reaction. The cells of each strain harvested from the culture in the VL medium were subjected to examination whether or not they carried out the STICKLAND reaction in the presence of leucine or glycine. All the strains, except Cl. tertium, catalyzed the STICKLAND reaction and some strains also metabolized leucine and glycine even when they were supplied singly.

魚類食品におけるボツリヌスE型菌の発育と毒素産生に関する研究-III

In order to establish the chemical nature of volatile acidic odors produced by Clostridium botulinum during growth, volatile fatty acids were determined by gas chormatography in the cultures of cooked meat broth inoculated with each strain of the six toxin-types. The results were as follows: 1. Acetic, propionic, n-butyric, iso-butyric, n-valeric, iso-valeric (iso-valeric and/or active α-methyl-butyric), and iso-caproic acids were identified in the cultures of type A, B and F. The branched acids except iso-caproic acid were deduced to be the metabolic products derived from some branched amino-acids by “Stickland reaction”. 2. Acetic, propionic, and n-butyric acids were identified in both the cultures of types Cβ and D. A high proportion of propionic acid among the resulting fatty acids suggests that these organisms appear to obtain some of their energy by propionic acid fermentation similar to that displayed by propionibacteria. 3. n-Butyric acid, in higher proportion, as well as acetic acid were identified in the cultures of type E, suggesting that this organism obtains most of its energy by butyric acid fermentation similar to that displayed by butyric acid bacteria. Further the chemical nature of the off-odor of foodstuffs contaminated with botulinal toxin was examined. An abnormally high proportion of n-butyric acid, being responsible for the “butyric” off-odor, was found in home-made “Izushi” of raw fish and rice cake, which had caused an episode of human type E botulism. Also a high proportion of propionic acid, as well as acetic and n-butyric acids were found in raw whale meat, which had caused an outbreak of type Cβ botulism in mink. Accordingly, the patterns of the volatile fatty acids presented by each type of Clostridium botulinum seem to be reflected in that of foodstuffs contaminated with respective type of the organisms.

PHYSIOLOGY OF THE SPORULATION PROCESS IN CLOSTRIDIUM BOTULINUM. II. MATURATION OF FORESPORES.

Day, Lawrence E., (Michigan State University, East Lansing) and Ralph N. Costilow. Physiology of the sporulation process in Clostridium botulinum. II. Maturation of forespores. J. Bacteriol. 88:695-701. 1964.-Clostridium botulinum, strain 62-A, did not sporulate endotrophically, but forespores matured to refractile, heat-resistant spores when replaced in solutions containing l-alanine and l-proline, l-isoleucine and l-proline, or l-alanine and l-arginine. Solutions of l-arginine or l-citrulline would not support the maturation process. Acetate, CO(2), and delta-amino valeric acid were produced during sporulation in a replacement solution of l-alanine and l-proline, indicating the operation of the Stickland reaction. There was no large uptake of either exogenous l-alanine or acetate during this process. Similarly, there was no apparent protein or nucleic acid synthesis, since high levels of chloramphenicol, 8-azaguanine, or mitomycin C failed to inhibit, and no significant amount of P(32) was incorporated into the spore nucleic acids. Dipicolinic acid (DPA) was synthesized during forespore maturation. It is believed that these final steps in sporulation of C. botulinum require only an exogenous source of energy which can be obtained via the Stickland reaction system, and that the synthesis of DPA and other unknown materials relies primarily on endogenous substrates.

Fermentative activities of control and radiation-"killed" spores of Clostridium botulinum.

Costilow, Ralph N. (Michigan State University, East Lansing). Fermentative activities of control and radiation-"killed" spores of Clostridium botulinum. J. Bacteriol. 84:1268-1273. 1962.-Spores of Clostridium botulinum 62-A exposed to high levels of ionizing radiation will undergo the initial steps in germination as rapidly as control spores. Manometric experiments demonstrated that these irradiated nonviable spores would ferment a tryptic digest of casein (Trypticase) at an increasing rate during germination, with the production of CO(2) and H(2). There was, however, an increasing lag in the development of rapid rates of fermentation as the radiation level was increased. Such a lag could be simulated by the addition of chloramphenicol, and the two effects were additive. By using high spore concentrations (about 8 mg/ml), it was possible to detect the fermentation of a number of individual amino acids and of glucose. Irradiation of spores at 8.3 x 10(5) rads had no measurable effect on the fermentation of l-arginine or glucose, or on the endogenous rate, but it partially inhibited the Stickland reaction with l-alanine and l-proline as substrates. The endogenous activity and the l-alanine, l-proline fermentation were also inhibited by chloramphenicol to basic levels which were the same in both control and irradiated spores. Neither the l-arginine nor glucose fermentation was inhibited by this antibiotic. The data indicate that spores of C. botulinum 62-A contain basic levels of the enzymes required for their primary catabolic processes, and that, as expected, ionizing radiation levels sufficient to render the spores nonviable do not significantly affect these enzymes. Furthermore, it is indicated that spores exposed to radiation levels of up to 1.25 x 10(6) rads retain the ability to synthesize new protein, although it appears to be somewhat impaired.

The Stickland reaction.

Introduction 16 Stickland reaction studied with cell suspensions 16 L-Amino acid dehydrogenase system 21 a-Keto acid dehydrogenase system 29 Acceptor amino acid reducing system 37

THE STICKLAND REACTION

Introduction 16 Stickland reaction studied with cell suspensions 16 L-Amino acid dehydrogenase system 21 a-Keto acid dehydrogenase system 29 Acceptor amino acid reducing system 37

Utilization of Amino-Acids by Clostridium botulinum.

Our knowledge of the metabolism of Clostridium botulinum is at present scanty. Wagner, Meyer and Dozier analyzed the products of the action of Cl. botulinum on complex media but obtained little information as to the reactions by which the products had been formed. Knight, summarizing the growth-requirements of Cl. botulinum, reports that the simplest medium which will support growth contains the amino-acids proline, glycine, leucine, alanine, lysine, and cystine, together probably with traces of tryptophan and of the “sporogenes growth factor.” He suggests that Cl. botulinum may obtain its energy through the “Stickland reaction,” a coupled oxido-reduction between pairs of amino-acids. Glycine and proline act as hydrogen acceptors while alanine and leucine act as hydrogen donators in the case of Cl. sporogenes (Stickland). Therefore these amino-acids were tested to determine whether or not they are attacked directly as is true for Cl. tetanomorphum with various amino-acids (Woods and Clifton) or by coupled reactions between pairs of amino-acids. All the experiments reported below were carried out with washed suspensions of Cl. botulinum, Type B (E-44). 800 ml of plain broth plus 0.1% yeast extract were inoculated with 0.5 ml of a beef-brain culture of this organism and incubated for 20 hours at 37°C in a McIntosh and Fildes anaërobic jar. The culture was then centrifuged, the cells were washed in saline and finally suspended in phosphate buffer of pH 7.4. The suspension so prepared was immediately deaërated in vacuo. The usual Thunberg-tube method was employed for the detection of hydrogen donators and it was found that alanine and leucine were the only amino-acids of those essential for growth which reduced methylene blue readily.