Richard S. Hanson

Formation of a factor lethal for S. Typhimurium TA1530 and TA1531 on incubation of aflatoxin B1 with rat liver microsomes

Abstract Strains TA1530 and TA1531, but not strains C207 and G46, of S. typhimurium (1) were killed when incubated with rat liver homogenate and aflatoxin B 1 but not with either component alone. The lethal effect required the microsomal fraction of liver, was dependent on O 2 and NADPH, and was inhibited by the addition of aniline, SKF-525A, or CO. Less of the lethal factor was formed with liver preparations from rats fed a low protein diet, and more was formed if the rats were administered phenobarbital. Aflatoxin M 1 (4-hydroxy aflatoxin B 1 ) was not toxic for these bacterial strains in the absence of liver preparations and was much less toxic than aflatoxin B 1 when incubated with rat liver.

Coarse and fine control of citrate synthase from Bacillus subtilis

Abstract The synthesis of citrate synthase (citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7) from Bacillus subtilis was found to respond to a dual control: A specific feedback repression caused by glutamate (or α-ketoglutarate), the biosynthetic end product of the first half of the tricarboxylic acid cycle, and a more general catabolite repression resulting from the accumulation of catabolic intermediates when cells were grown on glucose, a readily utilizable carbon source. The synthesis of aconitate hydratase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3, formerly known as aconitase) from this organism is sensitive to similar metabolic signals 1 . Because the kinetic patterns of repression and depression of the two enzymes are similar, and since a plot of the specific activities of aconitate hydratase vs. those of citrate synthase from B. subtilis cultures grown under differing nutritional conditions results in a straight line, the suggestion is made that citrate synthase and aconitate hydratase may be subject to coordinate regulation. Kinetic studies on a partially purified enzyme preparation show ATP to be an inhibitor of citrate synthase. ATP inhibition is competitive with CoASAc but not oxaloacetate. The results of this investigation lead to the conclusion that while the synthesis of citrate synthase is sensitive to both anabolic and catabolic signals, once the enzyme is formed, its activity is regulated primarily by catabolic effectors.

Enzyme Regulation in Methylobacterium organophilum

Summary: Several enzymes have been assayed in Methylobacterium organophilum grown on different substrates. The enzymes which are involved in growth on C1 compounds were induced by methanol and not repressed by succinate. When succinate-grown bacteria were resuspended in medium containing methanol, four enzymes unique to growth on C1 compounds (hydroxypyruvate reductase, serine-glyoxylate aminotransferase, methanol dehydrogenase and glycerate kinase) were fully induced by the time growth began. When methanol-grown bacteria were resuspended in medium containing succinate, all four enzyme activities decreased. Several mutants unable to grow on C1 compounds were examined for deficiencies in the enzymes specific for growth on these compounds. Seven of the mutants were pleiotropic, and six were not revertible by chemical mutagens, suggesting the possibility of genetic linkage or the presence of a regulon for the genes involved in C1 metabolism.

Regulation of isocitrate dehydrogenase from Thiobacillus thiooxidans and Pseudomonas fluorescens

Abstract The isocitrate dehydrogenases of T. thiooxidans and P. fluorescens have been studied. The apparent KMs for isocitrate are 1.2 × 10−5 M and 1.5 × 10−5 M respectively. The apparent KM for NAD+ is 2.9 × 10−4 M for the enzyme from T. thiooxidans while the apparent KM for the NADP-specific enzyme of P. fluorescens is 1.7 × 10−5 M. ADP and ATP were found to inhibit the isocitrate dehydrogenases of both organisms. Glyoxalate plus oxalacetate caused concerted inhibition of the enzyme from T. thiooxidans. Glyoxalate, when added alone, was an activator of the enzyme from P. fluorescens but increased the inhibition due to oxalacetate and reduced the KI for oxalacetate by three orders of magnitude.

Regulation of the tricarboxylic acid cycle in bacteria a comparison of citrate synthases from different bacteria

Abstract Citrate synthase (citrate oxalacetate-lyase (CoA-acetylating), EC 4.1.3.7) from Azotobacter vinelandii, Salmonella typhimurium, Rhodospirillum rubrum, Pseudomonas fluorescens and Corynebacterium lilium were examined in partially purified extracts. The enzymes differed in several respects including K m for substrates, stability, and response to modulators of activity (cations, ATP, AMP, NADH, and α-ketoglutarate). Citrate synthase from A. vinelandii was found to be activated by AMP and inhibited by ATP and NADH. The enzyme from R. rubrum was inhibited by ATP and that from S. typhimurium was inhibited by ATP and NADH. NADH inhibition was relieved by AMP. The activity of the enzyme from C. lilium , a glutamate excretor, was weakly inhibited by ATP and NADH. A comparison of the modulation of the activities of citrate synthase and from eight microorganisms representing different physiological groups is presented. It is indicated that the control of citrate synthase differs in organisms that are physiologically diverse. The regulation of isocitrate dehydrogenase, on the other hand, is similar in unrelated bacteria.

Bacterial citrate synthases: Purification, molecular weight and kinetic mechanism

Abstract Citrate synthase (citrate oxaloacetate-lyase, CoA-acetylating, EC 4.1.3.7) from cells of Azotobacter vinelandii and Acinetobacter anitratum was purified to apparent homogeneity. The molecular weight of native citrate synthase from A. anitratum was determined to be 242 000, and the enzyme from A. vinelandii had a molecular weight of 250 000. The subunit molecular weights of A. vinelandii and A. anitratum citrate synthases appeared identical. Each had a monomeric molecular weight, determined by sedimentation equilibrium studies, of 58 500–59 000. Purification of citrate synthase from Bacillus subtilis was hampered by low specific activity levels and poor enzyme stability and was not purified to homogeneity. The molecular weight of the active enzyme was determined to be 60 000–95 000 by acrylamide gel electrophoresis. The patterns observed from initial velocity and product inhibition studies of citrate synthase catalysis are consistent with those predicted for a random sequential reaction order mechanism. Extensively purified citrate synthase from each of the three genera studied gave similar kinetic patterns, despite variations in enzyme size and regulatory complexity.

Catabolite repression of aconitate hydratase in Bacillus subtilis

Abstract The synthesis of aconitate hydratase (citrate(isocitrate) hydro-lyase, EC 4.2.1.3) in Bacillus subtilis 168 is released from catabolite repression during nutrient-limited growth on media deficient in phosphate or nitrogen. Sulfate and tryptophan limitation lead to increased repression. The rate-limiting addition of adenosine increases the differential rate of aconitate hydratase synthesis in cultures of an adenine auxotroph of B. subtilis. The addition of adenosine to cultures gowing under nitrogen limitation causes a prolonged and complete repression of aconitate hydratase synthesis. It is concluded that restriction of anabolism itself does not cause an increase in catabolite repression in B. subtilis 168. The response of enzyme synthesis to nutrient deprivation depends on the nutrient limited. The data suggest that the effector of catabolite repression of aconitate hydratase is metabolically related to adenosine triphosphate.

LIGHT-ACTIVATED HYDROGENASE IN RHODOSPIRILLUM RUBRUM.

Abstract Reduction of 37.5 mM potassium ferricyanide by hydrogen with washed suspensions of Rhodospirillum rubrum cells in phosphate buffer requires light when calcium is added. Washed cells have little hydrogenase activity in Tris buffer, but they are activated by divalent metal-complexing anions, cadmium, zinc, and detergents. Light activation of hydrogenase activity in calcium-treated cells is inhibited by carbonyl-cyanide m-chlorophenylhydrazone and carbonylcyanide p-trifluoromethoxyphenyl-hydrazone. The reduction of 2,6-dichlorophenolindophenol and methylebe blue by hydrogen is not affected by these agents and occurs without the addition of anions. Evidence is presented which indicates that calcium causes structural changes in the cell membrane to render it impermeable to ferricyanide and that energy derived from photophosphorylation can alter membrane permeability to overcome calcium inhibition.

DORMANT AND RESISTANT STAGES OF PROCARYOTIC CELLS

Bacteria that form resting stages are the most probable survivors of probes to other planets, because of their ability to survive long periods of nutrient deprivation, and their resistance to temperature fluctuations, desiccation, irradiations, and mechanical stress. Resting stages, other than endospores, are formed by a variety of procaryotic organisms, including photosynthetic bacteria, methane oxidizing bacteria, and heterotrophic procaryotes. The various dormant cell types are compared. Dormancy is not as intense, and resistance to stresses is generally not as great as in exospores, myxospores, and cysts as it is in endospores. Resistance and dormancy of resting cells are dependent on growth conditions and these properties have not been well studied in recently discovered resting cells. An understanding of the variety and properties of resistant dormant stages of procaryotes is important in considering the possibility of inoculating extraterrestrial environments by bacteria on probes as well as in designing experiments to detect primitive organisms that may have evolved in extraterrestrial environments.