A Cytochemical Localization of Reductive Sites in a Gram-Negative Bacterium

Iterson, Woutera van; Leene, W.

doi:10.1083/jcb.20.3.377

Cited by 63 publications

(26 citation statements)

References 14 publications

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“…Bacterial cells resistant to tellurite often turn black when grown in liquid or solid media amended with K 2 TeO 3 , a phenotype that is also displayed by sensitive bacteria growing in media supplemented with sub-lethal concentrations of tellurite [18]. These deposits are of an insoluble material localized to specific subcellular compartments [19], [20], which X-ray diffraction studies have shown to be the less toxic, free metal, tellurium (Te°) [21], [22].…”

Section: Introductionmentioning

confidence: 99%

Catalases Are NAD(P)H-Dependent Tellurite Reductases

et al. 2006

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Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO3 2−) to the less toxic, insoluble metal, tellurium (Te°), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

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Section: Introductionmentioning

confidence: 99%

Catalases Are NAD(P)H-Dependent Tellurite Reductases

et al. 2006

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“…A number of similar experiments concerning dehydrogenase localization have been carried out on bacterial cells (2,6,9,14,15,16,18,19). A relationship between esterases and structures of the bacterial cell has not been studied by electron microscopy.…”

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confidence: 96%

Electron Microscopy of Alkaline Phosphatase of Escherichia Coli

Kushnarev¹,

Смирнова²

1966

Can. J. Microbiol.

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A method is described for determining the localization of alkaline phosphatase in the cells of E. coli B with the electron microscope. Enzyme activity, determined by deposition of inorganic phosphate, is located in the exterior layer of the cell wall.Electron microscopy is of great importance in the study of the relationship of enzymes and cell ultrastructures and this application has permitted the localization of some esterases and dehydrogenases (1,4,11). A number of similar experiments concerning dehydrogenase localization have been carried out on bacterial cells (2,6,9,14,15,16,18,19). A relationship between esterases and structures of the bacterial cell has not been studied by electron microscopy.The present paper concerns the distribution of alkaline phosphatase in the cells of Escherichia coli B. Materials a n d MethodsThe cells used were E . coli B. An 18-hour culture of E. coli B on nutrient agar was inoculated into a salts medium having limited inorganic phosphate (17) and grown for 24 to 48 hours. All the salts were recrystallized. Sodium P-glycerophosphate for determination of phosphatase was obtained from the Merck Company. No trace of free orthophosphate was found in this preparation.Coloration.-After growth, the cells were centrifuged and placed in a glycerophosphate medium (3) a t pH 9.0. The incubation was continued a t 37 OC for 10 minutes to 2 hours. After that the cells were again centrifuged and suspended in a 2y0 cobalt nitrate soiution, b u t in some experiments, suspension in cobalt solution was omitted. After 2-3 minutes the cell suspension in cobalt solution was centrifuged a t 3000 r.p.m. for 3 minutes and the supernatant was placed on formvar-coated grids for electron microscopy.Ultrathin sections.-The material obtained after cobalt nitrate treatment was fixed according to Ryter et al. (13), dehydrated in alcohols, and embedded in the mixture of butyl-and methyl-metacrylates (4: 1). The polymerization was performed a t 55' for 24-48 hours. The sections were obtained on the "Ultratome 4800" with glass knives and collected on formvar-covered copper grids strengthened with carbon. The material obtained was contrasted in 5yo uranyl acetate solution a t room temperature for 1 hour.Electron microscopy.-The preparations of whole cells and sections were looked over in UEMV-100B electron microscope a t 50 and 75 kV with a 40-p objective and recorded with photoplates MR. Electron optical magnification, 8000-62,000 X.

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“…Many microorganisms produce black intracellular deposits when grown in media supplemented with potassium tellurite (14,15,23,25,(28)(29)(30)(31). Even sensitive bacteria are capable of forming jet-black colonies when growing at subinhibitory tellurite concentrations (25).…”

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confidence: 99%

Purification and biochemical characterization of tellurite-reducing activities from Thermus thermophilus HB8

et al. 1988

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Cell-free extracts of Thermus thermophilus HB8 catalyze the in vitro, NADH-dependent reduction of potassium teilurite (K2TeO3). Three different protein fractions with tellurite-reducing activities were identified.Two exhibited high molecular weight and were composed of at least two different polypeptides. The protein in the third fraction was purified to homogeneity and had a single polypeptide chain of 53 to 54 kilodaltons, with an isoelectric point of 8.1. Each enzyme was thermostable, the temperature optimum was 75°C, and 30 mM NaCl, 1.5 M urea, or 0.004% sodium dodecyl sulfate caused 50% inhibition of the enzymes. However, 2% Triton X-100 did not have an inhibitory effect. The enzymes were also able to catalyze the reduction of sodium selenite and sodium sulfite in vitro. NADH was replaceable by NADPH. Divalent cations, such as Ca2' and

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A Cytochemical Localization of Reductive Sites in a Gram-Negative Bacterium

Cited by 63 publications

References 14 publications

Catalases Are NAD(P)H-Dependent Tellurite Reductases

Catalases Are NAD(P)H-Dependent Tellurite Reductases

Electron Microscopy of Alkaline Phosphatase of Escherichia Coli

Purification and biochemical characterization of tellurite-reducing activities from Thermus thermophilus HB8

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