Rapid Method for Detection and Quantitation of Hydroxylated Aromatic Intermediates Produced by Microorganisms

Wackett, Lawrence P.; Gibson, David T.

doi:10.1128/aem.45.3.1144-1147.1983

Cited by 30 publications

(33 citation statements)

References 20 publications

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“…A 100-l aliquot of 0.2% (wt/vol) tetrazotized o-dianisidine was added to 1 ml of a cell suspension that had been incubated with naphthalene. An extinction coefficient of 38,000 M Ϫ1 cm Ϫ1 for the naphthol-diazo dye was used for calibration (34). This coefficient was within 5% of the coefficients calculated from standard curves obtained with 1and 2-naphthols in this study.…”

Section: Methodssupporting

confidence: 71%

Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources

Chu

Alvarez‐Cohen

1999

Appl Environ Microbiol

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In this study we evaluated specific and nonspecific toxic effects of aeration and trichloroethylene (TCE) oxidation on methanotrophic bacteria grown with different nitrogen sources (nitrate, ammonia, and molecular nitrogen). The specific toxic effects, exerted directly on soluble methane monooxygenase (sMMO), were evaluated by comparing changes in methane uptake rates and naphthalene oxidation rates following aeration and/or TCE oxidation. Nonspecific toxic effects, defined as general cellular damage, were examined by using a combination of epifluorescent cellular stains to measure viable cell numbers based on respiratory activity and measuring formate oxidation activities following aeration and TCE transformation. Our results suggest that aeration damages predominantly sMMO rather than other general cellular components, whereas TCE oxidation exerts a broad range of toxic effects that damage both specific and nonspecific cellular functions. TCE oxidation caused sMMO-catalyzed activity and respiratory activity to decrease linearly with the amount of substrate degraded. Severe TCE oxidation toxicity resulted in total cessation of the methane, naphthalene, and formate oxidation activities and a 95% decrease in the respiratory activity of methanotrophs. The failure of cells to recover even after 7 days of incubation with methane suggests that cellular recovery following severe TCE product toxicity is not always possible. Our evidence suggests that generation of greater amounts of sMMO per cell due to nitrogen fixation may be responsible for enhanced TCE oxidation activities of nitrogen-fixing methanotrophs rather than enzymatic protection mechanisms associated with the nitrogenase enzymes.

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Section: Methodssupporting

confidence: 71%

Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources

Chu

Alvarez‐Cohen

1999

Appl Environ Microbiol

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“…The false-positives that were obtained in the initial screening steps are easily dismissed; the original plates were crowded and color development was extremely weak. It is possible that color formation was a consequence of dye reacting with other metabolites present (20). The frequency with which organisms grew without copper and without a naphthalene-degrading sMMO was unexpected and cannot be explained satisfactorily without a detailed look at the taxonomy of the methanotrophic isolates.…”

Section: Resultsmentioning

confidence: 99%

“…The procedure may have utility for (i) the isolation and enumeration of sMMO-bearing strains and (ii) the selection of mutant strains that can express sMMO in the presence of comparatively high Cu(II) concentrations. The procedure is an application of a method developed by Wackett and Gibson (20) for assessing monooxygenase activity in fungi and bacteria. It is based on observations that (i) some bacterial monooxygenases catalyze the conversion of naphthalene to 1-naphthol and (ii) naphthol formation can be monitored colorimetrically by the addition of certain aromatic diazo compounds to the reaction mixture (13).…”

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confidence: 99%

Applications of a colorimetric plate assay for soluble methane monooxygenase activity

Graham

Korich

LeBlanc

et al. 1992

Appl Environ Microbiol

100

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A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration ofMethylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications. Methanotrophic bacteria are capable of initiating the degradation of a variety of environmental pollutants including trichloroethylene, the isomers of dichloroethylene, vinyl chloride, and chloroform (9, 11, 12, 21). This phenomenon results from the broad substrate specificity of the initial enzyme in the pathway for methane catabolism, methane monooxygenase (MMO) (2, 12, 17). Methanotrophs possess two types of MMO, a soluble MMO (sMMO) and a particulate, membrane-bound MMO (pMMO). sMMO is considerably more effective than pMMO at catalyzing such nonenergy-yielding transformations; however, sMMO is expressed only in type II (and type X) methanotrophs that have been grown under severely copper-limited conditions (18, 19). Conversely, type I organisms appear to express only pMMO (6). Methylosinus trichosporium OB3b is the best studied of the type II methanotrophs (1, 2, 4, 12, 18). The sMMO of M.

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“…Estos microorganismos son ubicuos, habitan en el límite entre ambientes aeróbicos y anaeróbicos, donde metano y oxígeno están disponibles, jugando un papel esencial en el ciclo global del carbono al limitar la cantidad de metano que es liberado hacia la atmósfera (Hanson & Wattenburg 1991;Hanson & Hanson 1996). No obstante, los metanótrofos también son capaces de degradar un gran número de compuestos orgánicos considerados contaminantes, incluyendo compuestos halogenados de bajo peso molecular como el di y tri cloroetileno, di y tri cloroetano, cloruro de vinilo, cloroformo y diclorometano, entre otros (Wackett & Gibson 1983;Fogel et al 1986;Green & Dalton 1989;Brusseau et al 1990;Alvarez-Cohen & McCarty 1991;Koh et al 1994). La primera etapa de la oxidación de metano es llevada a cabo por la enzima metano monooxigenasa (MMO) que cataliza la hidroxilación de la molécula de metano a metanol, aumentando así su solubilidad.…”

Section: Introductionunclassified

Actividad enzimática de metanótrofos marinos y su uso potencial en biorremediación

2011

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Los metanótrofos son microorganismos con un importante rol en la biogeoquímica del metano en el océano. La enzima metano monooxigenasa soluble (MMOs) es la encargada de realizar la primera oxidación catabólica del metano, pero además es activa sobre compuestos aromáticos policíclicos y una gran variedad de substancias derivadas del petróleo consideradas como contaminantes. Muestras de sedimento fueron incubadas en medio líquido específi co con sales minerales de nitrato (NMS) y en presencia de metano como única fuente de carbono para determinar la actividad de MMOs de la comunidad de metanotrófos presentes en esta matriz ambiental. Las muestras fueron obtenidas desde un área con una emanación visible de metano (Isla Mocha) y de dos áreas sin emanación (Bahía Coronel y Golfo de Arauco). Los microorganismos incubados presentes en los sedimentos superfi ciales de Isla Mocha poseen una actividad de MMOs dos veces mayor en comparación a las actividades obtenidas en cultivos realizados en las mismas condiciones pero utilizando muestras recolectadas desde áreas sin emanación. Se observó además una relación inversa entre la distribución vertical

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Rapid Method for Detection and Quantitation of Hydroxylated Aromatic Intermediates Produced by Microorganisms

Abstract: Tetrazotized o-dianisidine was used for quantitative measurements of the amount of 1-naphthol formed from naphthalene by fungi in liquid cultures. The reagent was also used to detect the accumulation of phenols and cis-dihydrodiols in fungal and bacterial colonies on solid media.

Cited by 30 publications

References 20 publications

Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources

Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources

Applications of a colorimetric plate assay for soluble methane monooxygenase activity

Actividad enzimática de metanótrofos marinos y su uso potencial en biorremediación

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