Polyclonal antibodies recognizing the AmoB protein of ammonia oxidizers of the beta-subclass of the class Proteobacteria.
A 41-kDa protein of Nitrosomonas eutropha was purified, and the N-terminal amino acid sequence was found to be nearly identical with the sequence of AmoB, a subunit of ammonia monooxygenase. This protein was used to develop polyclonal antibodies, which were highly specific for the detection of the four genera of ammonia oxidizers of the beta-subclass of Proteobacteria (Nitrosomonas, including Nitrosococcus mobilis, which belongs phylogenetically to Nitrosomonas; Nitrosospira; Nitrosolobus; and Nitrosovibrio). In contrast, the antibodies did not react with ammonia oxidizers affiliated with the gamma-subclass of Proteobacteria (Nitrosococcus oceani and Nitrosococcus halophilus). Moreover, methane oxidizers (Methylococcus capsulatus, Methylocystis parvus, and Methylomonas methanica) containing the related particulate methane monooxygenase were not detected. Quantitative immunoblot analysis revealed that total cell protein of N. eutropha consisted of approximately 6% AmoB, when cells were grown using standard conditions (mineral medium containing 10 mM ammonium). This AmoB amount was shown to depend on the ammonium concentration in the medium. About 14% AmoB of total protein was found when N. eutropha was grown with 1 mM ammonium, whereas 4% AmoB was detected when 100 mM ammonium were used. In addition, the cellular amount of AmoB was influenced by the absence of the substrate. Cells starved for more than 2 months contained nearly twice as much AmoB as actively growing cells, although these cells possessed low ammonia-oxidizing activity. AmoB was always present and could even be detected in cells of Nitrosomonas after 1 year of ammonia starvation.
Pinck
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Institut für Allgemeine Botanik, Universität Hamburg, D-22609 Hamburg, Germany. pinck@microbiologie.uni-hamburg.de
Coeur
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Potier
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Bock
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D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D020562:Betaproteobacteria / Q000145:classification / Q000378:metabolism*; D003470:Culture Media; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009606:Nitrosomonas; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D021122:Protein Subunits
D000906:Antibodies; D001426:Bacterial Proteins; D003470:Culture Media; D021122:Protein Subunits; D000641:Ammonia; D010088:Oxidoreductases; C045582:ammonia monooxygenase