Polyclonal antibodies recognizing the AmoB protein of ammonia oxidizers ofthe 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 A moB, a subunit of ammonia monooxygenase. This protein was used to develop p olyclonal antibodies, which were highly specific for the detection of the f our genera of ammonia oxidizers of the beta -subclass of Proteobacteria (Ni trosomonas, including Nitrosococcus mobilis, which belongs phylogenetically to Nitrosomonas; Nitrosospira; Nitrosolobus; and Nitrosovibrio). In contra st, 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, Methylo cystis parvus, and Methylomonas methanica) containing the related particula te methane monooxygenase were not detected. Quantitative immunoblot analysi s revealed that total cell protein of N. eutropha consisted of approximatel y 6% AmoB, when cells were grown using standard conditions (mineral medium containing 10 mM ammonium). This AmoB amount was shown to depend on the amm onium concentration in the medium. About 14% AmoB of total protein was foun d when N. eutropha was grown with 1 mM ammonium, whereas 4% AmoB was detect ed 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 tha n 2 months contained nearly twice as much AmoB as actively growing cells, a lthough these cells possessed low ammonia-oxidizing activity. AmoB was alwa ys present and could even be detected in cells of Nitrosomonas after 1 year of ammonia starvation.