Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

Abstract

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the beta- and the gamma-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the beta-subdivision representatives but in single copies in the gamma-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the beta-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

“…The different IF labeling intensities of Nitrosomonas species might result from the differences between the N-terminal AmoA sequences, which could have caused the different binding efficiency of the antibodies. All Nitrosomonas strains showing high AmoA-sequence similarity to N. eutropha, such as N. europaea and N. halophila, were intensively stained, whereas strains with low AmoA-sequence similarity such as N. cryotolerans and N. marina were weakly stained [37,40]. Only Nitrosococcus mobilis, close relative of Nitrosomonas eutropha [39,40], could not be detected by AmoA antibodies.…”

“…The N-terminal AmoA sequence of N. cryotolerans differs from the sequence of N. eutropha [37], which might be the reason for the weak IF labeling using AmoA-antibodies. Recently, Norton and co-workers [37] described the AmoA-sequence of two new isolated strains of Nitrosomonas with N-terminal AmoA sequences, which further deviate from the sequence of N. eutropha and therefore might not be detected by AmoA antibodies. Although the N-terminal AmoA sequences of strains of the genera Nitrosospira, Nitrosovibrio, and Nitrosolobus correspond with the described sequence of N. cryotolerans [37], they did not react with the AmoA antibodies.…”

“…Only Nitrosococcus mobilis, close relative of Nitrosomonas eutropha [39,40], could not be detected by AmoA antibodies. The N-terminal AmoA sequence of N. eutropha is identical with the sequence of N. europaea [37]. Aside from the two Nitrosomonas species, only the sequence of the N terminus of AmoA of one strain of N. cryotolerans is known [37], whereas the N-terminal AmoA sequences of the other species of Nitrosomonas are known only partially (Nitrosococcus mobilis, SwissProt accession number AF037108) or not at all.…”

“…The N-terminal AmoA sequence of N. eutropha is identical with the sequence of N. europaea [37]. Aside from the two Nitrosomonas species, only the sequence of the N terminus of AmoA of one strain of N. cryotolerans is known [37], whereas the N-terminal AmoA sequences of the other species of Nitrosomonas are known only partially (Nitrosococcus mobilis, SwissProt accession number AF037108) or not at all. The N-terminal AmoA sequence of N. cryotolerans differs from the sequence of N. eutropha [37], which might be the reason for the weak IF labeling using AmoA-antibodies.…”

“…Aside from the two Nitrosomonas species, only the sequence of the N terminus of AmoA of one strain of N. cryotolerans is known [37], whereas the N-terminal AmoA sequences of the other species of Nitrosomonas are known only partially (Nitrosococcus mobilis, SwissProt accession number AF037108) or not at all. The N-terminal AmoA sequence of N. cryotolerans differs from the sequence of N. eutropha [37], which might be the reason for the weak IF labeling using AmoA-antibodies. Recently, Norton and co-workers [37] described the AmoA-sequence of two new isolated strains of Nitrosomonas with N-terminal AmoA sequences, which further deviate from the sequence of N. eutropha and therefore might not be detected by AmoA antibodies.…”