Species-specific probing for the polymerase chain reaction product of ammonia monooxygenase gene amplified from DNA mixtures of Nitrosomonas europaea and Nitrosolobus multiformis

Abstract

Y Ecological studies on chemolithotrophic ammonia-oxidizers have been limited because of their extremely slow growth rates in autotrophic life (Wood 1986). For these slowgrowing bacteria in environments, the PCR techniques have been introduced. For instance, 16S rDNAs of chemolithotrophic ammonia-oxidizers were amplified by PCR from environmental samples (Hiorns et al. 1995; Voytek and Ward 1995). At present, the use of amo which encodes ammonia monooxygenase (AMO) is a promising and more powerful tool than 16S rDNA for the precise differentiation of these bacteria (Rotthauwe et al. 1995) and for the specific analysis based on functions, because AMO plays a major role in the metabolism of halogenated hydrocarbons (Vannelli et al. 1990; Hyman et al. 1995) and in N20 production (Hynes and Knowles 1984) in addition to nitrification in soil. Except for Nitrosococcus oceanus, chemolithotrophic ammonia-oxidizers isolated belong to the \\gB-subdivision of Proteobacteria (Head et al. 1993; Teske et al. 1994). PCR primers which could be applied to amo of these bacteria have not been developed (Sinigalliano et al. 1995). DNA sequences of three open reading frames, namely amoC, amoA, and amoB in this order, have been accumulated in DNA data bases as the components of an amo operon. amoA encodes the active site of AMO and has been best studied. The products of amoB and amoC are functionally unknown. amoB is located downstream of amoA after internal sequences with variable length depending on the species. In this report, we designed universal PCR primers for targeting the region comprising the parts of amoA and amoB of chemolithotrophic ammonia-oxidizers of \\gB-Proteobacteria and identified the bacterial origin of PCR products with specific probes. We tested the efficiency of the technique using two representative species, Nitrosomonas europaea and Nitrosolobus multiformis. The former is the best studied species and the latter a representative species of the newly proposed genus, Nitrosospira, which consisted of the original genera, Nitrosospira, Nitrosolobus, and Nitrosovibrio (Head et al. 1993).