Amplification of the amoA gene from diverse species of ammonium-oxidizing bacteria and from an indigenous bacterial population from seawater

Abstract

Because the chemolithotrophic ammonium-oxidizing bacteria are an integral component of nitrogen biogeochemistry, a sensitive and accurate method to detect this ecologically important group of microorganisms is needed. The amoA gene of these organisms encodes the active site of ammonia monooxygenase, an enzyme unique to this group of nitrifying bacteria. We report here the use of the PCR technique to detect the amoA gene from pure cultures of chemolithotrophic ammonium-oxidizing bacteria, ammonium oxidizers introduced into filtered seawater, and the natural bacterial population of an unfiltered seawater sample. Oligonucleotide primers, based on the published amoA sequence from Nitrosomonas europaea, were used to amplify DNA from pure cultures of Nitrosomonas europaea, Nitrosomonas cryotolerans, and Nitrosococcus oceanus and from bacteria in seawater collected offshore near the Florida Keys. Partial sequencing of the amplification products verified that they were amoA. These primers, used in conjunction with a radiolabeled amoA gene probe from Nitrosomonas europaea, could detect Nitrosococcus oceanus inoculated into filter-sterilized seawater at 10(4) cells liter-1. Native marine bacteria containing amoA could also be detected at their naturally occurring titer in oligotrophic seawater. Amplification of the gene for ammonia monooxygenase may provide a method to estimate the distribution and relative abundance of chemolithotrophic ammonium-oxidizing bacteria in the environment.