Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment

Department of Genetics and Microbiology, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK Author for correspondence: Alan J. McCarthy. Tel: + 44 151 794 4413. Fax: +44 151 794 4401. e-mail: aj55m@liv.ac.uk Institute of Freshwater Ecology, Windermere Laboratories, Far Sawrey, Ambleside, Cumbria LA22 OLP, UK ABSTRACT Summary: Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas -specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed. Keywords: nitrification, ammonia-oxidation, Nitrosomunas , Nitrosospira , 16s ribosomal, RNA Present address: Newcastle Research Group in Fossil Fuels and Environmental Geochemistry, Drummond Building, University of Newcastle, NE1 7RU, UK.