AMPLIFICATION OF 16S RIBOSOMAL-RNA GENES OF AUTOTROPHIC AMMONIA-OXIDIZING BACTERIA DEMONSTRATES THE UBIQUITY OF NITROSOSPIRAS IN THE ENVIRONMENT

Oligonucleotide sequences selected from the 165 rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR a mplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were establishe d with sequence databases, and the primer pairs were used to amplify D NA from laboratory cultures and environmental samples. Eubacterial rRN A 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 we re analysed using a nested PCR technique in which eubacterial rRNA gen es were subjected to a secondary amplification with Nitrosomonas or Ni trosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybri dization 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 conta in DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 165 rRNA genes. This demonstrates th at Nitrosospira spp. are widespread in the environment. The implicatio ns of the detection of Nitrosomonas DNA only after enrichment culture are discussed.