Phylogenetic differentiation of two closely related Nitrosomonas spp. thatinhabit different sediment environments in an oligotrophic freshwater lake

The population of ammonia-oxidizing bacteria in a temperate oligotrophic fr eshwater lake was analyzed by recovering 16S ribosomal DNA (rDNA) from lake water and sediment samples taken throughout a seasonal cycle. Nitrosospira and Nitrosomonas 16S rRNA genes were amplified in a nested PCR, and the ide ntity of the products was confirmed by oligonucleotide hybridization. Nitro sospira DNA was readily identified in all samples, and nitrosomonad DNA of the Nitrosomonas europaea-Nitrosomonas eutropha lineage was also directly d etected, but during the summer months only. Phylogenetic delineation with p artial (345 bp) 16S rRNA gene sequences of clones obtained from sediments c onfirmed the fidelity of the amplified nitrosomonad DNA and identified two sequence clusters closely related to either N. europaea or N. eutropha that were equated with the littoral and profundal sediment sites, respectively. Determination of 701-bp sequences for 16S rDNA clones representing each cl uster confirmed this delineation. A PCR-restriction fragment length polymor phism (RFLP) system was developed that enabled identification of clones con taining N. europaea and N. eutropha 16S rDNA sequences, including subclasse s therein, It proved possible to analyze 16S rDNA amplified directly from s ediment samples to determine the relative abundance of each species compare d with that of the other. N. europaea and N, eutropha are very closely rela ted, and direct evidence for their presence in lake systems is limited. The correlation of each species with a distinct spatial location in sediment i s an unusual example of niche adaptation by two genotypically similar bacte ria. Their occurrence and relative distribution can now be routinely monito red in relation to environmental variation by: the application of PCR-RFLP analysis.