Abstract
Denitrification is an alternative type of anaerobic respiration in which nitrate is reduced to gaseous products via nitrite. The key step in this process is the reduction of nitrite to nitric oxide, which is catalyzed by two structurally different but functionally equivalent forms of nitrite reductase encoded by the nirK and nirS genes. Cultivation-independent studies based on these functional marker genes showed that in the environment there was a dominance of organisms with nirK and nirS genes presumably derived from organisms that have not been cultured yet. However, the phylogenetic affiliation of these organisms has not been resolved since the ability to denitrify is widespread in phylogenetically unrelated organisms. To unravel the phylogeny of the organisms from which the nitrite reductase (nirK) genes originated, one option is to use a special variant of whole-cell hybridization termed recognition of individual genes-fluorescence in situ hybridization (RING-FISH). In RING-FISH a multiply labeled transcript polynucleotide probe is used to detect a single gene on the bacterial chromosome during FISH. Here, RING-FISH was used with laboratory cultures and environmental samples, such as activated sludge. Furthermore, probe-based cell sorting using magnetic beads could also be carried out with mixtures of pure cultures, which led to effective depletion of the nirK-negative organism but capture of the nirK-positive organism, which was demonstrated by terminal restriction fragment length polymorphism analysis based on 16S rRNA genes. The results indicate that RING-FISH coupled with probe-based cell sorting could be used with environmental samples, which could provide a means for phylogenetic classification of nirK-type denitrifiers. Thus, the results of RING-FISH could increase our understanding of the phylogeny and function of denitrifying microorganisms in the environment.