Kultivierung von Denitrifikanten mit einem neuen nirK-Genotyp

Denitrification is an anaerobic respiration process with which oxidized nitrogen compounds are used as alternative electron acceptors. This process is of major importance in the global N-cycle and in addition it is of interest because it leads to an emission of nitrous oxide, which supports global warming and the destruction of the stratospheric ozone layer. The key enzyme of denitrification is nitrite reductase and the copper-containing form is encoded by nirK-gene. The phylogenetic tree of nirK consists of eight clusters. Two clusters (Cluster I and IV) are dominated by nirK-sequences from environmental samples and are so far not related to nirK-sequences from isolates. In particular, cluster I was of major interest since it is dominated by nirK-sequences of uncultivated denitrifiers from soil. So far cultivation techniques using nutrient rich media and anoxic conditions resulted in a limited variety of denitrifying bacteria with similar denitrification phenotypes. In contrast, cultivation-independent approaches using nirK as a functional marker gene showed a broad diversity and a dominance of sequences from denitrifiers of unknown phylogenetic affiliation. Denitrifiers are phylogenetically widespread and it is impossible to infer a phylogenetic affiliation based on nirK phylogeny, without pure cultures as references. In this study, advanced cultivation techniques were used that mimic the natural, oxic, low nutrient conditions in the environment to non-specifically enrich dominant microorganisms from soil. The cultivation procedure was performed with soil from a meadow located near to Marburg, Germany. A nirK-clone library was generated to prove the existence of nirK-containing denitrifiers in the soil. The nirK-sequences of the clone library grouped within the nirK-cluster I, II and VI. In an attempt to isolate representatives of these clusters, the cultivation procedure was performed. Soil samples were incubated for four weeks at the habitat-specific temperature under oxic conditions in minimal media. Eight colonies, 187, 406, 467, 471, 469, 373, 484 and 205 contained a nirK-gene and pure cultures were obtained for each strain. 16S rRNA gene sequence analysis indicated that the isolates belonged to various genera within the order Rhizobiales, specifically the genera Bradyrhizobium (isolates 406, 467, 469 and 471), Bosea (187), Mesorhizobium (373 and 484) and Devosia (205). The nirK-sequences of the isolates grouped within the clusters I, II, V and VII. The 16S rRNA gene phylogeny of these isolates provides the first information about the taxonomic identity of cluster I-type denitrifiers, which were previously unidentified. Here we show that cluster I-type nirK-sequences most probably belong to the Bradyrhizobium genus. The Bradyrhizobium isolates, as well as the Bosea isolate, also contained the other denitrification genes nap, nor and nos. An exception was isolate 471 in which the nosZ gene was not detectable. In addition to nirK, the genes narG and norB were detected in the Mesorhizobium isolates. For the Devosia isolate (205) no other gene of the denitrification besides nirK was detected. A phylogenetic comparison of the 16S rRNA gene and the functional marker genes nirK, norB and nosZ was performed for each of the pure cultures. The results showed congruent phylogenies of the different genes, but the nirK-phylogeny was the most suitable to mirror the 16S rRNA gene phylogeny. Despite the fact that the isolates of nirK-cluster I were identified as members of the genus Bradyrhizobium, they were only distantly related to the denitrifying Bradyrhizobium species B. japonicum. The conclusion might be drawn that the ability to denitrify is widespread within the Bradyrhizobium genus. Whereas Bradyrhizobium and Bosea isolates showed the genomic as well as the functional ability to denitrify, the Mesorhizobium isolates only contained the genomic potential and were not able to grow under oxygen limited conditions with nitrate as terminal electron acceptor. Furthermore, the Mesorhizobium isolates were only distantly related to known denitrifying Mesorhizobium species. Those isolates could be a novel denitrifying species within the genus Mesorhizobium that perform the process under unidentified conditions , or perhaps have lost the ability to denitrify.