Downloaded from www.microbiologyresearch.org by Characterization of the N 2 O-producing soil bacterium Rhizobium azooxidifex sp. nov. Undine Behrendt, 1 Peter K€ ampfer, 2 Stefanie P. Glaeser, 2 Jürgen Augustin 1 and Andreas Ulrich 1 Correspondence Undine Behrendt ubehrendt@zalf.de 1 Leibniz Centre for Agricultural Landscape Research (ZALF), Institute for Landscape Biogeochemistry, Eberswalder Str. 84, D-15374 Müncheberg, Germany 2 Department of Applied Microbiology, Justus-Liebig University Giessen, IFZ–Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany In the context of studying the bacterial community involved in nitrogen transformation processes in arable soils exposed to different extents of erosion and sedimentation in a long-term experiment (CarboZALF), a strain was isolated that reduced nitrate to nitrous oxide without formation of molecular nitrogen. The presence of the functional gene nirK, encoding the respiratory copper-containing nitrite reductase, and the absence of the nitrous oxide reductase gene nosZ indicated a truncated denitrification pathway and that this bacterium may contribute significantly to the formation of the important greenhouse gas N 2 O. Phylogenetic analysis based on the 16S rRNA gene sequence and the housekeeping genes recA and atpD demonstrated that the investigated soil isolate belongs to the genus Rhizobium. The closest phylogenetic neighbours were the type strains of Rhizobium. subbaraonis and Rhizobium. halophytocola. The close relationship with R. subbaraonis was reflected by similarity analysis of the recA and atpD genes and their amino acid positions. DNA–DNA hybridization studies revealed genetic differences at the species level, which were substantiated by analysis of the whole-cell fatty acid profile and several distinct physiological characteristics. Based on these results, it was concluded that the soil isolate represents a novel species of the genus Rhizobium, for which the name Rhizobium azooxidifex sp. nov. (type strain Po 20/26 T =DSM 100211 T =LMG 28788 T ) is proposed. The genus Rhizobium belongs to the family Rhizobiaceae, which has been subjected to several taxonomic re- evaluations (Willems, 2006). Thus, species of the genera Allorhizobium and Agrobacterium were reclassified in the genus Rhizobium based on comparative 16S rRNA gene sequence analysis (Young, 2004; Young et al., 2001). How- ever, this unification has been challenged and is not univer- sally accepted (Farrand et al., 2003; Lindström & Young, 2011). It was proposed to retain the genus Agrobacterium to accommodate predominantly phytopathogenic species. This recommendation was supported by multilocus sequence analysis of plant-associated members of the Rhizobiaceae (Mousavi et al., 2014, 2015) and the analysis of whole- genome averaged nucleotide identity of the family members that have been sequenced thus far (Ormeno-Orrillo et al., 2015). Furthermore, Mousavi et al. (2014, 2015) proposed reclassifying several Rhizobium species in the novel genera Pararhizobium and Neorhizobium and the revived genus Allorhizobium. Based on this approach, further strains cur- rently ascribed to the genus Rhizobium were identified that may represent as yet undescribed genera within the family Rhizobiaceae (Mousavi et al., 2015; Ormeno-Orrillo et al., 2015), and therefore further splitting of the genus can be expected that is not reflected in the 16S rRNA gene phylog- eny. Furthermore, this splitting based on genomic data is mostly unsupported by phenotypic differences that allow the differentiation of these genera on the basis of significant physiological or chemotaxonomic features. This situation makes it increasingly difficult to affiliate unknown strains at the genus level. Members of the genus Rhizobium are well known as endo- symbionts of diverse host legume species, where they con- tribute to significant nitrogen input into soils through biological N 2 -fixation (Olivares et al., 2013). Because of this The GenBank/DDJB/EMBL accession numbers for the 16S rRNA gene, atpD, recA and nirK gene sequences of Rhizobium azooxidifex Po 20/26 T are LN832063, LN868515, LN868516 and LN886516, respectively. The accession number of the atpD gene sequence of Rhi- zobium subbaraonis DSM 24765 T is LN868517. One supplementry table and two supplementry figures are available with the online Supplementry Material. 2354 001036 ã 2016 IUMS Printed in Great Britain International Journal of Systematic and Evolutionary Microbiology (2016), 66, 2354–2361 DOI 10.1099/ijsem.0.001036