Brief report Nitric oxide reductase (norB) gene sequence analysis reveals discrepancies with nitrite reductase (nir) gene phylogeny in cultivated denitrifiers Kim Heylen, 1 * Bram Vanparys, 1 Dirk Gevers, 1,2 Lieven Wittebolle, 3 Nico Boon 3 and Paul De Vos 1 1 Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. 2 Bioinformatics and Evolutionary Genomics, Ghent University/VIB, Technologiepark 927, B-9052 Gent, Belgium. 3 Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium. Summary Gene sequence analysis of cnorB and qnorB, both encoding nitric oxide reductases, was performed on pure cultures of denitrifiers, for which previously nir genes were analysed. Only 30% of the 227 denitrifying strains rendered a norB amplicon. The cnorB gene was dominant in Alphaproteobacteria, and domi- nantly coexisted with the nirK gene, coding for the copper-containing nitrite reductase. Both norB genes were equally present in Betaproteobacteria but no linked distributional pattern of nir and norB genes could be observed. The overall cnorB phylogeny was not congruent with the widely accepted organism phylogeny based on 16S rRNA gene sequence analy- sis, with strains from different bacterial classes having identical cnorB sequences. Denitrifiers and non-denitrifiers could be distinguished through qnorB gene phylogeny, without further grouping at a higher taxonomic resolution. Comparison of nir and norB phylogeny revealed that genetic linkage of both genes is not widespread among denitrifiers. Thus, independent evolution of the genes for both nitrogen oxide reductases does also occur. Introduction Nitric oxide (NO) in excess is toxic to bacteria, fungi, microbial parasites, tumour cells and viruses, because it can damage DNA, proteins and lipids. The detoxification of NO through reduction is innate to denitrifiers, which catalyse the sequential dissimilatory reduction of nitrate to nitrite, and further via NO to N 2O and N2 under oxygen- limited conditions. Non-denitrifying, mostly pathogenic strains can also contain a NO reductase, which is advan- tageous in the survival in oxygen-limited environments and confers protection against exogenous and endog- enous nitrosative stress (Philippot, 2005). Three nitric oxide reductases have been described to date. The best described is the membrane-bound dimer NORCB. All bacterial strains found with this nitric oxide reductase, designated NORB, cNORB or short-chain NOR (scNOR), could perform denitrification (Bartnikas et al., 1997; Philippot et al., 2001; Braker and Tiedje, 2003; Chan and McCormick, 2004). A second, single- component nitric oxide reductase has an N-terminal extension coding for quinol as electron donor and two- thirds of the catalytic region show homology with cNORB (Cramm et al., 1997). This nitric oxide reductase is termed qNORB, NORZ or long-chain NOR (lcNOR). The qnorB gene has been found to date in both denitrifiers and non-denitrifying strains (Cramm et al., 1999; Büsch et al., 2002; Philippot, 2005). A third nitric oxide reductase, puri- fied from Bacillus azotoformans, uses menaquinol as electron acceptor (Suharti and de Vries, 2005). The genes encoding this menaquinol nitric oxide reductase are hith- erto unknown. Because denitrifiers are not a monophyletic group of bacteria (Knowles, 1982; Tiedje, 1988), cultivation- independent studies on the denitrifying communities in the environment use the genes targeting the key enzymes of denitrification, nirS or nirK coding for nitrite reductase (Braker et al., 2000; Priemé et al., 2002; Yan et al., 2003; Yoshie et al., 2004; Sharma et al., 2005) and/or norB coding for nitric oxide reductase (Braker and Tiedje, 2003; Casciotti and Ward, 2005). However, cultivation-based Received 13 October, 2006; accepted 18 October, 2006. *For correspondence. E-mail Kim.Heylen@UGent.be; Tel. (+32) 92645101; Fax (+32) 92645092. Environmental Microbiology (2007) 9(4), 1072–1077 doi:10.1111/j.1462-2920.2006.01194.x © 2006 Ghent University Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd