title: Highly diverse nirK genes comprise two major clades that harbour ammonium-producing denitrifiers.
abstract: BACKGROUND
  Copper dependent nitrite reductase, NirK, catalyses the key step in denitrification, i.e. nitrite reduction to nitric oxide. Distinct structural NirK classes and phylogenetic clades of NirK-type denitrifiers have previously been observed based on a limited set of NirK sequences, however, their environmental distribution or ecological strategies are currently unknown. In addition, environmental nirK-type denitrifiers are currently underestimated in PCR-dependent surveys due to primer coverage limitations that can be attributed to their broad taxonomic diversity and enormous nirK sequence divergence. Therefore, we revisited reported analyses on partial NirK sequences using a taxonomically diverse, full-length NirK sequence dataset.
  
  
  RESULTS
  Division of NirK sequences into two phylogenetically distinct clades was confirmed, with Clade I mainly comprising Alphaproteobacteria (plus some Gamma- and Betaproteobacteria) and Clade II harbouring more diverse taxonomic groups like Archaea, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Nitrospirae, Firmicutes, Actinobacteria, Planctomycetes and Proteobacteria (mainly Beta and Gamma). Failure of currently available primer sets to target diverse NirK-type denitrifiers in environmental surveys could be attributed to mismatches over the whole length of the primer binding regions including the 3' site, with Clade II sequences containing higher sequence divergence than Clade I sequences. Simultaneous presence of both the denitrification and DNRA pathway could be observed in 67% of all NirK-type denitrifiers.
  
  
  CONCLUSIONS
  The previously reported division of NirK into two distinct phylogenetic clades was confirmed using a taxonomically diverse set of full-length NirK sequences. Enormous sequence divergence of nirK gene sequences, probably due to variable nirK evolutionary trajectories, will remain an issue for covering diverse NirK-type denitrifiers in amplicon-based environmental surveys. The potential of a single organism to partition nitrate to either denitrification or dissimilatory nitrate reduction to ammonium appeared to be more widespread than originally anticipated as more than half of all NirK-type denitrifiers were shown to contain both pathways in their genome.
authors.affiliation: Laboratory of Microbiology (LM-UGent), Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000, Ghent, Belgium. Helen.Decleyre@UGent.be.
authors.forename: Decleyre
authors.initials: D
authors.lastname: Helen
authors.affiliation: Laboratory of Microbiology (LM-UGent), Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000, Ghent, Belgium. Kim.Heylen@UGent.be.
authors.forename: Heylen
authors.initials: H
authors.lastname: Kim
authors.affiliation: Laboratory of Microbiology (LM-UGent), Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000, Ghent, Belgium. Bjorn.Tytgat@UGent.be.
authors.forename: Bjorn
authors.initials: B
authors.lastname: Tytgat
authors.affiliation: Laboratory of Microbiology (LM-UGent), Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000, Ghent, Belgium. Anne.Willems@UGent.be.
authors.forename: Willems
authors.initials: W
authors.lastname: Anne
mesh_terms: D000595:Amino Acid Sequence; D064751:Ammonium Compounds / Q000378:metabolism; D001105:Archaea / Q000201:enzymology / Q000235:genetics*; D019843:Archaeal Proteins / Q000235:genetics; D001419:Bacteria / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D058440:Denitrification / Q000235:genetics*; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism; D009572:Nitrite Reductases / Q000235:genetics*; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D064751:Ammonium Compounds; D019843:Archaeal Proteins; D001426:Bacterial Proteins; D017931:DNA Primers; D009566:Nitrates; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing