Detection and widespread distribution of the nrfA gene encoding nitrite reduction to ammonia, a short circuit in the biological nitrogen cycle that competes with denitrification

Abstract

Degenerate primers to detect nrfA were designed by aligning six nrfA sequences including Escherichia coli K-12, Sulfurospirillum deleyianum and Wolinella succinogenes. These primers amplified a 490 bp fragment of nrfA. The ability of these primers to detect nrfA was tested with chromosomal DNA isolated from a variety of bacteria: they could distinguish between bacteria in which the gene is known to be present or absent. The positive reference organisms spanned the various classes of Proteobacteria, suggesting that these primers are probably generic. The primer pair F1 and R1 was also used successfully to analyse nrfA diversity from community DNA isolated from a sulphate reducing bioreactor, and from two established Anammox reactors (for an aerobic ammonia oxidation, in which nitrite is reduced by ammonia to dinitrogen gas). The nrfA clones isolated from these three sources grouped with the Bacteroidetes phylum. The nrfA primers also amplified 570 bp fragments from the Anammox community DNA. These fragments encoded a protein with four haem-binding motifs typical of a c-type cytochrome, but were unrelated to the NrfA nitrite reductase. A BLAST search failed to reveal similarity to any known proteins. However, similarity was found to one sequence, which was annotated as rapC (response regulator aspartate phosphatase), in the genome of the planctomycete Rhodopirellula baltica. These sequences possibly belong to a new class of c-type cytochrome that might be specific to members of the order Planctomycetales. The data are consistent with the proposal that cytochrome c nitrite reductases, present in the periplasm of Gram-negative bacteria, are widely distributed in many different environments where they provide a short circuit in the biological nitrogen cycle by reducing nitrite directly to ammonia.

Propionate Oxidation by and Methanol Inhibition of Anaerobic Ammonium-Oxidizing Bacteria

“…4). Future studies will concentrate on the mechanism of nitrite reduction (14) and propionate oxidation via biochemical assays, as has been described for other bacteria (24,30).…”

“…Carbon dioxide was measured on an HP6890 gas chromatograph (80°C; poropack Q in combination with a 5-Å molsieve) equipped with a thermal conductivity detector. The bacterial population was monitored by FISH and immunofluorescence analysis as described previously (13,14,20). The oligonucleotide probes applied were as follows: AMX368 [S-G-Amx-0368-a-A-22] covering all anammox organisms, PLA46 [S-P-Planc-0046-a-A-18] covering all Planctomycetes, EUB338 mix [S-DBact-0338-a-A-18] covering almost all Bacteria, ALFA968 [S-P-Alph-968-a-A-18] covering most Alphaproteobacteria, BET42a [L-P-Beta-1027-a-A-17] covering most Betaproteobacteria, and GAM42a [L-P-Gamm-1027-a-A-17] covering most Gammaproteobacteria (11).…”

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria

“…PCR amplifications were carried out as previously described. All the primers used in this study were fully characterised in previous studies, in which they were used as primers or fluorescent in situ hybridisation (FISH) probes (Amann et al, 1990;Mobarry et al, 1996;Hovanec et al, 1998;Watanabe et al, 2001;Mohan et al, 2004;Geets et al, 2007;Smith et al, 2007). The PCR products were separated by electrophoresis on a 1% agarose gel and visualised by staining with 0.5 mg of ethidium bromide per mL of gel.…”

Diversity and Abundance of Nitrate Reductase Genes ( narG and napA ), Nitrite Reductase Genes ( nirS and nrfA ), and Their Transcripts in Estuarine Sediments

“…Previous PCRbased analysis of nrfA diversity in anammox and sulfate-reducing reactors showed that the majority of clones were most closely related to nrfA genes from Bacteroides spp. (40).…”