PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria

• DOI: 10.1128/aem.65.4.1652-1657.1999
• Publication Date: 2019-12-19T19:49:23Z
• Authors: Sara Hallin; Per-Eric Lindgren
• Keywords: 0303 health sciences; Nitrates; Nitrite Reductases; Base Sequence; Sewage; Molecular Sequence Data; Industrial Waste; Sequence Analysis, DNA; Polymerase Chain Reaction; 6. Clean water; Blotting, Southern; 03 medical and health sciences; Genes, Bacterial; Pseudomonas; Gram-Negative Bacteria; 11. Sustainability; Alcaligenes; DNA Probes; Water Microbiology

ABSTRACTUsing consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnirgenes were also established. Thecd1primers were designed to amplify a 778 to 799-bp region ofcd1-nirin the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nirsequences. Together, the two sets of PCR primers amplifiednirgenes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, butcd1-nirfragments were only obtained in five samples. PCR products of the expected sizes were verified asnirgenes after hybridization to DNA probes, except in one case. The sequencednirfragments were related to othernirsequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nirwere conserved, while broad-range primers targeting conserved regions ofcd1-nirseem to be difficult to find. We also report on the existence of Cu-nirinParacoccus denitrificansPd1222.