Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirKandnirS) To Detect Denitrifying Bacteria in Environmental Samples

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Abstract
A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirKandnirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the knownnirtype of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of thenirtype of five laboratory strains. ThenirKgene could be amplified fromBlastobacter denitrificans,Alcaligenes xylosoxidans, andAlcaligenessp. (DSM 30128); thenirSgene was amplified fromAlcaligenes eutrophusDSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the othernirtype. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for eachnirgene developed in this study to total DNA preparations from aquatic habitats.

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