DEVELOPMENT OF PCR PRIMER SYSTEMS FOR AMPLIFICATION OF NITRITE REDUCTASE GENES (NIRK AND NIRS) TO DETECT DENITRIFYING BACTERIA IN ENVIRONMENTAL-SAMPLES

A system was developed for the detection of denitrifying bacteria by t he amplification of specific nitrite reductase gene fragments with PCR , Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequenc e analysis. Whenever amplification was tried with these primers, the k nown nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five l aboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp, (DSM 3012 8); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698, For each of the two genes, a t least one primer combination amplified successfully for all of the t est strains. Specific amplification products were not obtained,vith no ndenitrifying bacteria or with strains of the other nir type. The spec ificity of the amplified products was confirmed by subsequent sequenci ng. These results suggest the suitability of the method for the qualit ative detection of denitrifying bacteria in environmental samples. Thi s was shown by applying one generally amplifying primer combination fo r each nir gene developed in this study to total DNA preparations from aquatic habitats.