Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples

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 (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 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 other nir type. 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 each nir gene developed in this study to total DNA preparations from aquatic habitats.

FIG. 1

Diagram showing the positions of primers used to amplify nir fragments: the positions for the nirK primers are correlated with the sequence of the nirK gene from Alcaligenes faecalis S-6 (top), and the positions for the nirS primers are correlated with the sequence of the nirS gene from Pseudomonas stutzeri ZoBell (bottom).

FIG. 2

Southern blot hybridization of nirK fragments obtained from environmental samples with the primer combination nirK1F-nirK5R to the digoxigenin-labeled fragment from Alcaligenes xylosoxidans. Lanes: 1 and 8, digoxigenin-labeled DNA size standard VII (Boehringer); 2, sediment from Lake Kleiner Plöner See (5 μl of PCR product), 3, water from Lake Plussee (7 μl); 4, water from Lake Kleiner Plöner See (7 μl); 5, activated sludge from the sewage treatment plant at Plön (10 μl); 6, enrichment culture for denitrifying methylotrophic bacteria (12 μl); 7, A. xylosoxidans positive control (5 μl).

FIG. 3

Southern blot hybridization of nirS fragments obtained from environmental samples with the primer combination nirS1F-nirS6R to the digoxigenin-labeled fragment from Pseudomonas stutzeri ZoBell. Lanes: 1 and 8, digoxigenin-labeled DNA size standard VII (Boehringer); 2, sediment from Lake Kleiner Plöner See (15 μl), 3, water from Lake Plussee (12 μl); 4, water from Lake Kleiner Plöner See (20 μl); 5, activated sludge from the sewage treatment plant at Plön (10 μl); 6, enrichment culture for denitrifying methylotrophic bacteria (20 μl); 7, P. stutzeri ZoBell positive control (1 μl).