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Applied and Environmental Microbiology, April 1999, p. 1652-1657, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria

Sara Hallin1,* and Per-Eric Lindgren2

Department of Microbiology, Swedish University of Agricultural Sciences, S-750 07 Uppsala,1 and Department of Physics and Measurement Technology, Molecular Microbiology Group, Linköping University, S-581 83 Linköping,2 Sweden

Received 25 September 1998/Accepted 15 January 1999

Using 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 amplify cd1- 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 of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes 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, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.

* Corresponding author. Mailing address: Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden. Phone: 46 18 67 32 88. Fax: 46 18 67 33 92. E-mail: Sara.Hallin{at}mikrob.slu.se.

Applied and Environmental Microbiology, April 1999, p. 1652-1657, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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