PCR detection of genes encoding nitrite reductase in denitrifying bacteria

Abstract

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.

FIG. 1

Consensus regions and PCR primers for the gene encoding Nir. Uppercase letters represent total homology, lowercase letters point to one deviating nucleotide, and hooks (˜) indicate no consensus (R, A and G; S, C and G; Y, C and T). (a) cd₁ primers and alignments of cd₁-nir genes. The positions of the first and last nucleotides in each primer corresponding to the gene sequence of P. stutzeri ATCC 14405 are in parentheses. (b) Cu primers and alignments of Cu-nir genes. The positions of the first and last nucleotides in each primer corresponding to the gene sequence of A. faecalis S-6 are in parentheses.

FIG. 2

Agarose gels of PCR fragments and Southern blot hybridizations. Arrows indicate fragments of the expected sizes. Panels: a, cd₁-nir primers; b, cd₁ probe; c, Cu-nir primers; d, Cu probe. The upper row has molecular size markers in lane 1, P. stutzeri ATCC 14405 in lane 2, P. stutzeri CCUG 29240 in lane 3, P. fluorescens ATCC 33512 in lane 4, P. fluorescens Mi32 in lane 5, R. eutropha CCUG 13724 in lane 6, A. eutrophus ATCC 17699 in lane 7, P. aeruginosa CCUG 241 in lane 8, P. aeruginosa Mi11 in lane 9, P. denitrificans Pd1222 in lane 10, P. denitrificans ATCC 19367 in lane 11, P. denitrificans ATCC 13867 in lane 12, P. denitrificans CCUG 2519 in lane 13, P. putida CCUG 2479 in lane 14, and molecular size markers in lane 15. The lower row has molecular size markers in lane 16, A. faecalis ATCC 8750 in lane 17, A. faecalis ATCC 19018 in lane 18, P. aureofaciens ATCC 13985 in lane 19, A. denitrificans ATCC 15173 in lane 20, A. cycloclastes ATCC 21921 in lane 21, sludge L1 in lane 22, sludge L2 in lane 23, sludge P1 in lane 24, E. coli TG1 in lane 25, S. aureus 8325-4 in lane 26, a negative control in lane 27, and molecular size markers in lane 28. The molecular size markers consisted of a 100-bp ladder with the 800-bp fragment twice as intense as the other bands (Pharmacia).

FIG. 3

Neighbor-joining analysis of partial nir gene sequences. Bootstrap values (percentages) are presented at the nodes. New sequences from this study are in boldface.