Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE.
We re-evaluated PCR primers targeting nirS, nirK and nosZ genes for denaturing gradient gel electrophoresis as a tool to survey denitrifying community composition in environmental samples. New primers for both nirS and nosZ were combined with existing primers, while for nirK the previously published F1aCu:R3Cu set was chosen for denaturing electrophoresis. All three sets yielded amplicons smaller than 500 bp and amplified the correct fragment in all environmental samples. The denaturing gradient gel electrophoresis worked satisfactorily for nirK and nosZ, but not for nirS. This was probably due to the multiple melting domains in this particular nirS fragment. From the excised and sequenced bands, only sequences related to the target genes were detected and tree analysis showed that the selected primers acted as broad range primers for each of the three genes. By use of the new nirS primers it was demonstrated that agricultural soil harbours a substantial diversity of nirS denitrifiers.
Throbäck
Ingela Noredal
IN
Department of Microbiology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden. Ingela.Throback@mikrob.slu.se
Enwall
Karin
K
Jarvis
Asa
A
Hallin
Sara
S
D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D017753:Ecosystem; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D009566:Nitrates / Q000378:metabolism*; D009572:Nitrite Reductases / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012722:Sewage / Q000382:microbiology*; D012988:Soil Microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D009566:Nitrates; D012722:Sewage; D010088:Oxidoreductases; D009572:Nitrite Reductases; C022763:nitrous oxide reductase