Detection of genes for periplasmic nitrate reductase in nitrate respiring bacteria and in community DNA

A nested PCR primed by four degenerate oligonucleotides was developed for t he specific amplification of sequences from the napA gene encoding the peri plasmic nitrate reductase. This approach was used to amplify fragments of t he napA gene from 10 Pseudomonas species and one Moraxella sp., previously shown to be able to express the periplasmic nitrate reductase activity, fro m Rhodobacter capsulatus and from community DNA extracted from a fresh-wate r sediment. Amino acid sequences encoded by the napA fragments were compare d to one another;md to the corresponding regions of related enzymes. This c omparison indicates that the amplification protocol is specific for its int ended target. The napA sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment commu nity. All tested Gram-negative strains capable of aerobic nitrate respirati on were found to have periplasmic nitrate reductase genes. However, some st rains which have and express the genes are incapable of aerobic nitrate res piration. The PCR primers and amplification protocols described will be use ful in future studies of nitrate respiring populations. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.