Antibody and DNA probes for detection of nitrite reductase in seawater.

Abstract

A polyclonal antiserum was produced by immunization with nitrite reductase (NiR) purified from Pseudomonas stutzeri (ATCC 14405) and tested for specificity among known denitrifying strains. The antiserum was nearly strain-specific, identifying NiR only in some, but not all, other P. stutzeri strains. Denitrifying isolates from water column and sediment environments were also screened; several isolates from an intertidal microbial mat reacted with the NiR antiserum. Activity assays for NiR in polyacrylamide gels demonstrated that strains with apparently very similar NiR proteins did not react with the antiserum. These results imply that the NiR protein is more variable even among closely related strains than previously suspected. A DNA probe for a 721 bp region of the NiR structural gene was obtained by PCR amplification of P. stutzeri (ATCC 14405) DNA and used to screen denitrifying strains and isolates. The probe hybridized with a greater variety of strains than did the antiserum, implying that the DNA probe may be a more broadly useful and functional probe in environmental samples, whilst the NiR antiserum is nearly strain- or, at most, species-specific. Limits for detection of the enzyme and gene in seawater were estimated and NiR DNA was detected in DNA extracted from natural seawater. The hybridization data imply that in the order of 1-10 in 1000 cells in natural seawater possess homology with the NiR gene probe.