ANTIBODY AND DNA PROBES FOR DETECTION OF NITRITE REDUCTASE IN SEAWATER

A polyclonal antiserum was produced by immunization with nitrite reduc tase (NiR) purified from Pseudomonas stutzeri (ATCC 14405) and tested for specificity among known denitrifying strains. The antiserum was ne arly strain-specific, identifying NiR only in some, but not all, other P. stutzeri strains. Denitrifying isolates from water column and sedi ment environments were also screened; several isolates from an interti dal microbial mat reacted with the NiR antiserum. Activity assays for NiR in polyacrylamide gels demonstrated that strains with apparently v ery similar NiR proteins did not react with the antiserum. These resul ts imply that the NiR protein is more variable even among closely rela ted strains than previously suspected. A DNA probe for a 721 bp region of the NiR structural gene was obtained by PCR amplification of P. st utzeri (ATCC 14405) DNA and used to screen denitrifying strains and is olates. The probe hybridized with a greater variety of strains than di d the antiserum, implying that the DNA probe may be a more broadly use ful and functional probe in environmental samples, whilst the NiR anti serum is nearly strain- or, at most, species-specific. Limits for dete ction of the enzyme and gene in seawater were estimated and NiR DNA wa s detected in DNA extracted from natural seawater. The hybridization d ata imply that in the order of 1-10 in 1000 cells in natural seawater possess homology with the NiR gene probe.