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

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.

Immunofluorescence detection of the denitrifying strain Pseudomonas stutzeri (ATCC 14405) in seawater and intertidal sediment environments.

A strain-specific immunofluorescence assay for enumeration of a marine denitrifying bacterium was developed and applied in the marine environment. The polyclonal antiserum for Pseudomonas stutzeri (ATCC 14405) did not react with other pseudomonads, other heterotrophs, or autotrophic nitrifying strains. The abundance of P. stutzeri in the shallow water column of Monterey Bay was less than 0.1% of the total bacterial abundance and decreased with depth, whereas the total bacterial abundance was variable and nearly constant with depth. P. stutzeri was also detected in the sediments of a microbial mat from Tomales Bay. The relatively low contribution of P. stutzeri to the total bacterial abundance in both environments implies that it is not a major component of the heterotrophic assemblage. This conclusion appears to hold for most other strains for which specific assays have been applied in the marine environment. The isolation of several different denitrifying strains from local marine environments implies that the culturable population is quite diverse, even in the absence of different selective enrichment media. Thus, strain specific immunofluorescence is of limited use in quantifying functional groups of bacteria. Conversely, they provide specific information on the diversity of natural populations and their relation to culturable strains.