Immunological method for direct assessment of the functionality of a denitrifying strain of Pseudomonas fluorescens in soil

This work describes an immunological method for detection and quantification in complex environments of the dissimilative nitrate reductase (NRA) responsible for the reduction of nitrate to nitrite, which plays an important role in ecosystem functioning. The alpha-catalytic subunit of the enzyme was purified from the denitrifying strain Pseudomonas fluorescens YT101 and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NRA by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. The specificity, detection threshold and reproducibility of the proposed method were evaluated. A soil experiment showed that. our method can be applied to complex environmental samples. (C) 2004 Elsevier B.V. All rights reserved.