Use of polyclonal antibodies to detect and quantify the NOR protein of nitrite oxidizers in complex environments.
In the approaches or models which aim to understand and/or predict how the functioning of ecosystems may be affected by perturbations or disturbances, little attention is generally given to microorganisms. Even when they are taken into account as indicators, variables which are poorly informative about the changes in the microbial functioning (microbial biomass or diversity or total number of microorganisms) are often used. To be able to estimate, in complex environments, the quantity of enzymes involved in key ecosystem processes may constitute a useful complementary tool. Here, we describe an immunological method for detecting and quantifying, in complex environments, the nitrite oxidoreductase (NOR), responsible for the oxidation of nitrite to nitrate. The alpha-catalytic subunit of the enzyme was purified from Nitrobacter hamburgensis and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NOR by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. They recognized the alpha-NOR of all the Nitrobacter species described to date, but no reaction was observed with members of other nitrite-oxidizing genera. The detection threshold and reproducibility of the proposed method were evaluated. The feasibility of its use to quantify NOR in a soil was tested.
Maron
Pierre-Alain
PA
Laboratoire d'Ecologie Microbienne, UMR CNRS 5557, Université Claude Bernard Lyon 1, 69622 Villeurbanne cedex, France. maron_pierre@voila.fr
Coeur
Caroline
C
Pink
Claudia
C
Clays-Josserand
Annie
A
Lensi
Robert
R
Richaume
Agnès
A
Potier
Patrick
P
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D015169:Colony Count, Microbial; D003470:Culture Media; D004777:Environment; D008163:Luminescent Measurements; D009572:Nitrite Reductases / Q000032:analysis* / Q000276:immunology; D009573:Nitrites / Q000378:metabolism; D009576:Nitrobacter / Q000201:enzymology* / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011817:Rabbits; D012988:Soil Microbiology
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D003470:Culture Media; D009573:Nitrites; D009572:Nitrite Reductases