Immunofluorescence Detection of Nitrogenase Proteins in Whole Cells

Abstract

Fluorescent antibodies (FA) prepared against the Mo-Fe and Fe proteins of nitrogenase from Klebsiella pneumoniae M5a1 were used to detect these protein components in toluene-treated whole cells that were actively reducing acetylene. The FA were highly specific, staining only nitrogenase component proteins originating from Klebsiella. Cross-reactions between the FA and purified nitrogenase proteins from other N2-fixing microorganisms [Azotobacter chroococcum, Clostridium pasteurianum, Bacillus polymyxa and Rhizobium japonicum] did not occur, except in the case of B. polymyxa. The tests rapidly and accurately assayed the component proteins in Klebsiella mutants and derivatives to which Klebsiella nif genes were transferred by plasmid or by other means. Cross-reactions also indicated the degree of relatedness between nitrogenase proteins from N2-fixing microorganisms of various origins.