Immunofluorescence detection and characterization of N2-fixing microorganisms from aquatic environments

The role of N2-fixing microheterotrophs in marine nitrogen budgets is poorly understood, primarily due to the absence of appropriate technology for assessing the in situ distribution and abundance of these microorganisms. We describe here the application of immunofluorescence microscopy for identifying and characterizing marine N2-fixing microorganisms. Polyclonal antibodies raised against the Fe-S protein of the nitrogenase enzyme complex from the bacterium Rhodospirillum rubrum were demonstrated to recognize nitrogenase from both marine and freshwater N2-fixing bacteria and cyanobacteria. Initial tests of the antibodies were conducted with immunoblots of cell lysates and indicated monospecificity for nitrogenase. We also conducted immunofluorescence assays on paraformaldehyde-fixed whole bacterial cells made permeable with lysozyme, allowing microscopic examination and identification of cells containing the enzyme. Immunofluorescence microscopy also revealed the localization of nitrogenase within cyanobacterial heterocysts or filaments. The approach and techniques described here provide reliable means to determine the presence, relative abundance, and distribution of N2-fixing microorganisms in natural microbial assemblages.