Whole-cell immunocytochemical detection of nitrogenase in cyanobacteria: improved protocol for highly fluorescent cells

Abstract

An improved immunocytochemical method was developed to detect nitrogenase in sin- gle cells of cyanobacteria using a unicellular diazotrophic strain (Gloeothece sp. 68DGA) and a non- diazotrophic strain (Synechocystis sp. PCC6714) as model organisms. Polyclonal antibodies raised against the Fe-protein and the MoFe-protein (α-subunit) of nitrogenase were used as probes. The antigenicity of nitrogenase was maintained for at least 6 mo when the cells were preserved in chilled methanol (-30°C) after paraformaldehyde fixation (3%). The cells were permeabilized for antibody penetration and non-specific binding was prevented by incubation in phosphate-buffered saline con- taining dimethyl sulfoxide (10%) and normal rabbit serum (15%). Antibody binding was visualized by a horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate 3-3'- diaminobenzidine tetrachloride because of the difficulty in discriminating between fluorescence from additive fluorochrome and natural autofluorescence from cyanobacteria. Almost all cells (>97%) were immunostained when they were grown diazotrophically and expressed nitrogenase (acetylene reduction) activity. Non-specific staining of both the diazotrophic strains grown with combined nitro- gen and the non-diazotrophic strain was negligible. Our protocol was able to detect nitrogenase in unicellular and filamentous non-heterocystous strains without modification.