Whole-cell immunolocalization of nitrogenase in marine diazotrophic cyanobacteria, trichodesmium spp.

The mechanism by which planktonic marine cyanobacteria of the genus Trichodesmium fix N2 aerobically during photosynthesis without heterocysts is unknown. As an aid in understanding how these species protect nitrogenase, we have developed an immunofluorescence technique coupled to light microscopy (IF-LM) with which intact cyanobacteria can be immunolabeled and the distribution patterns of nitrogenase and other proteins can be described and semiquantified. Chilled ethanol was used to fix the cells, which were subsequently made permeable to antibodies by using dimethyl sulfoxide. Use of this technique demonstrated that about 3 to 20 cells (mean +/- standard deviation, 9 +/- 4) consecutively arranged in a Trichodesmium trichome were labeled with the nitrogenase antibody. The nitrogenase-containing cells were distributed more frequently around the center of the trichome and were rarely found at the ends. On average 15% of over 300 randomly encountered cells examined contained nitrogenase. The percentage of nitrogenase-containing cells (nitrogenase index [NI]) in an exponential culture was higher early in the light period than during the rest of the light-dark cycle, while that for a stationary culture was somewhat constant at a lower level throughout the light-dark cycle. The NI was not affected by treatment of the cultures with the photosynthetic inhibitor dichloro 1,3'-dimethyl urea or with low concentrations of ammonium (NH4Cl). However, incubation of cultures with 0.5 &mgr;M NH4Cl over 2 days reduced the NI. The IF technique combined with 14C autoradiography showed that the CO2 fixation rate was lower in nitrogenase-containing cells. The results of the present study suggest that (i) the IF-LM technique may be a useful tool for in situ protein localization in cyanobacteria, (ii) cell differentiation occurs in Trichodesmium and only a small fraction of cells in a colony have the potential to fix nitrogen, (iii) the photosynthetic activity (CO2 uptake) is reduced if not absent in N2-fixing cells, and (iv) variation in the NI may be a modulator of nitrogen-fixing activity.