WHOLE-CELL IMMUNOLOCALIZATION OF NITROGENASE IN MARINE DIAZOTROPHIC CYANOBACTERIA, TRICHODESMIUM SPP

The mechanism by which planktonic marine cyanobacteria of the genus Tr ichodesmium fix N-2 aerobically during photosynthesis without heterocy sts is unknown. As an aid in understanding how these species protect n itrogenase, we have developed an immunofluorescence technique coupled to light microscopy (IF-LM) with which intact cyanobacteria can be imm unolabeled and the distribution patterns of nitrogenase and other prot eins 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 abo ut 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 freq uently around the center of the trichome and were rarely found at the ends. On average 15% of over 300 randomly encountered cells examined c ontained 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 t hat for a stationary culture was somewhat constant at a lower level th roughout 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, incubat ion of cultures with 0.5 mu M NH4Cl over 2 days reduced the NI. The IF technique combined with C-14 autoradiography showed that the CO2 fixa tion rate was lower ins nitrogenase-containing cells. The results of t he present study suggest that (i) the IF-LM technique may be a useful tool for in situ protein localization In cyanobacteria, (ii) cell diff erentiation occurs in Trichodesmium and only a small fraction of cells ire a colony have the potential to fix nitrogen, (iii) the photosynth etic activity (CO2 uptake) is reduced if not absent in N-2-fixing cell s, and (iv) variation in the NI mag be a modulator of nitrogen-fixing activity.