1887

Abstract

Nitrogenase is extremely sensitive to molecular oxygen (O), and unicellular diazotrophic cyanobacteria separate nitrogen (N)-fixation and photosynthesis to protect nitrogenase from O produced by photosynthesis. When grown under 12 h light/12 h dark cycles (LD), the marine unicellular diazotrophic cyanobacterium sp. 68DGA expressed the nitrogenase protein and its activity (acetylene reduction activity) only during the dark phase. However, this strain was able to grow diazotrophically under continuous light (CL). To determine whether nitrogenase synthesis and N-fixation are temporally separated from photosynthesis in the cells that have fully acclimated to CL, the proportion of cells containing nitrogenase (the Fe-protein of nitrogenase) in the culture was measured using an immunocytochemical technique. Cells were grown in a continuous-culture device to maintain constant cell density. Under LD, the cells showed diurnal oscillation of nitrogenase activity, photosynthesis, respiration and the expression and the abundance of the Fe-protein. The oscillation was gradually reduced after the transfer of the cells to CL, and was lost after 23–25 days of cultivation under CL. In CL-acclimated cultures, the Fe-protein was always detected in about 94 % of the cells, although the nitrogenase activity was about one-third of the maximum activity in LD-acclimated cultures. These results suggest that synthesis of nitrogenase proceeds without diurnal oscillation in the CL-acclimated cells of sp. 68DGA. As the respiration rate in CL-acclimated culture was as high as the maximum rate observed in LD-acclimated culture, O-uptake mechanism(s) may have been upregulated to maintain low intracellular pO.

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2008-07-01
2019-10-17
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. Left: oscillation of nitrogenase activity (acetylene reduction activity) in sp. 68DGA cultured under 12h light/12 h dark cycles (LD). Nitrogenase activity was observed only during the dark phase, with the peak around the middle of the dark phase. Right: immunocytochemical detection of nitrogenase (the Fe-protein) in individual cells grown under LD. Cells were collected at 3 h intervals from an LD-acclimated culture, fixed with paraformaldehyde and preserved in methanol at -30 °C. The fixed and preserved cells were treated with DMSO (for permeabilization) and non-immune rabbit serum (for blocking) and then incubated with an anti-Fe-protein antibody. The immunoreaction was visualized with horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate 3,3'-diaminobenzidine tetrachloride (DAB) in the presence of H O . A dark brown deposit of oxidized DAB was observed in nitrogenase-containing cells. The immunostained cells accounted for about 97% of the total cell population in the middle of the dark phase. The proportion of immunostained cells decreased to about 30% during the middle of the light phase, and the staining became faint. [ PDF] (572 kb) Left: nitrogenase activity (acetylene reduction activity) of sp. 68DGA in cells acclimated to continuous light (CL). Nitrogenase activity was observed constantly at the same level. Right: immunocytochemical detection of nitrogenase (the Fe-protein) in individual cells acclimated to CL. Cells were collected at 4.8 h intervals from a CL-acclimated culture. The conditions for immunodetection of nitrogenase were the same as those in Fig. S1. The dark brown deposit of oxidized DAB was always observed in almost all cells. The mean abundance of the immunostained cells in the CL-acclimated culture was 93.8±5.4% (mean for 72 h during CL). [ PDF] (791 kb)

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. Left: oscillation of nitrogenase activity (acetylene reduction activity) in sp. 68DGA cultured under 12h light/12 h dark cycles (LD). Nitrogenase activity was observed only during the dark phase, with the peak around the middle of the dark phase. Right: immunocytochemical detection of nitrogenase (the Fe-protein) in individual cells grown under LD. Cells were collected at 3 h intervals from an LD-acclimated culture, fixed with paraformaldehyde and preserved in methanol at -30 °C. The fixed and preserved cells were treated with DMSO (for permeabilization) and non-immune rabbit serum (for blocking) and then incubated with an anti-Fe-protein antibody. The immunoreaction was visualized with horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate 3,3'-diaminobenzidine tetrachloride (DAB) in the presence of H O . A dark brown deposit of oxidized DAB was observed in nitrogenase-containing cells. The immunostained cells accounted for about 97% of the total cell population in the middle of the dark phase. The proportion of immunostained cells decreased to about 30% during the middle of the light phase, and the staining became faint. [ PDF] (572 kb) Left: nitrogenase activity (acetylene reduction activity) of sp. 68DGA in cells acclimated to continuous light (CL). Nitrogenase activity was observed constantly at the same level. Right: immunocytochemical detection of nitrogenase (the Fe-protein) in individual cells acclimated to CL. Cells were collected at 4.8 h intervals from a CL-acclimated culture. The conditions for immunodetection of nitrogenase were the same as those in Fig. S1. The dark brown deposit of oxidized DAB was always observed in almost all cells. The mean abundance of the immunostained cells in the CL-acclimated culture was 93.8±5.4% (mean for 72 h during CL). [ PDF] (791 kb)

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