Immunolocalization and Western Blot Analysis of Nitrogenase in Oscillatoria limosa During a Light‐dark Cycle

Abstract

AbstractNitrogenase of the non‐heterocystous nitrogen‐fixing cyanobacterium Oscillatoria limosa was subjected to western blot analysis and immunogold electron microscopy using antisera raised against dinitrogenase (MoFe‐protein, Component I) and dinitrogenase reductase (Fe‐protein, Component II). O. limosa was grown diazotrophically under an alternating light‐dark cycle (16–8h light‐dark). Although nitrogenase activity (acetylene reduction) was found predominantly during the dark phase, being absent during most of the light period, immunogold electron microscopy revealed label of both subunits of nitrogenase in samples taken throughout the light‐dark cycle. It was also shown that the nitrogenase label was distributed homogeneously in the cell and that it was present in every cell of every trichome whether fixing nitrogen or not. On average, 34 (± 6) gold particles μm−2 thin section were detected. Nitrate‐grown cells did not contain nitrogenase label. Western blot analysis of the Fe‐protein in samples taken during the light phase, revealed a single band with an apparent molecular weight of 37 kDa. At the end of the light period, and during the dark phase when high nitrogenase activities were observed, an additional band of 36 kDa was found. The anti‐MoFe‐protein antiserum revealed a single band of 56 kDa which was present throughout the light‐dark cycle. Nitrate‐grown cells were not recognized by either antiserum. It is concluded that nitrogenase enzyme is present in O. limosa throughout the light‐dark cycle but that the Fe‐protein is modified (inactive form) during the light period when nitrogenase activity is absent.