Heterocyst formation and nitrogenase activity in I-tonB2 and I-tonB4. (A) Nitrogenase activity was determined under oxic and anoxic conditions in the wild type (WT), I-tonB2, and I-tonB4. Average values of 9 (wild type), 4 (I-tonB2), and 5 (I-tonB4) measurements on independent cultures are given, and error bars represent the standard deviation. Asterisks denote significant differences between each mutant and the wild type (Student’s t test, P < 0.05). (B) Light microscopy images of cultures grown for 3 days on BG110 medium plates; representative images are shown. (C) The peptidoglycan of cells grown for 7 days on BG110 medium plates was labeled with the fluorescent dye Van-FL that specifically binds to the cell wall (see Materials and Methods for details). Fluorescence images were recoded with a confocal laser scanning microscope; a merge of the Van-FL fluorescence (green) and the chlorophyll autofluorescence (red) is shown. For better visibility, the Van-FL signal and the overall intensity were enhanced.