Summary: Cultures of sp. strain 321 grown in a layer of soft agar on top of a layer of harder agar developed maximum nitrogenase activity [80 to 90 nmol acetylene reduced h (mg protein)] after 12 d in air at 28 °C. Electron microscopy of sections of cores of the soft agar layer showed differences in the morphology of rhizobia in colonies growing at the surface, in the middle or at the bottom of the layer. The properties of rhizobia growing in the colonies in the middle of the soft agar layer suggested that only these contained nitrogenase. These rhizobia were present as a distinct band of cells passing through individual colonies at a constant depth in the agar. They were larger than normal vegetative Rhizobium, were pleiomorphic and were similar in morphology to the nitrogen-fixing bacteroids formed by strain 321 in cowpea root nodules. Their location within the soft agar layer changed with the O concentration in the atmosphere above the agar during growth, and they were not found in cultures showing little or no nitrogenase activity. Loss of nitrogenase activity occurred when cultures were grown at 33 °C, in a O/Ar (1:4, v/v) atmosphere, or when the concentration of combined nitrogen in the agar medium was increased from 2 to 20 m-N.


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