Initiation and ontogeny of vesicles in cultured Frankia sp. strain HFPArI3

Removal of combined nitrogen from the medium of Frankia sp. strain HFPArI3 induced the formation of specialized structures, called vesicles, which are the proposed site of nitrogen fixation. After 5 to 6 h of culture on N-free medium, newly formed vesicles, termed provesicles, arose from the tips of some hyphae. These structures were spherical, phase dark, ca. 1.5 to 2.0 micron in diameter, and were not associated with acetylene reduction (nitrogenase) activity. Provesicles reached their greatest frequency after ca. 24 h of N-free culture. Provesicles increased in size to become mature vesicles which first appeared after 18 to 20 h of N-free culture. They were ca. 2.5 micron in diameter, phase bright, and reached their greatest frequency after 5 to 6 days, at which time nitrogenase activity peaked. Some vesicles eventually became damaged structurally and took on the appearance of ghosts. Transmission electron micrographs revealed an increase in size from provesicle to mature vesicle. Also evident with the micrographs were the presence of a septum between the young provesicle and parental hypha, the presence of glycogen in some young vesicles, the development of internal septations as vesicles matured, and the degradation of cytoplasm and internal septae in ghost vesicles. The extent to which the formation of vesicles is reversible by the addition of NH4+ was investigated. Commitment times of 3.2 and 6.5 h were obtained for provesicles and vesicles, respectively. A concentration-dependent inhibition of nitrogenase by NH4+ was demonstrated. The structure of preexisting vesicles was also affected by addition of NH4+ to the culture medium.