The function of vesicles in the actinomycete Frankia

The actinomycete Frankia is a symbiotic nitrogen fixer, living in root nodules of many non-leguminous plants. A typical characteristic of this endophytic organism is the formation of specialized swollen cell structures, called vesicles. Frankia vesicles have been brought In relation to nitrogen fixing activity, but many questions about their formation and their function still had to be answered. The present study was done to investigate the function of these vesicles. First, an electronmicros copy study has been initiated to investigate the ultrastructure of Frankia cells. The vesicle envelope was studied in detail, and shown to be a multilaminate structure, which was in accordance with other published results. The vesicle envelope was continuous with the hyphae cell wall. It was also shown that no difference was found in the ultrastructure of vesicles with, or without induced nitrogenase (chapter II). The formation of vesicles in different Frankia strains was investigated by comparing a strain with, and one without vesicle formation in the presence of ammonia. In the presence of ammonia, nitrogenase is generally repressed. The vesicles that were formed in the medium with ammonia did not contain nitrogenase (chapter III).Frankia nitrogenase could be detected by using antisera against nitrogenase from Rhizobium leguminosarum or from Azotobacter vinelandii. The latter appeared to be very specific, and was used for localization of nitrogenase on ultrathin cryosections. It could be shown that nitrogenase is present uniquely in the vesicles in Frankia strains Ccl.17 and EAN1pec (chapters IV-1, IV-2). In these cells, probably a reducing environment is maintained, thus preventing inhibition of nitrogenase by oxygen. In accordance herewith, it has been reported that one Frankia strain (Cc13) was able to fix nitrogen without forming vesicles, under microaerobic conditions. The Influence of microaerobic conditions on the localization of nitrogenase in strain Ccl. 17 was tested. In this strain, no change of the localization of nitrogenase in the vesicles with reduced oxygen partial pressure has been observed (chapter IV-3). From these results, it can be concluded that nitrogenase is generally located within the vesicles, although vesicle formation and nitrogenase induction can be controlled independently in some strains. The mechanism of regulation of the strict localization is still unknown. One of the possibilities of repression of nif- genes is the occurrence of DNA rearrangement during vesicle differentiation. This possibility has been investigated by comparing DNA from vesicle clusters from nitrogen fixing root nodules with DNA from hyphae from the corresponding pure culture. No differences could be detected in the nif -genes from these two preparations. The results mean that the question, how nif- gene expression i s repressed in the hyphae of Frankia, still need to be answered. The progress made in the past years in Frankia molecular genetics promises that many still open questions about regulation mechanisms in Frankia will be answered in the near future.