Effects of long-term preservation of frankia strains on infectivity, effectivity, and in vitro nitrogenase activity.

Frankia strain HFP ArI3 which had been preserved for 27 months by being lyophilized, frozen in glycerol, or stored in complex medium was successfully used as an inoculum after being subcultured for inducing nodulation and nitrogen fixation of Alnus rubra. Glycerol-preserved HFP ArI3 produced significantly lower rates of nitrogenase activity than did lyophilized or complex-medium-preserved inocula. Bacteria that had been preserved by all three methods were successfully induced to fix atmospheric nitrogen by being cultured in nitrogen-free medium. Subculturing of these cells in nitrogen-free medium a second and third time yielded increasing rates of nitrogenase activity. Initial nitrogenase activity was detected on days 5, 4, and 3 during the first, second, and third subcultures after preservation, respectively. Maximum activity was observed on days 11, 10, and 8 during the first, second, and third subcultures, respectively. A description is given of standard culture techniques used in our laboratory for Frankia isolates, and methods used to distribute Frankia cultures by mail are described.

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.

Oxygen protection of nitrogenase in Frankia sp. HFPArI3.

O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O2 uptake and acetylene reduction rates in response to substrate concentration. O2 of NH4+-grown cells showed an apparent KmO2 of approximately 1 microM O2. In N2-fixing cultures a second Km O2 of about 215 microM O2 was observed. Thus, respiration remained unsaturated by O2 at air-saturation levels. In vivo, the apparent Km for acetylene was more than 10-fold greater than reported in vitro values. These data were interpreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.