Developmental potential of Frankia vesicles

Abstract

The ability of nitrogenase-containing Frankia sp. strain CpIl vesicles to regrow vegetative hyphae is demonstrated. Vesicles attached to hyphae in N2-fixing Cpll cultures and sucrose gradient-isolated vesicles exhibited hyphal outgrowths when incubated in certain defined liquid media. Single or multiple hyphal extensions grew out from the vesicles.The onset of nitrogen fixation by Frankia strains in aerobic environments is preceded by the appearance of differentiated cellular structures, the frankial vesicles. Vesicles contain nitrogenase, both in culture (8,11,15) and in the N2-fixing root nodules formed on actinorhizal plants (13,18 screw-cap polycarbonate centrifuge tubes. The pellets were suspended in cold 10 mM potassium phosphate buffer (pH 6.5; buffer A) to a final volume of 6 ml and held on ice. Just prior to homogenization, 20 ml of cold 60% (wt/wt) sucrose in buffer A was added to each tube. The contents were homogenized with a Polytron tissue homogenizer in an ice bath for five cycles of 1-min homogenization followed by a 30-s rest to allow cooling of the suspension. Each suspension was examined microscopically to monitor the release of vesicles from the vegetative hyphae. Following homogenization, an additional 9 ml of cold 60% sucrose was added to each tube to yield a final concentration of 50% sucrose. The homogenates were overlayered with cold 10% (vol/vol) glycerol in buffer A, and the glycerol-sucrose gradients were centrifuged at 35,000 x g for 1 h at 4°C. Vesicles were removed from the glycerol-sucrose interface with Pasteur pipettes and washed by repeated centrifugation in buffer A at 40,000 x g for 15 min at 4°C.Regrowth potential of the isolated vesicles was tested by diluting the vesicles to about 105/ml in growth medium in 15-ml serum vials (Wheaton Scientific, Millville, N.Y.), and incubating them in air at 30°C. Vesicle concentration was kept low to avoid ambiguity in identification of the source of new hyphae. The percentage of vesicles with hyphal outgrowths was determined from direct counts as the ratio of the number of regrown vesicles to the total number of vesicles in several microscopic fields at 400x magnification with the phase-contrast optics of an Olympus BH-2 microscope (Olympus Corp., Woodbury, N.Y.). An Olympus OM-1 camera mounted on the microscope was used to document vesicle regrowth.Vesicles formed hyphal outgrowths when incubated as described above in the following media: succinate (0.5%) with and without NH4Cl (11), propionate (0.3%) or pyruvate (0.3%) with and without NH4Cl (9), QMOD medium (6) without lipid supplement, and R-2A broth (12). The percentages of regrown vesicles after 5 days were as follows: 8% and 10% for succinate medium with and without NH4Cl, respectively; 3% and 2% for propionate medium with and without NH4Cl, respectively; and <1% for the pyruvatecontaining media, the QMOD medium, and the R-2A broth. Regrowth was also observed in these same media solidified with agar (1.5%; Difco Laboratories, Detroit, Mich.) or with 1.0% agar and 6.0% gelatin (bo...

The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules

“…In contrast, hyphae accumulated in the gradient pellet when a polytron was used for mycelial disruption in Frankia CpIl (Noridge and Benson 1986). Previous workers have reported on a short hyphal piece attached to the isolated Frankia vesicles (Tisa and Ensign 1987;Puppo et al 1989;Schultz and Benson 1989). This was also observed in the present work with symbiotic material.…”

Biology of Frankia strains, actinomycete symbionts of actinorhizal plants.

“…During senescence, internal septa begin to become irregular, nitrogenase activity is lost, and they become ghosts which are recognizable in culture as deformed, thin-walled, nonfunctioning cells (60,88). Although the main physiological significance of vesicles is their nitrogenase function, a proportion of vesicles have also been observed to "germinate" and produce hyphae in culture (219). Both isolated vesicles and those still attached to parental hyphae can grow through the vesicle envelope or from the vesicle stem.…”

“…Vesicles isolated from N2-grown cultures of HFPCpI1 contain, in addition to nitrogenase, both forms of glutamine synthetase (GSI and GSII) and GOT but not GOGAT (218,219,292). The presence of GS activity and the absence of GOGAT activity suggest that vesicles may be capable of forming glutamine from ammonia and glutamate but may not be capable of converting glutamine to glutamate.…”

Producción de dos cepas de Frankia sp. aisladas de Alnus acuminata H.B.K. por fermentación fed-batch

“…Con el sobrenadante se realizó la lectura por conteo en cámara de Neubauer con profundidad de un 1 mm. El criterio de conteo de vesículas maduras fue por refracción y presencia de talo hifal (Schultz, & Benson, 1989…”