Strain CpI1 Research Articles

Isolation and nitrogen-fixing activity of Frankia sp. strain CpI1 vesicles.

Under N2-fixing conditions in aerobic culture and in symbiosis, frankiae produce spherical, multicellular structures that have been called vesicles. The vesicles have been proposed as the site of nitrogen fixation. We isolated vesicles by using density centrifugation in a single-step sucrose gradient. Vesicles migrated out of 50% (wt/vol) sucrose and banded at the 40 to 50% sucrose interface; they were intact, as assessed by transmission electron microscopy, and were free of hyphal contamination. Specific activities of nitrogenase in vesicles prepared anaerobically were up to 100-fold greater than the specific activity of the largely hyphal pellet, depending on the recovery of vesicles. All of the activity in the pellet could be accounted for by the number of vesicles present in the pellet. Glutamine synthetase activity in crude extracts of vesicles was extremely low.

14C]methylammonium transport by Frankia sp. strain CpI1.

We describe an NH4+-specific transport system in the N2-fixing symbiotic actinomycete Frankia sp. strain CpI1. [14C]methylammonium was used as an NH4+ analog. No specific transport process was detected when cells were grown on high concentrations of NH4+. A transport system with a high affinity for CH3NH3+ was synthesized after 3 to 4 h of nitrogen starvation. Methylammonium transport was not significantly inhibited by a variety of amino acids, primary amines, and polyamines. Ammonium completely eliminated CH3NH3+ transport. The Km for CH3NH3+ transport was around 2 +/- 1.8 microM with a Vmax of 4 to 5 nmol/min per mg of protein. The electron transport inhibitors cyanide and azide eliminated uptake, as did the uncoupler carbonyl cyanide-m-chlorophenylhydrazone. The sulfydryl reagent p-chloromercuribenzoic acid and the heavy metal thallium also inhibited uptake, suggesting the presence of an NH4+-specific permease. Concentration of CH3NH3+ across the membrane was demonstrated by conducting uptakes at low temperature to slow the metabolism of CH3NH3+ by glutamine synthetase. At 7 degrees C most of the label was concentrated inside the cells in a form that could be chased from the cells by adding excess NH4+ to the medium. At 30 degrees C most of the label was present as an impermeant metabolite. Thin-layer chromatography of cell extracts confirmed that the radioactivity inside the cells was mainly in the form of CH3NH3+ at 7 degrees C but was present as an unidentified metabolite at 30 degrees C. These studies demonstrate that Frankia sp. strain CpI1 has a high-affinity NH4+ transport system that is synthesized in response to NH4+ starvation.