Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis

Abstract

Incorporation of L_[14C]ornithine into gramicidin S by crude, unfractionated lysozyme extracts of Bacillus brevis ATCC 9999 was shown to represent the activity of the gramicidin synthetase complex. Frozen-thawed cells were the source of active extracts, but when cells were shaken in air at 370C, they rapidly lost activity in a first-order reaction with a half-life of 13 min. Protease inhibitors and inhibitors of energy metabolism had no effect on the inactivation process in frozen-thawed cells. Stabilization was achieved when the cells were shaken in nitrogen or helium instead of air. The addition of dithiothreitol produced a moderate degree of stabilization. The i-ornithineand i-phenylalanine-activating activities of the gramicidin S synthetase complex were also lost during aeration of the cells. Crude cell-free extracts also lost activity when they were shaken in oxygen, but, in this case, inactivation was slower (half-life of 80 min). Nitrogen also stabilized these cell-free extracts. 1010 on August 1, 2020 by guest

Control of antibiotic biosynthesis.

“…The molecular mechanism of inactivation of the antibiotic synthetases is unknown. In vitro incubation of the heavy gramicidin S synthetase at 37°C for 1 h resulted in an 80% loss of gramicidin S-synthesizing activity (46,100). Kleinkauf and Koischwitz (101) reported that this inactivation was due to the presence of a protease in the crude extract; they also observed that the heavy enzyme was divided into subunits of different sizes by proteolytic enzymes and that this could be prevented by adding a mixture of protease inhibitors.…”

“…It is interesting to note that removal of oxygen in vivo by sparging with nitrogen prevents inactivation of gramicidin S synthetase (46,47). Oxygen is required for growth, but it is possible that control of dissolved oxygen, to provide a high degree of oxygen transfer during exponential growth followed by a moderate degree of oxygen transfer during the stationary phase, could improve antibiotic production.…”