Characteristics of Penicillinase Release by Washed Cells of Bacillus licheniformis

Abstract

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly p H-dependent, occurring at a faster rate at alkaline p H in anionic or cationic buffers than at neutral p H. A strongly p H-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the p H to 6.0. Within 30 min at p H 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at p H 9.0 for 90 min. Penicillinase released at p H 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the p H to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the p H-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10 −4 m . Deoxycholate and Triton X-100 were active at 10 −3 m , but tungstate, arsenate, and molybdate had small effects at 10 −1 m . The rate of exopenicillinase release at p H 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at p H 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.