Two bacteriocins (boticins) were elaborated without induction by strain S5, a nontoxigenic variant of Clostridium botulinum type E. After separation of the two active entities by gel filtration on Sephadex G-50, a large particle with boticin activity was isolated by density gradient ultracentrifugation, and a small soluble boticin was purified by continuous curtain electrophoresis and chromatography on sulfoethyl-Sephadex. Large and small boticins were purified 200-and 3,000-fold, respectively, with yields of 50% or more. The small boticin, a basic substance with molecular weight under 30,000, was the predominant species; the large boticin, a negatively charged particle with particle weight greater than 40 x 106, represented less than 20% of the total activity. Both purified boticins were resistant to heat and were attacked by proteolytic enzymes, but the large boticin was less thermostable and less sensitive to proteolytic enzymes than was the smaller variety. The activity of the large boticin was not reduced by treatment with urea or deoxyribonuclease. Both boticins exhibited sporostatic and bactericidal activities for C. botulinum type E, strain 070. A suspension of type E strain 070 vegetative cells was rendered nonviable within 9 min by the small boticin. The lethal action of this bacteriocin was not reversed by trypsin.The production of bacteriocins (boticins) active against toxigenic Clostridium botulinum type E strains by several closely related but nontoxigenic organisms was described in an earlier report from this laboratory (25). Since the initial report, we have detected (unpublished data) bacteriocin activity against C. botulinum type E in filtrates of C. perfringens and C. botulinum types A, B, and E strains as well. Interference of these bacteriocin-producing organisms with methods for the detection or recovery (or both) of toxin-producing type E has been demonstrated (25).The status of our knowledge regarding the bacteriocins has been reviewed frequently (3,9,10,17,21,30,34); the recent reviews of Reeves (34) and Bradley (3) are especially pertinent. Bradley's broad definition of bacteriocins as "bactericidal particles which are unable to multiply in a sensitive indicator" is applied in this report.Many bacteriocinogenic organisms elaborate maximal quantities of bacteriocins only after induction with ultraviolet light, metabolic inhibitors such as mitomycin C, or other agents known to induce lysogenic phages (2,10,13,18,20,24,34,37). Some organisms release bacteriocin continuously (11,26); in other cases, an abrupt release of bacteriocin coincides with partial or complete lysis of the culture (11,14,17,20,24).As Bradley (3) pointed out, the bacteriocins studied to date fall into two distinct categories. One group consists of bacteriocins of small molecular size, which are usually thermostable and cannot be sedimented by ultracentrifugation; bacteriocins of very large size, which are usually thermolabile and can be sedimented easily, make up the second group. The production of a bacteriocin of each...