Purification and Some Properties of Two Boticins

Abstract

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 Since the initial report, we have detected (unpublished data) bacteriocin activity against C. botulinum type E in filtrates of C. perfringens and C. botulinum types A, B, and E strains as well.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...

Low-Temperature Irradiation of Beef and Methods for Evaluation of a Radappertization Process

“…Our results (Tables 2 and 3) reflect a complex phenomenon which may be due to one or more series of events occurring either simultaneously or sequentially: (i) the secretion into the food substrate by one or more strains of C. botulinum of a boticin-like substance, a bacteriocin, which is usually antagonistic against a limited number of strains of the same or closely related species of organism. Boticin production by type A, B, C, and E strains has been reported previously (1,12,22,37,40). A preliminary investigation in our laboratory seemed to indicate such a possibility with our strains.…”

Boticinogeny and Actions of the Bacteriocin

“…Activity spectrum against vegetative cells. Boticin E kills cells and/or lyses broth cultures of type E (6,12). The turbidity of cultures of six type E strains which had been isolated in this laboratory decreased when the crude boticin preparations were added to final 200 units/ml.…”

“…Trypsin rescue. Boticin E induces a rapid, irreversible lethal response in actively growing cells (6). When the culture is susceptible to clearing (OD decrease) by boticin, contact between cells and boticin results in a similarly rapid, irreversible commitment to cellular degeneration (Fig.…”

“…Immunity may explain the inability to demonstrate an action of the bacteriocin on vegetative cells of the producing strains, but an enzymatic factor could also be operative; i.e., in obtaining crude boticin, heat must be used to destroy a boticin-inactivating factor. The turbidity fluctuations observed in cultures which are producing boticin E could mean that boticin can lyse the producing cells (6) from an internal location.…”

“…The boticin is inactivated by trypsin but not by I hr of heating at 100 C (12). About 80% of the activity in a crude preparation is associated with a substance with a molecular weight less than 30,000 daltons; the remainder is associated with a particle weight greater than 40 x 106 daltons (6).…”