Purification of Two Clostridium Bacteriocins by Procedures Appropriate to Hydrophobic Proteins

Abstract

Two clostridocins distinguishable by their different modes of action on Clostridium pasteurianum have been isolated, namely, butyricin 7423 found in cultures of Clostridium butyricum NCIB 7423 and perfringocin 11105 produced by Clostridium perfringens type A, NCIB 11105. Both were trypsin-susceptible proteins which were soluble in concentrated aqueous ethanol and were able to bind large amounts of the nonionic detergent Triton X-100. In the presence of Triton X-100, butyricin 7423 behaved as a hydrophobic protein in being concentrated in the polyethylene glycol layer of a three-phase partition system of dextran-Ficoll-polyethylene glycol. Their capacity to bind Triton X-100 was exploited in a purification procedure applicable to both bacteriocins. After aqueous ethanol extraction of an ammonium sulfate-precipitated fraction (and, in the case of the perfringocin, a heat-treatment step), a bacteriocin-Triton X-100 adduct was purified by gel filtration through Sepharose 6B. The bacteriocin was then freed of Triton X-100 by chromatography on Sephadex LH-20. Samples of butyricin 7423 purified in this way from different sources contained variable amounts of carbohydrate. Yet sodium dodecyl sulfate-gel electrophoresis revealed the existence of a polypeptide component of 32,500 daltons (±10%), which displayed the biological activity of butyricin 7423 in the absence of any detectable associated carbohydrate (or lipid). Preparations of perfringocin 11105 contained no carbohydrate or lipid and migrated in sodium dodecyl sulfate-gel electrophoresis as a single protein component of 76,000 daltons (±10%). It was concluded that both bacteriocins behave as amphiphilic proteins, and some implications of this finding are considered.

Butyricin 7423: a Bacteriocin Produced by Clostridium butyricum NCIB7423

“…Its reactivation by treatment with Triton X-IOO suggests that this might be some multimeric aggregate or, and perhaps more likely, a complex of butyricin with material derived from the cell envelope of C. pasteurianum. In contrast, butyricin 7423 is secreted in an active form by its producer organism C. butyricum NCIB7423 (Clarke et al, 1975). The pattern of its adsorption suggests the existence of at least two classes of butyricin receptor sites, namely a number of irreversible binding sites supplemented by a larger number of reversible binding sites.…”

“…When secreted into the culture medium of the producer organism, butyricin 7423 is associated with carbohydrate, but it has been partially purified and shown to be a trypsin-sensitive, amphiphilic protein. Sodium dodecyl sulphate gel electrophoresis of purified butyricin 7423 revealed the existence of a polypeptide component of 32500 ( & 10 %) daltons, which displayed the biological activity of butyricin 7423 in the absence of any detectable carbohydrate or lipid (Clarke et al, 1975). In this paper we report the limited spectrum of activity of purified butyricin 7423 and describe its bactericidal action on C. pasteurianum, which is attributed to damage wrought at the cell membrane.…”

“…A concentration of 0.9 a.u./107 organisms was sufficient to cause complete inhibition of growth (Fig. I) and only when a large quantity of Sensitivity to butyricin 7423 was assayed by the diffusion zone method (Clarke et al, 1975) and is expressed as a percentage of the sensitivity displayed by the standard assay organism Clostridium pasteurianum ATCC~OI 3. butyricin (16 a.u./107 organisms) was added to a culture of low density in early exponential phase, was there any evidence of even slight bacterial lysis.…”

“…The culture was incubated at 37 "C under an atmosphere of N2-CO, (95 : 5, v/v) until early stationary phase, when the 35S-labelled butyricin 7423 in the culture supernatant was concentrated and purified as previously described by Clarke et al (1975).…”

“…We have recently discovered a bacteriocin produced by Clostridium butyricum ~~1~7 4 2 3 (and hence named butyricin 7423) whose action on Clostridium pasteurianum is readily distinguishable from that of perfringocin I I 105 (Clarke, Robson & Morris, 1975). When secreted into the culture medium of the producer organism, butyricin 7423 is associated with carbohydrate, but it has been partially purified and shown to be a trypsin-sensitive, amphiphilic protein.…”