Lactobacillus helveticus strain LP27 produced a bacteriocin, lactocin 27 Bacteriocins are antibiotic-like proteinaceous substances synthesized by certain strains of bacteria and are active against closely related species. The colicins produced by Escherichia coli and other species of Enterobacteriaceae have been studied extensively (15, 17). The recent studies with colicins (E1, E2, E., and K) suggest that the colicins manifest their biochemical effect(s) by interacting with their target directly (1,2,3,4,16,18). The specificity of colicins for E. coli and other closely related species, however, lies at the level of the receptor (19). Similar in vitro studies have not been conducted for the bacteriocins from grampositive bacteria.Little is known about the bacteriocins of lactobacilli. DeKlerk and Smit (8) characterized a bacteriocin from a heterofermentative Lactobacillus fermenti, but the mode of action was not studied. We previously characterized lactocin 27 from a homofermentative Lactobacillus helveticus, strain LP27 (20). The present paper describes aspects of production and the mode of action of lactocin 27. MATERIALS AND METHODSMedia, bacterial strains, isolation, and purification of lactocin. Isolation of lactocin 27, assay of its activity in arbitrary units, and lactocinogenic-and lactocin-susceptible strains have been described in a previous paper (20). In the experiments described here, partially purified lactocin (referred to as lactocin 27) was used rather than purified lactocin. The latter had an undetermined amount of bound sodium dodecyl sulphate (SDS). Portions of lactocin 27 in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 8.7), containing 1 mg of protein per ml, as determined by the Lowry method (14), were stored at -20 C. Once thawed, the solution could be stored in the refrigerator and used over several weeks without a significant drop in activity. The various bacterial suspensions used in this work were made up in sterile Ringer solution prepared by first dissolving 2.15 g of NaCl, 0.075 g of KCl, 0.12 g of CaCl2, and 0.5 g of Na,S20, .5H20 per liter of distilled water, then diluting fourfold before use.Permeation of lactocin. A section of thin-wall dialysis tubing (Union Carbide, Chicago. Ill.) was cut open to give a flat piece of dialysis membrane (3 by 10 cm), washed with distilled water, and sterilized by autoclaving. The dialysis membrane was placed aseptically on an APT (Difco, Detroit, Mich.) agar plate containing 0.01% sodium azide. The azide is not essential but greatly reduces the chances of mold contamination. Two sterilized cylinders (inner diameter 2 cm) were placed 3 to 4 cm apart over the dialysis membrane. Lactocin 27 (0.1 ml) and an exponentially growing culture of L. helveticus strain LP27 (0.1 ml) were mixed separately with 3 ml of APT soft agar (APT broth containing 0.75% agar). Two drops of soft agar containing lactocin 27 and L. helveticus strain LP27 cells, respectively, were placed inside the hollow cylinders over the dialysis membrane and also on t...

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Nearly 100 isolates of Lactobacillus were obtained from human and animal sources. Screening tests with the isolates revealed seven possible bacteriocinogenic strains and 26 strains sensitive to one or more of these inhibitory strains.

The hydrolytic action of the bee venom phospholipase A2 on phosphatidylcholine bilayers is studied under a variety of conditions that introduce alterations in the packing, such as those induced by sonication, gel to liquid crystalline phase transition, and osmotic shock. Two phases of hydrolysis could be resolved under a wide range of experimental conditions. With the various forms of the bilayers one observes only a partial hydrolysis of the total available substrate during the first phase. However, the fraction of the substrate hydrolyzed in the first phase changes with the form of the available substrate, with the amount of the enzyme added, with the temperature, with the phase transition characteristics of the substrate, and by the sonication of the substrate. The second phase of hydrolysis is generally observed when a certain concentration of the products has been produced during the first phase of hydrolysis. These obervations are interpreted to suggest that the bee venom phospholipase A2 preferentially catalyzes hydrolysis of the substrate available at or near the defects in the organization of the substrate in the bilayers.

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