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...
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 bacteriocin produced by a strain of Staphylococcus aureus has been isolated and designated staphylococcin (414), and a study was made of its chemical, physical, and biological properties. The staphylococcin is released in appreciable quantities after breakage of the cells and can be purified through differential centrifugation and column chromatography. In the native state, it appears to be a lipoprotein-carbohydrate complex with a molecular weight in excess of 200,000. The complex can be dissociated by sodium dodecyl sulfate into smaller subunits which retain activity. The gross chemical and physical properties of the bacteriocin closely resemble those ascribed to certain preparations of cell membranes. Staphylococcin (414) is not a lytic enzyme like lysostaphin and does not have the same spectrum of activity. Like other bacteriocins from gram-positive microorganisms, it does not inhibit any gram-negative bacteria, but does inhibit several other genera.
Staphylococcus aureus 462 is one of three bacteriocin-producing strains selected for study from 200 isolates of staphylococci of animal origin. These bacteriocins are specific in their activity, inhibiting the growth of certain strains of S. aureus and other gram-positive species, but not gram-negative organisms. Staphylococcin 462 was not found in significant concentrations in the supernatant fluid of broth cultures, nor was it released into the suspending liquid when the cells were mechanically disrupted. However, extraction of the cells with 7 M urea resulted in the liberation of much of the activity. The material was purified by gel permeation chromatography by using Sephadex G-200 and by preparative electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Chemical analysis showed that the material consisted of roughly 90% protein and 3% lipid. The molecular weight of sodium dodecyl sulfate-dissociated staphylococcin 462 was calculated to be about 9,000.This investigation is part of a program aimed at characterizing various bacteriocins, especially the staphylococcins, obtained from grampositive bacteria so that they may be compared with each other and with the well-studied colicins. Reeves (11) has recently reviewed the nomenclature, chemistry, genetics, and mode of action of bacteriocins.In a previous paper (4), the screening of 200 isolates of Staphylococcus aureus for bacteriocin production and sensitivity was described. From these cultures, three producing strains (263, 414, and 462) and three indicator strains (19, 140, and 698) were selected for study. An examination of the bacteriocin isolated from strain 414 revealed that it was a lipoprotein complex chemically similar to membranes isolated from S. aureus (4). This paper describes the isolation and characterization of another of these staphylococcins, that produced by strain 462.MATERIALS AND METHODS Source of microorganisms. S. aureus isolates were kindly provided by J. B. Wilson of this department. Cultures were maintained for daily use on brain heart infusion (BHI, Difco Laboratories, Detroit, Mich.) agar slants. Stock cultures were kept in a dried state on porcelain beads (5). Most of the investigation was done with S. aureus 462, but for comparison certain experiments were performed with S. aureus 414 as well.Bacteriocin assays. The killing strength of various preparations was estimated by serially diluting the 634 material in BHI broth and then spotting 0.01 ml of each dilution onto sections of an agar plate. After the drops had dried, the surface of each plate was exposed to chloroform vapor for sterilization and was overlaid with indicator cells as described previously (4). The highest dilution of bacteriocin which inhibited growth, i.e., gave a clear spot in the indicator culture, was considered to contain 1 U of bacteriocin per ml. Thus, the titer of the assayed preparation was the reciprocal of this dilution.Enzymatic studies. Five enzymes at a concentration of 1 mg/ml were tested individually for their ability t...
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