Production and Mode of Action of Lactocin 27: Bacteriocin from a Homofermentative Lactobacillus

Abstract

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...

Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481

“…A narrow inhibitory spectrum, proteinaceous nature, and bactericidal mode of action are typical characteristics of antimicrobial agents classified as *bacteriocins (23). Very few lactobacifli have been confirmed to produce true bacteriocins within these criteria (3,4,6,25,26). Only a single bacteriocin has been identified and characterized within the L. helveticus species.…”

Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J

“…Bacteriocin genes are typically plasmid borne (43), but the genetic determinants encoding helveticin J appear to reside on the chromosome (16). Evidence for chromosomally located bacteriocin genes has also been reported for lactacin B produced by Lactobacillus acidophilus N2 (2) and lactocin 27 produced by L. helveticus LS18 (44).…”

Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp

“…Barefoot and Klaenhammer (4) reported that 63% of the Lactobacillus acidophilus strains they examined produced bacteriocins. Bacteriocins produced by L. acidophilus 11088, L. acidophilus N2, Lactobacillus helveticus 481, and L. helveticus LP27 have been purified and shown to constitute a heterogeneous class of antimicrobial peptides (5,25,33,42). Although efforts have been made to characterize the proteinaceous nature of these bacteriocins, limited information is available about the genetic determinants of bacteriocins from lactobacilli or other LAB.…”