A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides

Abstract

A lactococcal bacteriocin, termed lactococcin G, was purified to homogeneity by a simple four-step purification procedure that includes ammonium sulfate precipitation, binding to a cation exchanger and octyl-Sepharose CL4B, and reverse-phase chromatography. The final yield was about 20%k, and nearty a 7,000-fold increase in the specific activity was obtained. The bacteriocin activity was associated with three peptides, termed al, a2, and 13, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, al and a2 were the same gene product. Differences in their configurations presumably resulted in a2 having a slightly lower affinity for the reverse-phase column than al and a reduced bacteriocin activity when combined with 13. Bacteriocin activity required the complementary action of both the a and the 1 peptides. When neither al nor 1 was in excess, about 0.3 nM al and 0.04 nM 13 induced 50%1 growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, a1has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 104 M-1 cm-', and a molecular weight of 4,346 (39 amino acid residues long). Similarly, 13 has an isoelectric point of 10.4, an extinction coefficient of 2.4x 104 M-1 cm'1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for al and (, respectively, were obtained by mass spectrometry. The N-terminal halves of both the a and the 13 peptides may form amphiphilic a-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism. The C-terminal halves of both peptides consist largely of polar amino acids.Bacteriocins are proteins that show bactericidal activity towards bacteria that are closely related to the bacteriocinproducing species (25). Because of their potential use as antibacterial agents, bacteriocins have been the subject of much research. In recent years, there has been considerable interest especially in bacteriocins from lactic acid bacteria (LAB), because of their potential as food and feed additives.The LAB bacteriocins appear to be structurally quite different from the colicins of Escherichia coli (7,14). LAB bacteriocins are usually small peptides, seldom containing more than 60 amino acids, while colicins are proteins of 300 to 800 amino acids. On the basis of their structure, LAB bacteriocins may be divided into two groups. The first group contains the so-called lantibiotics, which have been known for a long time (6, 11). Lantibiotics consist of a polypeptide chain that has been posttranslationally modified. The modified amino acids are lanthionine and methyllanthionine and their precursors dehydroalanine and dehydrobutyrine. Among the lantibiotics, nisin is by far the most studied (1, 4, 12), although three new LAB lantibiotics recently have been purified and characterized (15,16,21,24 which does not show any apparent amino acid sequence homology to other isolated LAB bacteri...

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

“…Binding of lactococcin G to cationic exchangers is generally not as efficient as one would expect from the cationic character of the two peptides that constitute lactococcin G, possibly due to interactions between lactococcin G and other components (33). A recovery similar to that obtained with lactococcin G was also obtained when a culture of E. faecium CTC492 was passed through a cation exchanger.…”

“…not as high as that obtained for the pediocin-like bacteriocins, it was higher than that obtained (about 35% binding to the column) in the cation-exchange chromatography step of the earlier standard purification procedure (33). Binding of lactococcin G to cationic exchangers is generally not as efficient as one would expect from the cationic character of the two peptides that constitute lactococcin G, possibly due to interactions between lactococcin G and other components (33).…”

Structure-Function Analysis of Immunity Proteins of Pediocin-Like Bacteriocins: C-Terminal Parts of Immunity Proteins Are Involved in Specific Recognition of Cognate Bacteriocins

“…Bacteriocin assay. Bacteriocin activity was measured by using a microtiter plate assay system, essentially as described previously (24). Each well of a microtiter plate contained 200 l of culture medium with bacteriocin fraction at twofold dilutions and an indicator strain at an OD 610 of about 0.01 (inoculated from a 16-to 20-h overnight culture at 30°C).…”

High-Throughput Isolation of Bacteriocin-Producing Lactic Acid Bacteria, with Potential Application in the Brewing Industry

“…The precipitated proteins were retrieved through centrifugation at 8,000 g for 30 min and resuspended in 20 mM sodium phosphate buffer (pH 6). This suspension was fractionated using three successive chromatography steps: cation exchange, hydrophobic interaction and reversed phase chromatography 31 . Reversed phase chromatography was performed using a Beckman Ultrasphere ODS (5 µm, 4.6 mm × 25 mm) C 18 column on a HPLC system (Beckman).…”