A NOVEL LACTOCOCCAL BACTERIOCIN WHOSE ACTIVITY DEPENDS ON THE COMPLEMENTARY ACTION OF 2 PEPTIDES

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 CL-4B, and reverse-phase chromatography. The final yield was about 20%, and nearly a 7,000-fold increase in the specific activity was obtained. The bacteriocin activity was associated with three peptides, termed alpha-1, alpha-2, and beta, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, alpha-1, and alpha-2 were the same gene product. Differences in their configurations presumably resulted in alpha-2 having a slightly lower affinity for the reverse-phase column than alpha-1, and a reduced bacteriocin activity when combined with beta. Bacteriocin activity required the complementary action of both the alpha and the beta-peptides. When neither alpha-1, nor beta was in excess, about 0.3 nM alpha-1 and 0.04 nM beta-induced 50% growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, alpha-1, has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 10(4) M-1 cm-1, and a molecular weight of 4,346 (39 amino acid residues long). Similarly, beta has an isoelectric point of 10.4, an extinction coefficient of 2.4 x 10(4) M-1 cm-1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for alpha-1, and beta, respectively, were obtained by mass spectrometry. The N-terminal halves of both the alpha and the 13 peptides may form amphiphilic alpha-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.