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 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 α1, α2, and β, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, α1 and α2 were the same gene product. Differences in their configurations presumably resulted in α2 having a slightly lower affinity for the reverse-phase column than α1 and a reduced bacteriocin activity when combined with β. Bacteriocin activity required the complementary action of both the α and the β peptides. When neither α1 nor β was in excess, about 0.3 nM α1 and 0.04 nM β induced 50% growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, α1 has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 104 M-1 cm-1, and a molecular weight of 4,346 (39 amino acid residues long). Similarly, β has an isoelectric point of 10.4, an extinction coefficient of 2.4 x 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 α1 and β, respectively, were obtained by mass spectrometry. The N-terminal halves of both the α and the β peptides may form amphiphilic α-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.