A novel lactococcal bacteriocin whose activity depends on the complementary action of two 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 beta 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.
Nissen-Meyer
J
J
Laboratory of Microbial Gene Technology, NLVF, As, Norway.
Holo
H
H
Håvarstein
L S
LS
Sletten
K
K
Nes
I F
IF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001430:Bacteriocins / Q000737:chemistry*; D013294:Lactococcus lactis / Q000472:pathogenicity*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D001430:Bacteriocins; D046911:Macromolecular Substances