Nukacin ISK-1, a bacteriostatic lantibiotic

Abstract

We determined the mode of action of nukacin ISK-1. It did not cause membrane potential dissipation or the efflux of ATP or K(+) ions from the cells of a sensitive bacterial strain; however, it blocked the membrane depolarization activity of nisin. Nukacin ISK-1-treated cells had single arrangements of cells without the formation of a complete septum. A remarkable reduction in cell wall width was also observed, but cytoplasmic content was unaffected. We concluded that nukacin ISK-1 is bacteriostatic.

FIG. 1.

Structure of nisin A (A) and nukacin ISK-1 (B). A-S-A, lanthionine; Abu-S-A, 3-methyllanthionine; Abu, aminobutyrate; Dha, dehydroalanine; Dhb, dehydrobutyrine.

FIG. 2.

Bacteriostatic or bactericidal nature of nukacin ISK-1. CFU were counted (A) and optical density (OD) determined (B). The indicator bacterial strain L. sakei JCM 1157^T was grown to the mid-log phase, and the culture was divided into three aliquots. Control cells (•) were not treated, and cells in the other two aliquots were treated with either nukacin ISK-1 (○) or nisin (▴). The lantibiotics (10× MIC) were added, the turbidity (OD) measured, and the CFU determined by the spread plate method after plating the cells on MRS agar in 10-fold serial dilutions. The arrow indicates the point at which nukacin ISK-1/nisin was added. This experiment was performed twice with high reproducibility. Representative data were used for the figure.

FIG. 3.

Antibacterial action of nukacin ISK-1. (A) Dissipation of the cell membrane potential by nukacin ISK-1 in normal cells (a), nisin A in normal cells (b), and nisin A in nukacin ISK-1-treated cells (c). The dissipation was examined by observing the fluorescence emitted by DiS-C₃ (3,3′-dipropylthiadicarbocyanine iodide). (B) Efflux of ATP molecules from nukacin ISK-1-treated cells (a) and nisin A-treated cells (b), detected by using a Lucifer-HS ATP detection kit. (C) Efflux of K⁺ ions from nukacin ISK-1-treated cells (a) and nisin A-treated cells (b), detected by using potassium-binding benzofuran isophthalate-AM (PBFI). K⁺ efflux is expressed in relation to the total amount of K⁺ released after the addition of 1 μM nisin (100% value). These experiments were carried out with the lantibiotics at 10× MIC, using B. subtilis JCM 1465^T as an indicator. These experiments were performed twice with high reproducibility. Representative data were used for the figure.

FIG. 4.

Overview of transmission electron micrographs of B. subtilis JCM 1465^T cells. (A) The control cells were not treated with any lantibiotics. (B and C) Micrographs of cells that were treated with nukacin ISK-1 (B) and nisin A (C) at 10× MIC for 90 min are shown. (D1 and 2) Enlarged cross sections of control cells (D1) and nukacin ISK-1-treated cells (D2). Bars in panels A and D1, 500 nm.