Cardiolipin domains in Bacillus subtilis marburg membranes

Abstract

Recently, use of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) revealed CL-rich domains in the Escherichia coli membrane (E. Mileykovskaya and W. Dowhan, J. Bacteriol. 182: 1172-1175, 2000). Staining of Bacillus subtilis cells with NAO showed that there were green fluorescence domains in the septal regions and at the poles. These fluorescence domains were scarcely detectable in exponentially growing cells of the clsA-disrupted mutant lacking detectable CL. In sporulating cells with a wild-type lipid composition, fluorescence domains were observed in the polar septa and on the engulfment and forespore membranes. Both in the clsA-disrupted mutant and in a mutant with disruptions in all three of the paralogous genes (clsA, ywjE, and ywiE) for CL synthase, these domains did not vanish but appeared later, after sporulation initiation. A red shift in the fluorescence due to stacking of two dye molecules and the lipid composition suggested that a small amount of CL was present in sporulating cells of the mutants. Mass spectrometry analyses revealed the presence of CL in these mutant cells. At a later stage during sporulation of the mutants the frequency of heat-resistant cells that could form colonies after heat treatment was lower. The frequency of sporulation of these cells at 24 h after sporulation initiation was 30 to 50% of the frequency of the wild type. These results indicate that CL-rich domains are present in the polar septal membrane and in the engulfment and forespore membranes during the sporulation phase even in a B. subtilis mutant with disruptions in all three paralogous genes, as well as in the membranes of the medial septa and at the poles during the exponential growth phase of wild-type cells. The results further suggest that the CL-rich domains in the polar septal membrane and engulfment and forespore membranes are involved in sporulation.

FIG. 1.

Staining of wild-type B. subtilis cells with NAO. Wild-type B. subtilis 168 cells were cultivated in DSM. The cells were harvested during exponential and in the sporulation phase (at T₂and T₄) and stained with 100 nM NAO (Molecular Probes) for 20 min at room temperature to visualize CL. Fluorescence images of exponential-phase cells (A) and of sporulation-phase cells at T₂ (B) and T₄ (C) were taken by using the GFP(R)-BP filter unit (excitation at 460 to 500 nm and emission at 510 to 560 nm) as described in Materials and Methods. Corresponding phase-contrast images (D, E, and F) are also shown. The exposure times used for the fluorescence and phase-contrast images were 0.3 and 0.02 s, respectively. The single arrow indicates a sharp fluorescent band in the center of a cell. Two-fluorescent-dot structures in a cell center are indicated by a pair of arrowheads. Regions of NAO-stained nascent poles in cells that just separated are indicated by pairs of arrows.

FIG. 2.

Staining of clsA-disrupted mutant BFS219 cells with NAO. BFS219 cells were cultivated in DSM, harvested during exponential growth and in the sporulation phase at T₄, and stained with 100 nM NAO for 20 min. Fluorescence images of exponential-phase cells (A) and sporulation-phase cells at T₄ (B) were obtained as described in Materials and Methods. Corresponding phase-contrast images (C and D) are also shown. The exposure times used for the fluorescence and phase-contrast images were 0.3 and 0.02 s, respectively.

FIG. 3.

Staining of triply disrupted mutant SDB206 cells with NAO and FM4-64. Wild-type strain 168 (A-1 to A-3 and D-1 to D-3), strain BFS219 (B-1 to B-3 and E-1 to E-3), and strain SDB206 (C-1 to C-3 and F-1) cells were cultivated in DSM, harvested during exponential growth(A-1, B-1, C-1, D-1, E-1, and F-1) and in the sporulation phase at T₂ (A-2, B-2, C-2, D-2, and E-2) and at T₄ (A-3, B-3, C-3, D-3, and E-3), and stained with NAO (A-1 to A-3, B-1 to B-3, and C-1 to C-3) or FM4-64 (D-1 to D-3, E-1 to E-3, and F-1) as described in the legend to Fig. 1. Corresponding phase-contrast images are below the stained images. The exposure times used for the NAO fluorescence and phase-contrast images were 0.2 to 0.8 and 0.02 s, respectively. The exposure times used for the FM4-64 fluorescence images were 3.3 to 4.5 s. The images of the wild-type cells (G-1-N and G-1-F) stained with both NAO and FM4-64 were obtained by using a GFP(R)-BP filter unit (G-1-N) and a G-2A filter unit (G-1-F) to detect the fluorescence of NAO and FM4-64, respectively. The exposure times used for the NAO and FM4-64 fluorescence images in panels G-1-N and G-1-F were 0.8 and 1.9 s, respectively.

FIG. 4.

Red fluorescence from NAO bound to CL of stationary-phase cells of mutant SDB206. Wild-type strain 168 cells during vegetative growth (A-1 to A-3) and in the sporulation phase at T₄ (B-1 to B-3) and SDB206 cells in the sporulation phase at T₄ (C-1 to C-3) were harvested and stained with NAO as described in the legend to Fig. 1. Green fluorescence (emission at 525 nm) (A-1, B-1, and C-1) and red fluorescence (emission at 640 nm) (A-2, B-2, and C-2) were detected by using a GFP(R)-BP filter unit (excitation at 460 to 500 nm and emission at 510 to 560 nm) and a filter unit with excitation at 450 to 490 nm and emission at 610 nm, respectively, as described in Materials and Methods. The exposure times used for green and red fluorescence were 0.3 and 7.7 s, respectively. (A-3, B-3, and C-3) Colocalization of green and red fluorescence images.