Phosphatidylethanolamine domains and localization of phospholipid synthases in Bacillus subtilis membranes

Abstract

Application of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange has recently revealed CL-rich domains in the septal regions and at the poles of the Bacillus subtilis membrane (F. Kawai, M. Shoda, R. Harashima, Y. Sadaie, H. Hara, and K. Matsumoto, J. Bacteriol. 186:1475-1483, 2004). This finding prompted us to examine the localization of another phospholipid, phosphatidylethanolamine (PE), with the cyclic peptide probe, Ro09-0198 (Ro), that binds specifically to PE. Treatment with biotinylated Ro followed by tetramethyl rhodamine-conjugated streptavidin revealed that PE is localized in the septal membranes of vegetative cells and in the membranes of the polar septum and the engulfment membranes of sporulating cells. When the mutant cells of the strains SDB01 (psd1::neo) and SDB02 (pssA10::spc), which both lack PE, were examined under the same conditions, no fluorescence was observed. The localization of the fluorescence thus evidently reflected the localization of PE-rich domains in the septal membranes. Similar PE-rich domains were observed in the septal regions of the cells of many Bacillus species. In Escherichia coli cells, however, no PE-rich domains were found. Green fluorescent protein fusions to the enzymes that catalyze the committed steps in PE synthesis, phosphatidylserine synthase, and in CL synthesis, CL synthase and phosphatidylglycerophosphate synthase, were localized mainly in the septal membranes in B. subtilis cells. The majority of the lipid synthases were also localized in the septal membranes; this includes 1-acyl-glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, diacylglycerol kinase, glucolipid synthase, and lysylphosphatidylglycerol synthase. These results suggest that phospholipids are produced mostly in the septal membranes and that CL and PE are kept from diffusing out to lateral ones.

FIG. 1.

Visualization of PE-rich domains in B. subtilis cells with Ro. (A) Analysis with mutant cells lacking PE. Wild-type B. subtilis 168 cells (A-1) and the cells of the mutant strains lacking PE, SDB01 (psd1::neo) (A-2) and SDB02 (pssA10::spc) (A-3), were cultivated in DSM at 37°C and harvested in the early stationary phase to visualize PE. The cells were fixed in 4.4% (wt/vol) paraformaldehyde, washed with PBS, applied onto poly-l-lysine-coated microscope slides, washed with PBS, and treated with lysozyme (2 mg/ml). The slides were incubated in 0.5% (wt/vol) BSA-PBS containing 2 μg of biotinylated Ro/ml for 20 min, washed, incubated in BSA-PBS containing 5 μg of tetramethyl rhodamine/ml conjugated with streptavidin for 30 min, then washed, and subjected to microscopic observation. Fluorescence images were taken using a G-2A filter unit (510- to 560-nm excitation and 590-nm emission) as described in Materials and Methods. Corresponding phase-contrast images are also shown. Exposure times for fluorescence and phase-contrast images were 0.35 and 0.025 s, respectively. (B) PE-rich domains in sporulating B. subtilis cells. Wild-type B. subtilis 168 cells were cultivated in DSM at 37°C. Cells were harvested in the late exponential growth phase (B-1), and the sporulation phase at T2 (B-2), T3 (B-3), and T4 (B-4). The cells were fixed and processed to visualize the localization of PE as described above. Corresponding phase-contrast images are also shown. Exposure times for fluorescence and phase-contrast images were 0.35 and 0.025 s, respectively.

FIG. 2.

Double staining with NAO and Ro of B. subtilis cells. Wild-type 168 cells in late vegetative growth were harvested and processed with Ro as described in the legend to Fig. 1. After the last wash, NAO at a final concentration of 1 mM was directly added to the washed slides. After incubation for 20 min at room temperature, cells were washed and subjected to microscopic observation. Fluorescence images of NAO (A-1) and tetramethyl rhodamine (A-2) were taken by using a GFP(R)-BP filter unit (460- to 500-nm excitation and 510- to 560-nm emission) and a G-2A filter unit (510- to 560-nm excitation and 590-nm emission), respectively, as described in Materials and Methods. Exposure times for green and red fluorescence were 0.35 and 0.45 s, respectively. (A-3) Colocalization of green (A-1) and red (A-2) fluorescence images.

FIG. 3.

Distribution of PE-dependent fluorescence in E. coli cells. (A) Analysis with mutant cells lacking PE and cells with a reduced PE content. Wild-ype E. coli strain W3110 (A-1), strain GN10 lacking PE (A-2), strain UE81 (A-3), and strain S107 (A-4) cells were cultivated in LB containing MgCl₂ to the early stationary phase. UE81 (ΔpssA10::cat ParaBAD-pssA) cells were cultivated in the absence of l-arabinose to reduce PE content to ca. 20% of total phospholipids. S107 (pssA1) cells were cultivated at 42°C to reduce PE content to ca. 30%. These cells were harvested and processed to visualize PE as described in the legend to Fig. 1. Corresponding phase-contrast images are also shown. Exposure times for fluorescence and phase-contrast images were 0.45 and 0.035 s, respectively. (B) Analysis with deconvolution microscopy. Wild-type E. coli strain W3110 (B-1 and B-2) and B. subtilis 168 strain (B-3) cells were treated with Ro and processed as described in Materials and Methods. The processed samples were subjected to deconvolution microscopy with the ECLIPS TE2000-U fluorescence laser microscope system C1 (Nikon). Nine (E. coli) and seven (B. subtilis) optical z-axis sections (0.1-μm intervals) were collected as raw data and deconvoluted with Metamorph software. The resulting images were processed with Adobe Photoshop, version 6.0.

FIG. 4.

Septal localization of phosphatidylserine synthase, CL synthase, and phosphatidylglycerophosphate synthase in B. subtilis cells. (A) Typical images of localization of GFP fusions. Cells of the B. subtilis strains harboring the gfp fusions in the amyE locus were cultivated in DSM. They were induced with 0.5 mM citrate for PssA-GFP (A-1) or 0.1% xylose for GFP-PgsA (A-2), GFP-ClsA (A-3), and ClsA⁺⁹-GFP (A-4). The cells were harvested in the late logarithmic growth phase and subjected to fluorescence microscopy as described in Materials and Methods. Green fluorescence from the GFP fusions was detected by using a standard GFP(R)-BP filter unit. Exposure times were 3 to 5 s. Cells of SDB1220 harboring the PssA-GFP fusion under the natural promoter were cultivated to late log phase (B-1 and B-2), stage T2 (B-3 and B-4), and stage T3 (B-5). Cells of SDB1209 harboring the ClsA-GFP fusion under the natural promoter were cultivated to late log phase (C-1 and C-2). Exposure times were 2 to 3 s.

FIG. 5.

Septal localization of other lipid synthases in B. subtilis cells. Cells of the B. subtilis strains harboring gfp fusions in the amyE locus were cultivated in DSM up to the late logarithmic growth phase. The cells were harvested and subjected to fluorescence microscopy as described in Materials and Methods. The pDHCMGFP vector (A) and the fusions GpsA-GFP (B) and UgtP-GFP (H) were induced with 0.1 mM citrate. Fusions GFP-YhdO (C), GFP-CdsA (D), GFP-Psd (E), GFP-MprF (F), and GFP-DgkA (G) were induced with 0.1% xylose.

FIG. 6.

FtsZ-dependent localization of PE- and CL-rich domains and phospholipid synthases in B. subtilis cells. (A) Cells of the SDB1010F [amyE::(PcitM-pssA-gfp) Pspac-ftsZ)] strain harboring the fusion gene for PssA-GFP were cultivated in DSM containing 3 mM IPTG and 0.5 mM citrate. Depletion of FtsZ by removal of IPTG gave rise to filamentous cells. The cells were harvested at 0 h (A-1), 2 h (A-2), and 3 h (A-3) after removal of IPTG and subjected to fluorescence microscopy as described in Materials and Methods. The filamentous cells of ASK510 (Pspac-ftsZ) harvested at 4 h after removal of IPTG were subjected to the process for visualization of PE (A-4) as described in Materials and Methods. (B) Cells of the strain SDB1109T [ts1(ftsZ1) amyE::(Pxyl-clsA⁺⁹-gfp)] were cultivated in DSM containing 0.1% xylose. The cells were harvested at 2 h after the temperature shift to 49°C, and localization of ClsA⁺⁹-GFP (B-1) was observed as described in Materials and Methods. The filamentous cells of the ts1 strain harvested at 1 h (B-2) and at 2 h (B-3) after the temperature shift were subjected to the process for visualization of CL. For visualization of PE (B-4), the filamentous cells harvested at 2 h after the temperature increase were processed as described in Materials and Methods.