Kawai Fumitaka, Shoda Momoko, Harashima Rie, Sadaie Yoshito, Hara Hiroshi, Matsumoto Kouji
Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-ohkubo, Sakura, Saitama, Saitama 338-8570, Japan.
J Bacteriol. 2004 Mar;186(5):1475-83. doi: 10.1128/JB.186.5.1475-1483.2004.
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.
最近，使用心磷脂（CL）特异性荧光染料10-N-壬基吖啶橙（NAO）揭示了大肠杆菌膜中富含CL的结构域（E. Mileykovskaya和W. Dowhan，《细菌学杂志》182: 1172 - 1175，2000年）。用NAO对枯草芽孢杆菌细胞进行染色显示，在隔膜区域和两极存在绿色荧光结构域。在缺乏可检测到的CL的clsA破坏突变体的指数生长期细胞中，这些荧光结构域几乎检测不到。在具有野生型脂质组成的芽孢形成细胞中，在极隔膜以及吞噬膜和前芽孢膜上观察到荧光结构域。在clsA破坏突变体以及CL合酶的所有三个同源基因（clsA、ywjE和ywiE）均被破坏的突变体中，这些结构域并未消失，而是在芽孢形成起始后稍后出现。由于两个染料分子的堆积导致的荧光红移以及脂质组成表明，突变体的芽孢形成细胞中存在少量CL。质谱分析揭示了这些突变体细胞中存在CL。在突变体芽孢形成的后期阶段，热处理后能够形成菌落的耐热细胞频率较低。这些细胞在芽孢形成起始后24小时的芽孢形成频率为野生型频率的30%至50%。这些结果表明，即使在所有三个同源基因均被破坏的枯草芽孢杆菌突变体中，在芽孢形成阶段，富含CL的结构域也存在于极隔膜膜以及吞噬膜和前芽孢膜中，在野生型细胞的指数生长期，也存在于中间隔膜膜和两极的膜中。结果进一步表明，极隔膜膜以及吞噬膜和前芽孢膜中的富含CL的结构域参与芽孢形成。
