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.The bacterial cell membrane is widely recognized as a matrix in which lipid molecules are homogeneously distributed. However, it has been noticed that lipid molecules are heterogeneously distributed in bacterial membranes, and the observations increasingly include those obtained by using fluorescent lipophilic probes. Immunoelectron microscopic observations showing the polar localization of the chemoreceptor complexes in Caulobacter crescentus and Escherichia coli cells provided early indications of membrane heterogeneity (1, 27). By using the lipophilic fluorescent styryl dye FM4-64, laterally uneven distribution of the fluorescence, which could be an indication of heterogeneous distribution of phospholipids in E. coli membranes, was then discovered (14). Recently, the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to demonstrate that there are CL-containing domains in E. coli membranes, which were observed mostly in the septal regions and at the poles of the cells (31, 32). The hypothesis that there are CL-containing domains in these regions of E. coli cells was supported by an analysis of the li...

Examination of the lipid composition of spore membranes of Bacillus subtilis Marburg, extracted after treatment of spores with dithiothreitol/urea and NaOH followed by lysozyme digestion, revealed that the spore membranes had significantly higher cardiolipin (CL) content than the membranes of exponentially growing cells. Analysis of the membranes of coat-defective, cotE :: cat and gerE :: cat mutant spores, which are susceptible to lysozyme digestion without chemical treatment, confirmed that spore membranes contain a high level of CL. After addition of the germinants L -alanine or AGFK (a combination of asparagine, glucose, fructose, and KCl), the turbidity of wild type spore suspensions decreased to 50% within 30 min. Suspensions of spores with only trace amounts of CL, however, showed no decrease in turbidity when L -alanine was added and the initial decrease in turbidity with AGFK was slight (14% after 60 min). These results indicate that CL is involved in an early step of germination, related to the functioning of germinant receptors. This is the first conspicuous in vivo evidence that CL in bacterial membranes has a specific role, in which it cannot be replaced by other anionic phospholipids.

These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter.