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.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...

Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry

“…This binding pattern was consistently observed for all samples incubated with RB and fairly correlates with previously published reports on 10-nonyl-acridine-orange (NAO). NAO, which shares similar physico-chemical properties with RB (anionic, hydrophobic), was shown to label anionic phospholipid rich domains (phosphatidyland diphosphatidyl-glycerol) of the bacterial membranes, precisely located at the cellular poles or at regular intervals along cells [35][36][37].…”

FisB mediates membrane fission during sporulation in Bacillus subtilis

“…Thus, we expect the dynamics of FisB and CL to be intimately coupled (see below). In this regard, it is noteworthy that during sporulation, the levels of CL increase, and CL is enriched in the engulfing membranes (Kawai et al 2004). B. subtilis possesses three genes that encode proteins homologous to CL synthases: clsA (ywnE), which encodes the major CL synthase; clsB (ywjE); and ywiE (Kunst et al 1997;Kawai et al 2004).…”

“…In this regard, it is noteworthy that during sporulation, the levels of CL increase, and CL is enriched in the engulfing membranes (Kawai et al 2004). B. subtilis possesses three genes that encode proteins homologous to CL synthases: clsA (ywnE), which encodes the major CL synthase; clsB (ywjE); and ywiE (Kunst et al 1997;Kawai et al 2004). The expression of both clsA and clsB increases during sporulation, consistent with the idea that CL plays an important role during this process.…”

“…The expression of both clsA and clsB increases during sporulation, consistent with the idea that CL plays an important role during this process. Cells lacking all three genes are viable and have undetectable levels of CL during vegetative growth (Kawai et al 2004). However, CL is readily detectable in the triple mutant during the early stages of sporulation, indicating that an additional sporulation-specific CL synthase exists or that CL is produced as a side reaction by a lipid synthesis/modification enzyme (Kawai et al 2004(Kawai et al , 2006Tan et al 2012).…”

“…Cells lacking all three genes are viable and have undetectable levels of CL during vegetative growth (Kawai et al 2004). However, CL is readily detectable in the triple mutant during the early stages of sporulation, indicating that an additional sporulation-specific CL synthase exists or that CL is produced as a side reaction by a lipid synthesis/modification enzyme (Kawai et al 2004(Kawai et al , 2006Tan et al 2012). In accordance with the presence of CL during engulfment in cells lacking ClsA, ClsB, and YwiE, we found that the triple mutant was able to undergo fission at a frequency comparable with wild type (data not shown).…”