Fusion of Spiroplasma floricola cells with small unilamellar vesicles is dependent on the age of the culture

Abstract

Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells. The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S. floricola membranes because of lipid mixing upon fusion. The progression of S. floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles. Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle. Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures. Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle. Nonetheless, upon aging of S. floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed. A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected. The possible influence of these changes on the fusogenic activity of the cells is discussed.We recently demonstrated that Mycoplasma capricolum and Spiroplasma floricola cells are capable of fusing with lipid vesicles (25,26,31). This observation opened the way for the development of a fusion-mediated system for the delivery of genetic material into S. floricola cells (26). Nonetheless, exponential-phase cells showed high fusion activity, whereas low fusion activity was obtained with stationary-phase cells (26). It was therefore suggested that the fusion activity of S. floricola cells depends upon the growth phase of the culture. The low fusion activity of aged S. floricola cells may reflect changes in the chemical composition and biophysical characteristics of the cell membranes upon aging. Changes in the ratios of phospholipid to protein, saturated to unsaturated fatty acids, and phosphatidylglycerol (PG) to diphosphatidylglycerol (DPG) in the cell membranes upon aging of Acholeplasma laidlawii (11, 12), M. hominis (20), M. capricolum (10), and M. canadense cells (16) have been reported elsewhere.Mycoplasmas and spiroplasmas are fatty acid and cholesterol auxotrophs (17, 18), requiring a mixture of saturated and unsaturated fatty acids as well as unesterified cholesterol for growth. The de novo-synthesized phospholipids of these organisms are rather simple, being composed primarily of negatively charged phospholipids, such as PG and DPG (18,30). When the organisms are grown in a serum-containing medium, significant amounts of exogenous lipids, mainly phosphatidylcholine (PC), sphingomyelin (SPM), and cholesteryl esters, are incorporated into the...

Alternative to fluorescence assays to monitor fusion between Acholeplasma laidlawii cells and liposomes

“…Consequently, an interaction between liposomes and acholeplasma cells creates a condition, which in principle, could lead to fusion between the two membranes. However, the number of studies concerning, mollicutes-liposome fusion are few (Grant and McConnel 1973;Salman et al 1991;Tarshis et al 1991;Salman et al 1993;Tarshis et al 1993;Ben-Menachem et al 2001). There are two classic methods for monitoring fusion between liposomes and bacterial cells both based on the use of fluorescence dequenching assays.…”