Lipoteichoic Acid Localization in Mesosomal Vesicles of Staphylococcus aureus

Abstract

Mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P have been prepared and examined for the presence of lipoteichoic acid. Lipids were first removed by treatment with pyridine-acetic acid-butanol (22:31:100, vol/vol/vol) and chloroform-methanol (2:1, vol/vol). Subsequently, lipoteichoic acid was removed with 40%o phenol in water. The lipoteichoic acid from mesosomal vesicles was characterized by (i) equimolar glycerol and phosphate, (ii) alanine upon hydrolysis (2 N NH,OH, 18 h, 22 C), and (iii) fatty acids, diglycerol triphosphate, glycerol monophosphate, and glycerol diphosphate upon alkaline hydrolysis (1 N NaOH, 3h, 100 C). The plasma membranes contained no lipoteichoic acid. The presence in mesosomal vesicles of 18% of the dry weight as lipoteichoic acid and its absence from plasma membranes provide the first major chemical differences between these organelles. A study of the lipoteichoic acid content in various fractions of the cell showed that the mesosomal vesicles were the major and probably the sole site for the localization of the lipoteichoic acid in these organisms. A new method for the preparation of mesosomes in increased yields is reported. A theory for the control of cell division involving lipoteichoic acid and the mesosome is proposed. This paper describes the isolation of lipoteichoic acid from mesosomal vesicles of S. aureus 209P (ATCC 6538P). Plasma membranes from this bacterium are shown to lack lipoteichoic acid. The data demonstrate a major qualitative chemical difference between mesosomal vesicles and plasma membranes and show further that most and probably all of the intracellular teichoic acid (membrane or lipoteichoic acid) is 273 on July 31, 2020 by guest http://jb.asm.org/ Downloaded from 274

Membrane and cytoplasmic location of streptolysin S precursor

“…Electron microscopy of membrane and periplasm sediment fractions revealed that the former comprised large flattened spheres or irregular sheets and fragments of membrane, most from 600 to 750 nm in diameter, interspersed with some ribosomes and debris; the latter consisted predominantly of much smaller (15to 30nm) membrane-bounded spheres, although some larger membranous fragments (up to 200 nm in diameter) and debris were also present. The composition ofthese fractions is in conformity with findings for similar crude fractions prepared previously from Staphylococcus aureus (12). Electron microscopy of the periplasmic supernatant fraction showed only a rather uniform background of what is assumed to be aggregated protein, and the cytoplasmic fraction contained predominantly ribosomes.…”

“…However, the highest specific activity of CPH was found in the sediment of the periplasmic function. This fraction corresponds to the crude mesosomal vesicle fraction isolated by similar procedures from other gram-positive bacteria such as staphylococci (12), and electron microscopy verified the predominance therein of small membrane-bounded vesicles as well as a minor degree of contamination with somewhat larger membrane fragments. Because of minimal yields of this fraction, it could not be purified on sucrose density gradients in adequate quantity for further study, as were membranes.…”

Lipoteichoic Acid, a Major Amphiphile of Gram-Positive Bacteria That Is Not Readily Extractable

“…Lipoteichoic acid (LTA) is a major membrane-associated amphiphilic molecule found in most gram-positive organisms. In prior studies, LTA was extracted by using acid (5), aqueous phenol (6,9,16), enzymatic lysis (11,14,15), mechanical disruption (1,3), hot water (21), or a combination of these (13). Cells at different stages of growth, disrupted and nondisrupted, were used for such extractions.…”

“…However, there has been little experimental work directed at the yield of LTA as a function of extraction procedure (21), stage of growth (16), or cellular disruption (18). The cellular yield of LTA on a weight basis was reported in only a few instances (2,14).…”

“…Some change in the cell wall or membrane must occur which makes the cell FIYLOCOCCUS AUREUS (LaffUty) more permeable to LTA or to the extracting * phenol during exponential-phase growth. These ._________ -----_a changes do not seem to be due to changes in the membrane, since treatment of cells with phenol X at 80°C (after p-PAB), a procedure which effects extraction of LTA from exponential- (14) and stationary-phase mesosomes and protoplasts (Huff, unpublished (7).…”