Release of Membrane Components from Viable Haemophilus parainfluenzae by Ethylenediaminetetraacetic Acid-Tris(hydroxymethyl)-aminomethane

Abstract

Logarithmically growing Haemophilus parainfluenzae lost 15 to 20% of the phospholipids, demethyl vitamin K2, cytochrome b, and cytochrome c, and 50% of the lipopolysaccharide when incubated in ethylenediaminetetraacetic acid (EDTA)-tris-(hydroxymethyl)aminomethane (Tris) for 10 min. This loss of membrane components occurred without loss in viability, and the lost components were recovered as membrane fragments in the surrounding buffer. The phospholipids recovered in the membrane fragments had a slightly lower specific activity than the phospholipids in the residue. Lysis of a portion of the cells could not account for the release of membrane components, as the cells lost neither glucose-6-phosphate dehydrogenase activity nor deoxyribonucleic acid. The treated cells were osmotically stable and contained the same proportions of the individual phospholipids as pretreatment cells. Prolongation of the EDTA-Tris treatment did not induce further loss of phospholipid or demethyl vitamin K2, but caused a decrease in viability. If the cells were returned to the growth medium after 10 min, the cells immediately resumed growth at the pretreatment rate. During growth in the recovery period, the phospholipids increased logarithmically in the pretreatment proportions, although there was a marked decrease in the turnover and a shift from the use of extracellular lipid precursors to the use of intracellular pools of precursors.

Consequences of the Inhibition of Cardiolipin Metabolism in Haemophilus parainfluenzae

“…After 1 hr with the inhibitors, the entire sample (cells plus medium) was extracted and the lipids were analyzed. By analyzing the entire sample, complications which have been observed in H. parainfluenzae (16)(17)(18) resulting from the loss of membrane fragments were eliminated. During exponential growth the cells lost about 20% of the 32P from the total lipids in I hr.…”

“…Tetramethyldipicrylamine (TMPA) was supplied from Naval Dental Research Institute. Rutamycin was gift from the Lilly Laboratories, Eli Lilly & Co., Indianapolis, Inc. Other reagents were as described previously (14)(15)(16)(17)(18)(19)(20)(21)(22).…”

“…Growth of H. parainfluenzae. Cells were grown and harvested as described previously (16). Growth was measured as the absorbance at 750 nm in 13-mm round test tubes.…”

“…Growth was measured as the absorbance at 750 nm in 13-mm round test tubes. An absorbance of 0.50 corresponded to a dry wt of 0.28 mg per ml (16).…”

Isolation of Outer Membranes with an Ordered Array of Surface Subunits from Acinetobacter

“…The use of EDTA has not been necessary at any stage in the preparation, and this is considered to be an important feature of the method. Since (22), many effects of EDTA on the outer layers of gram-negative bacteria have been described; they include the extraction of large amounts of lipopolysaccharide and other components from such diverse organisms as Escherichia (22,23), Pseudomonas (16), and Haemophilus (40), and the selective removal of certain proteins from Spirillum (11). It follows, therefore, that the outer membranes from any gram-negative bacterium are more likely to remain unaltered in composition during isolation if the use of EDTA is avoided.…”