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 K(2), 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 not 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 K(2), 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 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.