Effect of ethylenediaminetetraacetate on phospholipids and outer membrane function in Escherichia coli

Abstract

Treatment of Escherichia coli K-12 strain S15, containing a normal amount of phospholipase A, with ethylenediaminetetraacetate (EDTA) resulted in an increase in sensitivity of the organism to actinomycin D. Strain S17, a mutant deficient in both detergent-resistant phospholipase A and detergent-sensitive phospholipase A, was considerably less sensitive to the antibiotic after the treatment. Both strains released lipopolysaccharide after EDTA treatment, indicating that this outer membrane component alone is not the barrier to actinomycin in these organisms. The phospholipase A-deficient strain released less alkaline phosphatase, a periplasmic enzyme. EDTA treatment of S15 resulted in the accumulation of free fatty acids, indicative of phospholipase A activation. Cells briefly treated with EDTA regained the barrier to actinomycin when incubated in growth media, and the cessation of the accumulation of free fatty acids was in approximate temporal agreement with restoration of the barrier. Cells in which phospholipase A was activated by brief exposure to EDTA synthesized relatively more phosphatidylethanolamine than did untreated cells in the initial period after dilution into growth media. These experiments suggest that the EDTA-induced loss of outer membrane barrier function of E. coli K-12 is mediated through the activation of phospholipase A.

Colicin E2 release: lysis, leakage or secretion? Possible role of a phospholipase.

“…Here we try to discriminate between three possible modes of colicin E2 export: release resulting from cell lysis; the specific secretion (excretion) of colicin independent of, but usually accompanied by lysis; and a semi-selective increase in membrane permeability allowing some proteins, including colicin, to leak out of the cell. The results presented support the last alternative, and we therefore examined the possibility that envelope permeability changes were caused by the activation of host cell phospholipase(s), as is known to occur upon EDTA treatment or bacteriophage infection Hardaway et al, 1975;Hardaway and Buller, 1979).…”

“…The major E. coli K-12 phospholipase is the detergent-resistant PldA phospholipase Al-A2 (Scandella and Kornberg, 1971;Ohki et al, 1972;Nishijima et al, 1977;de Geus et al, 1983). PldA phospholipase activity is increased when cells are treated with membrane perturbants or are subjected to heat shock (Hardaway and Buller, 1979;de Geus et al, 1983, and data not shown). Direct measurements of phospholipase activity in crude cell extracts (Materials and methods) indicated a 30to 40-fold increase starting shortly before the onset of protein release and quasi-lysis, irrespective of whether the lysis gene was under the control of the colicin gene or lacZ promoters.…”

Outer membrane phospholipase is not the mediator in the bactericidal or outer membrane permeability-increasing action of polycations

“…The results in this paper showed that the unshielding does not require the presence of OM phospholipase. In this respect, the action of EDTA on the OM seems to be different: it also releases LPS [23], but the OM permeability increase is reported to be dependent on the presence of OM phospholipase [7].…”

“…However, Escherichia coli mutants deficient in this phospholipase are still fully sensitive to polymyxin [16,17], BPI [11] or immune bacteriolysis [13] indicating that under these conditions the role of phospholipase is secondary. On the other hand, a phospholipase-deficient mutant was resistant to the increase of the permeability of the OM caused by EDTA, indicating that phospholipase is an important mediator in the action of EDTA on the OM [7].…”

“…One possible mechanism for the increase of the permeability of the OM caused by the polycations would be an activation of the phospholipase present in the OM of gram-negative bacteria [5,6], with a resultant hydrolysis of the phospholipids in the OM. Such an activation is known to take place during outer membrane perturbations caused by EDTA [7,8], polymyxin [9,10], cationic leukocyte protein BPI [9,11 ], immune bacteriolysis [ 12,13] or multiple phage attachment [14,15]. However, Escherichia coli mutants deficient in this phospholipase are still fully sensitive to polymyxin [16,17], BPI [11] or immune bacteriolysis [13] indicating that under these conditions the role of phospholipase is secondary.…”