An equilibrium between two fractions of lipopolysaccharide in Escherichia coli.

When cells of Escherichia coli are exposed briefly to ethylenediaminetetraacetate (EDTA), they release approximately half their lipopolysaccharide (LPS).1, 2 The amount lost for a given strain appears fixed, since it cannot be increased by several changes in procedure, including increase in the time of exposure and concentration of EDTA.2 Because these results suggested that the released LPS might differ from the remainder, the metabolic relationship between these two fractions was investigated. Lipopolysaccharide was labeled with galactose-C'4 for various periods of time, using a mutant that can incorporate this sugar into only LPS, and the amount of labeled LPS released by EDTA treatment was determined. The results show that in the growing cell the fraction of LPS that is released by EDTA is in rapid equilibrium with that which is retained. Newly synthesized LPS is initially part of the retained fraction but rapidly enters this equilibrium. Materials and Method&.E. coli J5, a mutant derived from E. coli 0111:B4, was used. This mutant lacks UDP-galactose-4-epimerase and cannot form complete LPS without galactose in its growth medium; with galactose, it forms a cell wall having the same structure as that of its parent.' This organism was the gift of Dr. Edward Heath. Cells were grown to mid-exponential phase (5 X 108 cells/ml) in Trypticase soy broth containing 0.25% galactose and 0.25%o glucose. Where indicated, LPS was labeled with galactose-1-C"4 (New England Nuclear Corporation). When labeled cells were diluted into nonradioactive medium and grown for various lengths of time, there was no loss of galactose label from TCA-precipitable material. EDTA treatment was performed as previously described,l 2 except that cells were washed with 0.85% NaCl at 40 before treatment; exposure to EDTA (2-6 X 10-4 M) was terminated after 4-6 min by addition of MgCl2 (2-6 X 10-3 M), and the cells were centrifuged. The amount of LPS in the supernatant and cell-pellet fractions was estimated by assaying for colitose as previously described.'1 4 This sugar is present only in LPS and its precursors,5 and its per cent release by EDTA in the parent strain is the same as that of total cell LPS.2 The amount of labeled galactose in LPS was estimated by precipitating aliquots of the supernatant and cell-pellet material with 5% trichloroacetic acid (TCA) at 40, filtering on Millipore filters (pore size 0.22 JA), washing once with 2.0 ml cold 5% TCA and three times with 5.0 ml water, and measuring the radioactivity in a scintillation counter as previously described.' In mutants of Salmonella lacking UDP-galactose-4-epimerase, virtually all acidprecipitable galactose is in LPS.6 The same is true for this E. coli mutant as shown by the following control experiments: (1) Cells labeled for 2 min and for 3 hr (four generations) were extracted with phenol and the LPS was purified as previously described.2 In several experiments 65-100% of the original TCA-precipitable counts were recovered in the purified LPS with no systematic differences between the 2-min and the 3-hr sample. For both times of labeling, the ratio of TCA-precipitable galactose-C14 to total colitose in the cells was the same as the ratio of total galactose-C'4 to colitose in the purified LPS. (2) When fully labeled cells were treated with EDTA, the ratio of colitose to TCA-precipitable galactose-C'4 was the same for the whole cell, the released material, and the material retained in the cells. (3) Highly labeled galactose-1-C'4 and UDP-galactose-C'4