Abstract
Diglyceride kinase mutants of Escherichia coli contain about 50- to 100-fold more 1,2-diglyceride than wild type cells. We now report that monoglyceride and triglyceride also accumulate in these strains. In mutant RZ60 (dgk-6) these compounds represent about 1 and 0.2%, respectively, of the total lipid fraction, while diglyceride represents 5-8% under most conditions. Monoglyceride accumulates predominantly in the outer membrane, while triglyceride builds up together with diglyceride in the cytoplasmic membrane. Under typical growth conditions about two-thirds of the diglyceride in E. coli arises in conjunction with synthesis of the membrane-derived oligosaccharides (Raetz, C.R.H., and Newman, K.F. (1979) J. Bacteriol. 137, 860-868). Inhibition of membrane-derived oligosaccharides (MDO) synthesis also curtails the accumulation of monoglyceride and triglyceride. However, there appears to be at least one other MDO-independent source of diglyceride and related metabolites. Since MDO synthesis is suppressed by high osmolarity (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S. A. 79, 1092-1095), we have examined the effects of osmolarity on diglyceride accumulation in RZ60 (dgk-6). As expected, if MDO synthesis and diglyceride formation are coupled, the diglyceride level in RZ60 is higher at low osmolarity, while at high osmolarity the level of diglyceride is reduced to that observed in double mutants defective both in MDO synthesis and diglyceride kinase. Since dgk mutants do not grow at very low osmolarity, we have isolated several spontaneous phenotypic revertants that do. One class regains diglyceride kinase and has low diglyceride levels under all conditions. The other class remains defective in diglyceride kinase but tolerates higher diglyceride levels which amount to 13% of the total lipid during maximal induction of MDO synthesis at low osmolarity.
Highlights
Diglyceride kinase mutantsof Escherichia coli con- [3,4] have shown that most of the phosphatidic acid of E. coli tain about 50- to 100-fold more 1,a-diglyceride than is generated by the acylation of sn-glycerol3-phosphate
Inthe presentstudy we have further characterized the relationship between diglycerideaccumulation in dgk mutants and thebiosynthesis of membrane-derived oligosaccharides (MDO)
A. 79, 1092-1095), we have examined the effects of oglyceride and triglyceride in certain E. coli mutants implies osmolarity on diglyceride accumulation in RZ60
Summary
The upper phase was RZR6O-1 zdg::Tn 10 l ~ p - 2transductant of This study " RZ60 dried and spotted on a Kodak cellulose thin layer plate, which was developed in the system 1-butanol/acetic acid/water, 25:4:10,v/v. RZ60 by a Silica Gel 60 plate developed in ethanol/ammonium hydroxide, RZR75-1 zdg::Tn 10 lpp-2 transductant of This study. The fatty acid methyl esters were separated at 170 "C on CRZ23-12 malB+ dgk-23 CRZ29-13 m l B + d ~ k - 2 9 This study This studv a 10-foot glass column packed with 10% S P 2330 on Chromasorb. The MDO,was dried under Nz and re-extracted using the Bligh-Dyer cells (10-50 ml of culture) were harvested by a 10-min centrifugation method to remove traces of lipid. L ml of chloroform and l ml of the acidic sodium chloride solution were added, whereupon the phases were thoroughly mixed. After a brief low speed centrifugation the resulting chloroform phase was analyzed for the individual lipids
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