Abstract
Growth of a temperature-sensitive general fatty acid synthesis mutant of Escherichia coli K12 at its restrictive temperature in the presence of exogenous palmitate results in lysis of the bacterium. Under these conditions, palmitate is incorporated into membrane phospholipid to a high level. Mutants of bacteria restricting this incorporation (having a palmitate-resistant phenotype) have been isolated and one such mutant, strain L8-2/3, has been further characterized. This mutant has lowered acyl-CoA synthetase (fadD) activity (25-33% of normal) and consequently is defective in fatty acid uptake. This lowered uptake could explain the palmitate-resistant phenotype of strain L8-2/3. However, both in vivo (fatty acid composition and positional distribution data) and in vitro (acyltransferase activity measurements) experiments suggest that this mutant is also altered in its acyltransferase activities. The mutation(s) of strain L8-2/3 appears to allow increased (approximately 2-fold) incorporation of myristate (and possible unsaturated fatty acids) into position 2 of 1-acyl-sn-glycerol 3-phosphate but normal palmitate incorporation into the same position. The incorporation of palmitate, myristate, and oleate into position 1 of sn-glycerol 3-phosphate by strain L8-2/3 is also higher than that observed with the parent, strain L8-2. Replacing the partially defective fadD gene of strain L8-2/3 with a wild type allele conferred on this strain the palmitate sensitivity and the acyltransferase activity of the parent strain L8-2. This finding, taken together with other data, suggests that acyl-CoA synthetase interacts with the acyltransferase(s) in some manner to influence the fatty acid specificity of the acyltransferase.
Highlights
Growth of a temperature-sensitive general fatty acid(4)
Fatty acids were added in small volumes of 80% (v/v) ethanol, neutralized with KOH, such that the final ethanol concentration in each flask was 0.5% (v/v)
Position 1 represents the fatty acids remaining on lysophosphatidylethanolamine after phospholipase A, treatment; position 2, the fattyacids released by phospholipase A,; total, the fatty acids derived from phosphatidylethanolamine without prior exposure to phospholipase A,; SFA and UFA, saturatedand unsaturated fatty acids, respectively; (T),total SFA or total UFA in each position or in total phosphoatidylethanolamine
Summary
F- fabE fadE strA fadD* his man Chemicals-ATP, sn-glycerol 3-phosphate, fatty acid-free bovine L8-2/3C2. Acyl-CoA synthetase was assayed using the hydroxamate reaction as described by Overath et al (I).Late exponential phase cells were harvested by centrifugation, washed once in 0.1 M phosphate buffer, pH 6.8, and resuspended in the same buffercontaining 10 mM 2mercaptoethanol. The cells were broken, as described for acyltransferase preparations, in a Aminco French pressure cell at 20,000 p.s.i. The lysate was centrifuged at 3,000 X g for 5 min, and the supernatant was used for determination of acyl-CoA synthetase activity. Bacteria were cultured at 30 "C in minimal medium containing 1% (w/v) Triton X-100, sampled during exponential growth at an absorbance of 100 Klett units, and transferred to flasks at 30 "C which contained [3H]palmitateof [3H]oleate.The final concentration of both fatty acids was 10 pg/ml and the radioactive concentrations ods have been describedpreviously (21). Blanks (no bacteria present) were performed to monitor the efficiency of the washing procedure.The fikers were dried and counted in a scintillation counter (scintillation fluid 3a70 of RPI Corporation, Elk Village, IL; Packard Tri-carb counter)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
