Membrane phospholipid synthesis and phenotypic correlation of an Escherichia coli pss mutant

Abstract

A pair of putatively isogenic pss (Ts) and pss+ (phosphatidylserine synthetase structural gene) strains was constructed and analyzed, together with the revertants, for the physiological consequences of cessation of the optimal synthesis of phosphatidylethanolamine (PE). Their in vivo and in vitro abilities to synthesize PE and the growth rates at different temperatures were determined. The rate of PE synthesis by OS2101 pss(Ts) was inversely related to the culture temperature. OS2101 in a low-salt broth medium stopped division and formed filamentous cells with declining viability upon the elevation of culture temperature from 27 to 42 or 44°C, whereas the syntheses of deoxyribonucleic acid, ribonucleic acid, and protein were not affected. Proper concentrations of cations such as Na+, Kt, NH4+, and Mg2or of sucrose could remedy the division and growtlh of OS2101 at the restrictive temperature without restoring normal PE synthesis. A remedial effect other than osmotic protection of these effectors and an adaptive regulatory mechanism for PE formation are suggested.

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase

“…isopropyl-13-D-thiogalactoside (IPTG) and collected at 200 Klett units, and the enzyme activities in membrane and soluble fractions, as well as phospholipid compositions, were determined. OS2101 harboring pWSK29 (vector) showed no synthase activity in either fraction and contained a low level of phosphatidylethanolamine as previously described (32). On the other hand, OS2101 harboring pBM22 showed a Mn2+_ dependent phosphatidylserine synthase activity in the membrane fraction, and its phosphatidylethanolamine content increased to 74% of the total phospholipids.…”

Alteration of phospholipid composition by combined defects in phosphatidylserine and cardiolipin synthases and physiological consequences in Escherichia coli

“…The E. coli K-12 strains obtained in this work are listed in Table 1 together with the strains used in their construction. They were constructed by standard mating and P1 vir transduction procedures (23,27,28) so as to carry, besides several usual genetic markers, the mutations that allow the future manipulation of the intracellular sn-glycerol-3-phosphate pool by added glycerol in the presence of glucose, i.e., glpD3 glpR2 glpKphoA8 and glpKp (3,22). BB20-14 and GN1908 were first mated, and an F-Thy' Strr recombinant carrying gpsA20 in addition to the genotype of SD12 was chosen for the further construction.…”

“…Culture media and growth determination. A broth medium, NBY (27), containing 8 g of nutrient broth (Difco), 5 g of Polypeptone, 2 g of yeast extract (Difco), and 1 g of NaCl per liter, adjusted to pH 7.2 with NaOH, was routinely used. This medium had a relatively low osmolarity (114 osmol/kg) and phosphorus content (6.7 mM) and was conveniently used in testing the temperature sensitivity of pss-J mutants and in labeling the cells with [32P]phosphate.…”

“…To facilitate the understanding of the specific functions of major phospholipids in E. coli membranes (i.e., phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin), desirable features for a manipulated cell will be (i) a radically altered composition of major phospholipids; (ii) no accumulation of unusual lipids that will result in complication of analysis; (iii) normal membrane structures except for an altered phospholipid polar headgroup composition; and (iv) the ability to grow steadily to allow function analysis. Among the reported mutations in E. coli phospholipid synthesis, temperature-sensitive mutations in phosphatidylserine synthase (EC 2.7.8.8) that catalyzes the committed step in phosphatidylethanolamine synthesis (27,39) and a defective mutation in cardiolipin synthase (36) are the only useful mutations from the above criteria. The growth defect of phosphatidylserine synthase mutants at nonpermissive temperature is remediable by appropriate culture supplements without correcting the phospholipid abnormality (27).…”

“…Among the reported mutations in E. coli phospholipid synthesis, temperature-sensitive mutations in phosphatidylserine synthase (EC 2.7.8.8) that catalyzes the committed step in phosphatidylethanolamine synthesis (27,39) and a defective mutation in cardiolipin synthase (36) are the only useful mutations from the above criteria. The growth defect of phosphatidylserine synthase mutants at nonpermissive temperature is remediable by appropriate culture supplements without correcting the phospholipid abnormality (27). These two mutations have not been studied in combination.…”