Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane

Abstract

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20-30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell's phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.

Keywords: archaea; bacteria; ether lipids; hybrid membranes; lipid biosynthesis.

Fig. 1.

TLC-based quantitation of in vivo archaeal lipid synthesis. (A) TLC of lipid extracts from wild-type E. coli [JM109 (DE3)] heterochiral mixed membrane E. coli (MEP/DOXP⁺EL⁺) induced early during growth (OD₆₀₀ = 0.0) with different IPTG concentrations and incubated until stationary phase, and the E. coli strain harboring the entire ether lipid pathway but lacking the araM gene (MEP/DOXP⁺EL⁺AraM⁻) treated similarly. (B) Relative quantitation of the spots detected in the TLC. AG, archaetidylglycerol; CL, cardiolipin; PE, phosphatidylethanolamine; PG, phosphatidylglycerol.

Fig. 2.

Stereochemistry of the ether lipid biosynthesis in E. coli and stereoselectivity of the archaeal GGGPS. Specificity of archaeal M. maripaludis GGGPS (A) and the bacterial E. coli PlsB (B) enzymes toward G1P and G3P. Total ion counts are normalized using n-dodecyl-β-d-maltoside (DDM) detergent as internal standard. Results are the averages of two experiments ±SEM. (C) NMR spectra of Mosher’s ester derivatized AG. Synthetic AG with G3P configuration (I), synthetic AG with G1P configuration (II), a mixture of both (III), AG from the E. coli strain expressing the whole ether lipid biosynthetic pathway (IV), and from the E. coli strain harboring the AraM gene deletion (V). The dashed red boxes highlight the diagnostic signals.

Fig. 3.

Growth and cell morphology analysis of the heterochiral mixed membrane strains. (A) Growth of the E. coli MEP/DOXP⁺EL⁺ strain with all of the ether lipid enzymes [not induced (orange)], induced with 10 μM (red), and induced with 100 μM (black) of IPTG added early during growth (OD₆₀₀ = 0.0) compared with two negative control strains: E. coli JM109(DE3) wild-type (blue) and E. coli MEP/DOXP⁺ strain with the integrated MEP-DOXP operon (green). The data are the averages of three biological replicates ±SEM. (B) SEM of wild-type E. coli and the heterochiral mixed membrane strain induced at a late (0.3 OD₆₀₀) and early (0.03 OD₆₀₀) growth phase using 100 μM of IPTG. (I) Altered cell shape and length. (II) aberrant cell division and formation of bulges and shreds. (C) Statistical analysis of the cell length of the E. coli JM109(DE3) (Top), E. coli MEP/DOXP⁺EL⁺ induced with 10 μM IPTG (Middle), and 100 μM IPTG (Bottom).

Fig. 4.

Effect of mixed heterochiral membranes on E. coli cells detected by double staining with FM4-64 and DAPI. Lipid staining showing elongated and thinner cells in the engineered strain compared with the control, the presence of membrane associated spots in the engineered strain. Double staining with FM4-64 and DAPI showing the presence of appendages surrounded by a lipid layer and the presence of DNA. Presence of irregular division sites in engineered cells compared with the symmetrical division septum present in the wild-type cells. (Scale bar: 5 μm.)

Fig. 5.

Robustness of E. coli with a heterochiral mixed membrane. The E. coli strain with all ether lipid enzymes. MEP/DOXP⁺EL⁺ [not induced, orange; induced with 10 μM IPTG at early growth phase (OD₆₀₀ = 0.0, red)] was compared with the wild-type strain JM109(DE3) (blue) and the strain harboring the integrated MEP-DOXP operon MEP/DOXP⁺ (green) for survival against exposure to different environmental stresses. (A) Heat shock. (B) Freezing at −80 °C. (C) l-butanol tolerance. The data were normalized against the cfu of untreated samples. The results are the averages of four biological replicates ±SEM.