Engineering archaeal membrane-spanning lipid GDGT biosynthesis in bacteria: Implications for early life membrane transformations

Abstract

Eukaryotes are hypothesized to be archaeal-bacterial chimeras. Given the different chemical structures of membrane phospholipids in archaea and bacteria, transformations of membranes during eukaryogenesis that led to the bacterial-type membranes of eukaryotic cells remain a major conundrum. One of the possible intermediates of eukaryogenesis could involve an archaeal-bacterial hybrid membrane. So far, organisms with hybrid membranes have not been discovered, and experimentation on such membranes has been limited. To generate mixed membranes, we reconstructed the archaeal membrane lipid biosynthesis pathway in Escherichia coli, creating three strains that individually produced archaeal lipids ranging from simple, such as DGGGOH (digeranylgeranylglycerol) and archaeol, to complex, such as GDGT (glycerol dialkyl glycerol tetraether). The physiological responses became more pronounced as the hybrid membrane incorporated more complex archaeal membrane lipids. In particular, biosynthesis of GDGT induced a pronounced SOS response, accompanied by cellular filamentation, explosive cell lysis, and ATP accumulation. Thus, bacteria seem to be able to incorporate simple archaeal membrane lipids, such as DGGGOH and archaeol, without major fitness costs, compatible with the involvement of hybrid membranes at the early stages of cell evolution and in eukaryogenesis. By contrast, the acquisition of more complex, structurally diverse membrane lipids, such as GDGT, appears to be strongly deleterious to bacteria, suggesting that this type of lipid is an archaeal innovation.

Keywords: SOS response; archaeal lipid GDGT; cellular filamentation; eukaryogenesis; hybrid membrane.

Figure 1

Biosynthesis of archaeal membrane lipids DGGGOH, archaeol, and GDGT in Escherichia coli. (A) Identification of archaeal lipids in E. coli strains. The left column is the liquid chromatography‐mass spectrometry (LC‐MS) extracted ion chromatograms (EICs) of lipid extracts from various E. coli transformants. The right column is the tandem mass spectrometry (MS/MS) spectra of the corresponding compounds from the left column. The intact polar lipids (IPLs) extracted from DGGGOH‐producing strain and the core lipids (CLs) extracted from archaeol‐ and GDGT‐producing strains are analyzed. (B) The archaeal lipid biosynthetic pathway reconstructed in E. coli. CarS, CDP‐archaeol synthase; DGGGOH, digeranylgeranylglycerol; DGGGPS, digeranylgeranylglyceryl phosphate synthase; G1PDH, G‐1‐P dehydrogenase; GGGPS, geranylgeranylglycerol phosphate synthase; GGPPS, geranylgeranyl pyrophosphate synthase; GGR, geranylgeranyl reductase; Tes, tetraether synthase.

Figure 2

GDGT synthesis induces cellular filamentation in E. coli. (A) More complex archaeal lipid synthesis resulting in significantly longer filamentous cells. Cell morphology of various E. coli transformants with corresponding archaeal lipid synthesis was observed by the scanning electron microscope (SEM). (B) GDGT‐producing strain exhibiting slightly slower growth compared to the wild type during the early logarithmic phase. The growth curves of the GDGT‐producing strain and the wild‐type strain were determined by optical density measurement under anaerobic conditions at 37°C. However, both strains ultimately achieve similar cell densities by the stationary phase. (C) The reversible changes in cellular morphology of GDGT‐producing strain. The single‐cell sorting system was used to isolate rod and filamentous cells from the GDGT‐producing strain for subsequent cultures.

Figure 3

Explosive cell lysis and cell division inhibition in the GDGT‐producing strain. (A) The elongation process of the GDGT‐producing strain ending with explosive cell lysis illustrated by the live‐cell time‐lapse microscopy imaging. (B) The cell swelling site containing high DNA content shown by DNA staining with DAPI. (C) The Z‐rings formation inhibited in the GDGT‐producing strain. The GFP‐fused FtsZ proteins aggregated at the middle of the cell, forming a Z‐ring to facilitate cell division in the wild‐type strain, but they were distributed along the whole filamentous cell without Z‐ring formation in the GDGT‐producing strain.

Figure 4

Biosynthesis of GDGT in bacteria triggers SOS response and ATP accumulation. (A) The relative expression levels of the SOS response‐related genes in the DGGGOH‐, archaeol‐ and GDGT‐producing strains compared to the wild‐type strain revealed by qRT‐PCR analysis. The housekeeping gene rssA was used as the internal reference. (B) The intercellular ATP concentrations of the GDGT‐producing strain and the wild‐type strain at different growth time points. Data were calculated from three biological replicates, and the error bars stand for standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C) The schematic diagram of the proposed mechanism for induction of SOS response by GDGT synthesis in E. coli.

Figure 5

Phylogenetic tree of Asgard lineages and their distribution of the tes gene. The tree topology was modified from Eme et al. and the bars on the right show the fraction of the examined Asgard archaeal genomes in each lineage that contained the tes gene responsible for GDGTs biosynthesis. FA, fatty acids.