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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.

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overex- pression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting dif- ferent codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool.

The cytoplasmic membrane of a prokaryotic cell consists of a lipid bilayer or a monolayer that shields the cellular content from the environment. In addition, the membrane contains proteins that are responsible for transport of proteins and metabolites as well as for signalling and energy transduction. Maintenance of the functionality of the membrane during changing environmental conditions relies on the cell’s potential to rapidly adjust the lipid composition of its membrane. Despite the fundamental chemical differences between bacterial ester lipids and archaeal ether lipids, both types are functional under a wide range of environmental conditions. We here provide an overview of archaeal and bacterial strategies of changing the lipid compositions of their membranes. Some molecular adjustments are unique for archaea or bacteria, whereas others are shared between the two domains. Strikingly, shared adjustments were predominantly observed near the growth boundaries of bacteria. Here, we demonstrate that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria. Electronic supplementary material The online version of this article (doi:10.1007/s00792-017-0939-x) contains supplementary material, which is available to authorized users.

Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102107 pfu.mL1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and under the scope of the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). Franklin L. Nobrega and Ana Rita Costa acknowledge FCT for grants SFRH/BD/86462/2012 and SFRH/BPD/94648/2013, respectively. Melvin F. Siliakus acknowledges funding from the Biobased Ecologically Balanced Sustainable Industrial Chemistry (BE-BASIC) foundation. Electron microscopy work was performed at the Wageningen Electron Microscopy Centre (WEMC) of Wageningen University.