The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, their biosynthetic pathway remains undefined since several key eukaryotic ceramide synthesis enzymes have no bacterial homologue. Here we used genomic and biochemical approaches to identify six proteins comprising the complete pathway for bacterial ceramide synthesis. Bioinformatic analyses revealed the widespread potential for bacterial ceramide synthesis leading to our discovery of the first known Gram-positive species to produce ceramides. Biochemical evidence demonstrated that the bacterial pathway operates in a different order than in eukaryotes. Furthermore, phylogenetic analyses support the hypothesis that the bacterial and eukaryotic ceramide pathways evolved independently.

Abstract— With accumulating evidence that the membrane is an important site for near‐UV inactivating events, we have investigated the effects of near‐UV radiation on the unsaturated fatty acid auxotroph E. coli K1060 following incorporation into the membrane phospholipids of fatty acids with varying degrees of unsaturation. Sensitivity, lipid peroxidation and membrane damage, as determined by 86Rb+ leakage, have been found to increase with increasing unsaturation in log‐phase cells. Similar experiments carried out in D2O also show an increase in sensitivity, lipid peroxidation and membrane damage, indicating that singlet oxygen may be responsible for such near‐UV‐radiation‐induced damage

Background The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “enhancer” (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. Methods and results Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call “CON_G”) that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. Conclusions The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.