Changes in the distribution of membrane lipids during growth of Thermotoga maritima at different temperatures: Indications for the potential mechanism of biosynthesis of ether-bound diabolic acid (membrane-spanning) lipids.

Membrane-spanning lipids are present in a wide variety of archaea but they are rarely in bacteria. Nevertheless, the (hyper)thermophilic members of the order Thermotogales harbor tetraester, tetraether, and mixed ether/ester membrane-spanning lipids mostly composed of core lipids derived from diabolic acids, C30, C32 and C34 dicarboxylic acids with two adjacent mid-chain methyl substituents. Lipid analysis of Thermotoga maritima across growth phases revealed a decrease of the relative abundance of fatty acids together with an increase of diabolic acids with independence of growth temperature. We also identified isomers of C30 and C32 diabolic acids, i.e. dicarboxylic acids with only one methyl group at C-15. Their distribution suggests they are products of the condensation reaction but preferably produced when the length of the acyl chains is not optimal. In comparison with growth at the optimal temperature of 80°C, an increase of glycerol ether-derived lipids was observed at 55°C. Besides, our analysis only detected diabolic acid-containing intact polar lipids with phosphoglycerol (PG) headgroups. Considering these findings, we hypothesize a biosynthetic pathway for the synthesis of membrane-spanning lipids based on PG polar lipid formation, suggesting that the protein catalyzing this process could be a membrane protein. We also identified, by genomic and protein domain analyses, a gene coding for a putative plasmalogen synthase homologue in T. maritima, which is also present in other bacteria producing sn1-alkyl ether lipids but not plasmalogens, suggesting it could be involved in the conversion of the ester to ether bond in the diabolic acids bound in membrane-spanning lipids. Importance Membrane-spanning lipids are unique compounds found in most archaeal membranes, but they are also present in specific bacterial groups like the Thermotogales. The synthesis and physiological role of membrane-spanning lipids in bacteria represent an evolutionary and biochemical open question that points to the differentiation of the membrane lipids composition. Understanding the formation of membrane-spanning lipids is crucial to solving this question and identifying the enzymatic and biochemical mechanism performing this procedure. In the present work, we found changes at the core lipid level, and we propose that the growth phase drives the biosynthesis of these lipids rather than temperature. Our results identified physiological conditions influencing the membrane-spanning lipids biosynthetic process which can further clarify the pathway leading to the biosynthesis of these compounds.