An iso-15 : 0 O-alkylglycerol moiety is the key structure of the E-signal in Myxococcus xanthus

Abstract

The E-signal is one of five intercellular signals (named A- to E-signal) guiding fruiting body development in Myxococcus xanthus, and it has been shown to be a combination of the branched-chain fatty acid (FA) iso-15 : 0 and the diacylmonoalkyl ether lipid TG1. Developmental mutants HB015 (Δbkd MXAN_4265::kan) and elbD (MXAN_1528::kan) are blocked at different stages of fruiting body and spore formation as they cannot form the required iso-FA or the actual ether lipid, respectively. In order to define the structural basis of the E-signal, different mono- and triglycerides containing ether or ester bonds were synthesized and used for complementation of these mutants. Here, the monoalkylglyceride dl-1-O-(13-methyltetradecyl)glycerol exhibited comparably high levels of complementation in both mutants, restoring fruiting body and spore formation, identifying iso-15 : 0 O-alkylglycerol, part of the natural lipid TG1, as the 'signalophore' of E-signalling.

Fig. 1.

Glycerolipids synthesized and tested, featuring 13-methyltetradecanol (iso-15 : 0 alcohol; 1–6), tetradecanol (14 : 0 alcohol; 7–9), 13-methyltetradecanoic acid (iso-15 : 0; 10, 11, 14) or tetradecanoic acid (14 : 0; 12, 13). See the Supplementary Material for further details on the structure and synthesis of these compounds.

Fig. 2.

Complementation of M. xanthus mutant HB015 with various lipids in different concentrations. A 40 μl aliquot of a cell suspension with a density of 5 × 10⁹ ml^− 1 was allowed to develop on TPM agar plates for 72 h at 30 °C. Data were acquired by graphical analysis of fruiting bodies using ImageJ (Rasband, 1997–2014). The number in the upper right corner of each image indicates the level of complementation, which was calculated as a product of number, size and density of fruiting bodies. The WT at 72 h was set to 100.

Fig. 3.

Sporulation and germination data for complementation of M. xanthus mutants HB015 and elbD. Lipids were added to a concentration of 40 and 20 nmol per 10⁹ cells for HB015 and elbD, respectively. Myxospores from complementation assays with HB015 (a) and elbD (c) were isolated and counted. Afterwards spore suspensions of HB015 (b) and elbD (d) were plated on CTTYE germination medium and incubated for 7 days at 30 °C. Values were calculated with regard to DK1622 WT set to 100 % and are the result of triplicates.

Fig. 4.

Fruiting body formation in M. xanthus WT DK1622 and elbD complemented with various lipids. Five microlitres of cell suspensions with a density of 5 × 10⁹ ml^− 1 were allowed to develop on TPM agar plates for 48 h at 30 °C. Lipids were added to a concentration of 20 nmol per 10⁹ cells. Numbers indicate the level of complementation. For details see Fig. 2.

Fig. 5.

Proposed formation of the E-signal in M. xanthus development. Branched-chain FA precursor isovaleryl-CoA is synthesized either from leucine or by the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) pathway. Iso-15 : 0 FA is used in either method of lipid biosynthesis leading to TG1 (4). Lipid bodies (or lipid vesicles) containing 4 are released into the extracellular space by an unknown mechanism or by cell lysis. Subsequent uptake of these lipid bodies (or lipid vesicles) containing 4 by the receiving cell (bottom), hydrolysis to 1 and further modifications then possibly form the E-signal. Bkd, branched-chain-2-keto-acid dehydrogenase.