Download An iso-15:0 O-alkylglycerol moiety is the key structure of the E

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An iso-15:0 O-alkylglycerol moiety is the key structure of the E-signal in
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Myxococcus xanthus
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Tilman Ahrendt[a]1, Christina Dauth[a]1, Helge B. Bode[a,b]*
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[a]
Merck Stiftungsprofessur für Molekulare Biotechnologie
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Fachbereich Biowissenschaften
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Max-von-Laue-Str. 9, 60438 Frankfurt am Main (Germany)
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*E-mail: [email protected]
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[b]
Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität
Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main (Germany)
1Authors
contributed equally to this work
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Keywords: fruiting body development, E-signal, lipid signaling, ether lipid,
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myxospore, germination
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Contents category: Physiology and metabolism
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Word count: abstract (137); main text (2884)
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Running title: iso-15:0 O-alkylgylcerol is the signalophore in myxobacterial E-
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signaling
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1
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Abstract
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The E-signal is one of five intercellular signals (named A to E-signal) guiding fruiting
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body development in Myxococcus xanthus and it has been elucidated as a
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combination of the branched chain fatty acid (FA) iso-15:0 and the diacylmonoalkyl
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ether lipid TG1. Developmental mutants HB015 (Δbkd MXAN_4265::kan) and elbD
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(MXAN_1528::kan) are blocked at different stages of fruiting body and spore
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formation as they cannot form the required iso-FA or the actual ether lipid,
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respectively. In order to define the structural basis of the E-signal, different mono-
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and triglycerides containing ether or ester bonds were synthesized and used for
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complementation
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methyltetradecyl)glycerol (1) exhibited comparably high levels of complementation in
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both mutants, restoring fruiting body and spore formation identifying iso-15:0 O-
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alkylglycerol as the “signalophore” of E-signaling that is also part of the natural lipid
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TG1.
of
these
mutants.
Here,
monoalkylglyceride
DL-1-O-(13-
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Introduction
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The myxobacterium Myxococcus xanthus is well known for forming spore containing
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fruiting bodies as a response to amino acid starvation (Zusman et al., 2007). Genetic
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regulations during this development, (Müller et al., 2010) as well as the morphology
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of fruiting bodies (Kaiser & Welch, 2004) have been studied intensively in the past.
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However, much less is known about the signaling processes required for the
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formation of these fruiting bodies. The development is guided by extracellular signals
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subdivided into complementation groups (A-E) and the actual signaling molecules are
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only known for the A signal being a mixture of amino acids and small peptides
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(Kuspa & Kaiser, 1989; Garza et al., 2000). E-signal mutants are defective in the
2
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branched-chain-2-keto-acid
dehydrogenase
(BCKAD)
complex
resulting
in
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incomplete aggregation and decreased spore yield (Downward & Toal, 1995). The
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enzyme complex catalyzes deamination and decarboxylation of branched-chain
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amino acids and by this provides isovaleryl-CoA needed as starter units for the
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biosynthesis of iso-branched fatty acids (FA) and secondary metabolites (Ring et al.,
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2006). However, M. xanthus also produces isovaleryl-CoA using a second pathway
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via 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) (Bode et al., 2006; Mahmud et al.,
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2005; Li et al., 2013). Insertion of mutations into both pathways results in the double
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mutant HB015, which hardly produces any isovaleryl-CoA and is therefore incapable
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of forming aggregates, not to mention myxospores (Bode et al., 2009). This defect
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can be complemented by isovalerate providing the starting unit for branched-chain
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fatty acid biosynthesis or directly by adding iso-15:0 fatty acid during development
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(Bode et al., 2009). Beyond that, the monoalkyl diacylglycerol TG1 (1-iso15:0-alkyl-
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2,3-di-iso15:0 acyl glycerol) that accumulates during development is also capable of
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rescuing fruiting body formation and sporulation (Bhat et al., 2014). In M. xanthus
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ether lipids are synthesized by the elb biosynthesis gene cluster coding for the
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multidomain enzyme ElbD with similarity to polyketide synthases (Lorenzen et al.,
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2014). A knockout of elbD results in a mutant with delayed fruiting body formation
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and a defect in sporulation indicating the ether lipids importance to complete
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development (Lorenzen et al., 2014). Both iso-15:0 fatty acid and TG1 have already
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been referred to as signals, or at least precursors for a molecule functioning in E-
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signaling (Bhat et al., 2014). However, it remains unclear which structural moieties of
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both molecules are actually forming the signalophore for aggregation, fruiting body
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formation and sporulation.
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In this study various lipids were tested for their potential to complement HB015 or an
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elbD mutant, deficient in producing iso-FAs (Bode et al., 2009) or ether lipids
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(Lorenzen et al., 2014), respectively. Thus, glycerolipids with iso-branched or
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straight-chain fatty acids or alcohols linked were synthesized as esters or ethers. As
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HB015 and elbD are apparently impaired in different stages of development, the level
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of complementation of different lipids compared to the wild type allowed the
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identification of the structural moieties that are actually relevant for development.
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Material and methods
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Bacterial strains and culture conditions
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Myxococcus xanthus DK1622 and its mutants HB015 (Δbkd MXAN_4265::kan)(Bode
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et al., 2009) and elbD (MXAN_1528::kan) (Lorenzen et al., 2014) were grown in CTT
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media (Kroos et al., 1986) supplemented with 40 µg/ml kanamycin (Km) (Carl
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Roth,Karlsruhe, Germany) if necessary. Fruiting body and spore development was
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performed on TPM agar plates (Bretscher & Kaiser, 1978). For the isolation of
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myxospores fruiting bodies were harvested after 72 hours and washed with water
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twice. Fruiting bodies were incubated for 2 h at 60°C to inactivate remaining
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vegetative cells before treating with sonification. Germination experiments were
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performed on CTTYE agar plates prepared by adding 0.2% yeast extract to CTT.
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Sporulation and germination experiments were performed in triplicates.
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Chemical complementation, sporulation and germination assays
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The complementation with lipids was performed according to Bhat et al., 2014 with
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modifications. Bacteria were harvested at OD600 of 1-1.2 and concentrated to a
4
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density of 5x109 cells/mL. 5 and 40 µl cell suspension were mixed with lipids of
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different concentrations from 1 to 200 nmol per 10 9 cells and put on TPM agar. The
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plates were incubated at 30°C. Developing spots were photographed at different time
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points at 25x magnification. In order to determine number and size of fruiting bodies,
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ImageJ 1.48v (Rasband, 1997-2014) was used. Pictures were transformed into a
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binary format. The size of the fruiting bodies was measured in square pixels and
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density was determined by the mean grey value of the fruiting bodies. The auto
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threshold method was used to reduce the background and to foreground the fruiting
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bodies. Automated particle analysis was performed using the following parameters:
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Size: 2,500 to infinity. Circularity: 0,75 to 1,00. Show outline activated. Number, mean
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size and mean density of the fruiting bodies were set to 100 for wild type and the
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level of complementation was calculated as the product of all three factors and set to
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100 for wild type for each time point. Complementation levels for mutants were
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calculated accordingly and were set in reference to the wild type values for each time
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point. Harvesting fruiting bodies and germination assay was performed as described
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previously (Lorenzen et al., 2014). The spore number was determined using a
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Neubauer counting chamber. Sporulation and germination experiments were
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performed in triplicates.
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General methods for chemical synthesis
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All reagents and anhydrous solvents were purchased from Sigma-Aldrich and used
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without further purification. Analytical TLC was performed on precoated silica gel
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plates (Macherey-Nagel, Polygram® SIL G/UV254) using a KMnO4 solution as a
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staining reagent. Flash column chromatography was carried out on a Biotage SP1
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Flash Purification System using prepacked silica gel (particle size: 40 - 65 µm)
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cartridges from Biotage (Flash 12+M KP-Sil 12 x 150 mm (flow rate: 12 mL/min) or
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Flash 25+M KP-Sil 25 x 150 mm (flow rate: 25 mL/min)). 1H NMR spectra were
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recorded either on a Bruker AV400 (400 MHz) or a Bruker AV300 (300 MHz)
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spectrometer.
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(75 MHz) spectrometer. Chemical shifts are reported in ppm () with respect to the
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residual solvent signal of CDCl3 ( = 7.24 ppm (1H);  = 77.0 ppm (13C)). Further
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information on lipid synthesis can be found in the supplementary information.
13C
NMR spectra (Fig. S10-S29) were recorded on a Bruker AV300
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Results and Discussion
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Iso-15:0 is required for complementation of fruiting body development in M.
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xanthus mutant HB015
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Glycerolipids with 13-methyltetradecanol (iso15:0 alcohol; 1-6), tetradecanol (14:0
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alcohol; 7-9) 13-methyltetradecanoic acid (iso15:0; 10,11,14) and tetradecanoic acid
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(14:0; 12-13) (Fig. 1) were synthesized according to standard procedures exhibiting
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different chemical features in order to define the actual signalophore responsible for
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E-signaling in fruiting body and spore formation of M. xanthus. Lipids were dissolved
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in DMSO in different concentrations and were added directly to vegetative cells
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before allowing them to develop on TPM agar plates (Bhat et al., 2014). DMSO alone
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did not influence the development (Fig. S1). The first complementation assay was
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performed with the mutant strain HB015, which only forms loose aggregates but
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neither fruiting bodies nor spores (Fig. 2) due to its loss of iso-FAs. The cells were
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allowed to develop on TPM agar for 72 hours and the level of complementation was
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determined as the product of number, size and density of the fruiting bodies
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determined by ImageJ (Rasband, 1997-2014) in comparison to developing wild type
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cells. The negative control palmitic acid (16:0) indeed showed no complementation
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(Fig. S2). This is not surprising since C16:0 is only present in minute amounts in the
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fatty acid profile of M. xanthus (Bode et al., 2006; Garcia et al., 2011) and was never
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described to exhibit any signaling function. The positive controls isovalerate and iso-
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15:0 FA were capable of promoting fruiting body formation as already described (Fig.
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S2) (Bode et al., 2009; Bhat et al., 2014). Here, higher amounts of isovalerate also
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lead to a higher level of complementation (Fig. 2, Fig. S3). Unexpectedly, iso-15:0 FA
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concentrations higher than 40 nmol per 109 cells lead to a total loss of aggregates
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(Fig. 2). The same observation was made for lipids 1 and 10. Lipids 7 and 12 carrying
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C14:0 chains did not complement the mutant at any concentration indicating that
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glycerol alone is not promoting fruiting body formation at the tested concentrations
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although it is well known that the addition of glycerol can induce spore formation in M.
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xanthus from vegetative cells at concentrations of 0.5 M and higher (Dworkin &
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Gibson, 1964; Komano et al., 1980). This strongly supports the already described
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signaling character of iso-15:0 required at a specific concentration with a loss of
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development being the result of a signal “overdose”. However, it is not crucial
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whether iso-15:0 is bound to the glycerol backbone by an ester or an ether linkage
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since both compounds 1 and 10 lead to a similar level of complementation (Fig. 2).
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The important factor for complementation of HB015 is iso-15:0. Addition of
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isovalerate did not lead to a reduction in development at high concentrations as it is
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only the precursor required for the biosynthesis of iso-15:0 (Bode et al., 2006; Bode
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et al., 2009) and therefore the amount of signal produced can be regulated during the
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biosynthesis leading to a physiological amount of signals and the expected saturation
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(Fig. S3). Other complementing lipids were 2, 3, and 11 (Fig. S4 and S5) all bearing
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an iso-15:0 chain. In contrast to 1 and 10, these lipids did not complement at the
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same level (3) or they did not lead to an overdose (2 and 11). That might be due to
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decreased uptake efficiency by the cells, as these lipids carry acetyl (2) or an
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acetonid moiety (3 and 11) instead of free hydroxyl group. Moreover, triglycerides
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used in this work did only show small or no effects in complementation. Presumably,
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the hydrophobic properties of these lipids impede the uptake by the cells as they
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bear the same acyl residues as the MAGs but have no or little effect on
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complementation. Especially TG1 (4), which was identified as a signaling factor
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before (Bhat et al., 2014) was expected to have a much higher effect on
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development. Also 14 was expected to have a higher complementing effect as it
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theoretically provides the threefold amount of iso-15:0 to the cells compared to 10
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showing a strong effect on complementation in HB015 (Fig. 2).
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Spores from complemented HB015 fruiting bodies are defective in germination
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In order to test whether the complementation of fruiting bodies correlates with the
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formation of viable myxospores, fruiting bodies from HB015 developed with 40 nmol
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lipids per 109 cells were harvested and disrupted to give individual spores.
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Subsequently, isolated spores were counted (= total spores), plated on CTTYE plates
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and allowed to develop for seven days (= viable spores). Total spore counts showed
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results similar to the fruiting body complementation (Fig. 3A). As expected, cells
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treated with DMSO or palmitic acid did not develop any spores, as they were also not
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capable of forming fruiting bodies (Fig. 2, S1 and S3). While the addition of
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triglycerides (4, 5, 6 and 14) did only lead to very low levels of complementation (Fig.
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S2-S6) with only a few fruiting bodies, spore formation was restored to about 5 % of
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the wild type level. The addition of isovalerate, iso-15:0 FA and the monoglycerides
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with iso-15:0 caused obvious improvements in spore formation. Especially the
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monoglycerides as 1 and 10 exhibited strong complementation potential of 42 and 67
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%, respectively (Fig. 2, Fig. S3 and S5). The subsequent germination experiments
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showed that even if some lipids were able to restore the formation of myxospores,
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only a very small number of these spores were actually viable and could germinate
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(Fig. 3B). A possible explanation for the discrepancy between total and germinating
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spores could be the defect of strain HB015: Recent studies showed that during
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germination all iso-15:0 FAs are synthesized de novo (Ahrendt et al., 2015). Hence,
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the loss of IV-CoA biosynthesis in HB015 might prevent or delay spore germination
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as iso-FAs already present in the spore membranes cannot be used (Ahrendt et al.,
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2015). However, addition of 1 mM isovalerate to the germination media did not
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increase the number of germinating cells indicating a fine tuned regulation of iso-FA
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biosynthesis or function during germination that might be dependent on a very
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specific iso-FA concentration.
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An iso-15:0 ether lipid is capable of complementing the elbD mutant
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The elbD mutant is delayed in fruiting body formation and barely forms spores
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(Lorenzen et al., 2014). Figure 4 illustrates fruiting body formation in M. xanthus wild
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type DK1622 and elbD mutant over 48 hours. Mutant cells with DMSO exhibited
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almost no fruiting body formation within the first 20 hours and only reached about 57
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% of wild type development after 24 hours. This indicates no complementing effect of
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DMSO in early fruiting body formation in elbD. Nevertheless, no matter which lipid
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was added to the mutant, fruiting body formation was comparable to wild type after
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48 hours. The focus was therefore set on complementation during early fruiting body
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formation between 12 and 20 hours. As an initial assay showed the best
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complementation for 1 at 20 nmol per 109 cells (Fig. S7 and S8), this concentration
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was used for all lipids in this assay. The best level of complementation in early
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fruiting body formation was observed for 1, being an iso-15:0 ether lipid (Fig. 4; 18h).
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The most striking observation in this assay was the strong complementing effect of
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the C14:0 ether lipid 7 (Fig. 4) showing the second highest level of complementation.
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7 had no effect on complementation for HB015 (Fig. 2). As C14:0 ether lipids have
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never been found in M. xanthus and the absence of any effect by the glycerol
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backbone is demonstrated with 12, the ether bond must be responsible for the effect.
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Lipid 10 had only little effect on complementation in early fruiting body development
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and 12 did not have any effect (Fig. 4 and S9). Comparison of the complementing
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effect for iso-15:0 lipids 1 and 10, as well as the C14:0 lipids 7 and 12 emphasize the
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importance of the ether linkage for elbD complementation. Ether lipids 1 and 7 have a
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higher effect on complementation in early development than 10 and 12, which do not
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bear an ether linkage (Fig. 4, 18 h). However, iso-15:0 supports complementation as
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1 and 10 both show a higher effect than their C14:0 equivalents 7 and 12. In contrast
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to previous results (Lorenzen et al., 2014), elbD was able to form about 10 % of the
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wild type spore level (Fig. 3C). The addition of 1, which has already exhibited the
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highest effect on fruiting body formation, improved sporulation to over 40 % of wild
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type level. However, surprisingly most added lipids lead to a similar spore yield of 20
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to 30 %. The germination assay produced a more clear result, as the amount of
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viable spores was indeed higher for 1, 7 and 10, similar to their complementation
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potential in early fruiting body formation (Fig. 3D). The results indicate that ether
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lipids play a more important role in early fruiting body formation than in sporulation.
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The addition of different lipids could have provided a new energy source required for
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spore formation resulting in a higher amount of total and viable spores.
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DL-1-O-(13-methyltetradecyl)glycerol 1 includes the pharmacophore of the E-
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signal
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E-signal has been described as a combination of iso-15:0 FA and TG1 (4) (Bhat et
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al., 2014). The fact that complementation of HB015 development is only dependent
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on iso-15:0 and complementation of elbD is promoted by ether bonds, identifies
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these features as the actual pharmacophore of the E-signal, both present in 1.
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However, in contrast to TG1, no such monoglycerides as 1 have ever been detected
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in any lipid analysis in M. xanthus. Nevertheless, both molecules 1 and 4 exhibit the
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same molecular features, aside from the acyl chains in 4, which obviously hindered
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an efficient uptake by the cells resulting in only low complementing effects by 4.
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Previous research has shown that developing cells form TG1-filled lipid bodies and
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secret them into the fruiting body where they disappear gradually during development
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(Hoiczyk et al., 2009). This would argue for a mechanism, which transmits TG1, or a
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TG1-like lipid, as the actual E-signal. Figure 5 shows a proposed mechanism for E-
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signal transmission. During development ether lipid biosynthesis produces 4 leaving
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the cell through lipid body formation by an unknown mechanism. Probably lipid
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bodies are also released by cell lysis as the majority of cells are lysed during fruiting
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body formation in M. xanthus. Once the lipid bodies or the lipids they contain are
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taken up by a receiving cell 4 is hydrolyzed to 1, which might be further modified to
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yield the actual E-signal. Although uptake of lipids or lipid bodies is unknown so far,
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the proposed intermediate 1 can indeed been taken up directly as we have shown in
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this work. Nevertheless, the fate of the ether bond leading to the E-signal after
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degradation of the lipid bodies remains unexplored and there is no known
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mechanism for the degradation of ether lipids in myxobacteria. Although previous
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work has identified TG1 as the only physiological neutral lipid containing the E-signal
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pharmacophore identified in this work, more work is needed to identify the true
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structure of the E-signal as well its underlying mechanisms for signal detection and
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integration.
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Acknowledgement
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This work was funded by the DFG. The authors are grateful to Wolfram Lorenzen and
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Michael Ring for their pioneering work on ether lipids in myxobacteria.
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Figure legends
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Figure 1. Glycerolipids synthesized and tested featuring 13-methyltetradecanol
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(iso15:0 alcohol; 1-6), tetradecanol (14:0 alcohol; 7-9) 13-methyltetradecanoic acid
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(iso15:0; 10,11,14) or tetradecanoic acid (14:0; 12-13). See supplemental material for
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further details on the structure and synthesis of these compound .
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Figure 2. Complementation of M. xanthus mutant HB015 with various lipids in
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different concentrations. 40 µl of a cell suspension with a density of 5x10 9/mL were
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allowed to develop on TPM agar plates for 72 hours at 30°C. Data were acquired by
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graphical analysis of fruiting bodies using ImageJ (Rasband, 1997-2014). Numbers in
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the upper right corner of each image indicate the level of complementation that was
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calculated as a product of number, size and density of fruiting bodies. The wild type
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at 72h was set to 100.
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Figure 3. Sporulation and germination data for complementation of M. xanthus
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mutants HB015 and elbD. Lipids were added to a concentration of 40 and 20 nmol
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per 109 cells for HB015 and elbD, respectively. Myxospores from complementation
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assays with HB015 (A) and elbD (C) were isolated and counted. Afterwards spore
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suspensions of HB015 (B) and elbD (D) were plated on CTTYE germination media
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and incubated for 7 days at 30°C. Values were calculated with regard to DK1622 wild
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type set to 100 and are the result of triplicates.
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Figure 4. Fruiting body formation in M. xanthus wild type DK1622 and elbD
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complemented with various lipids. 5 µl of cell suspensions with a density of 5x10 9/mL
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were allowed to develop on TPM agar plates for 48 hours at 30°C. Lipids were added
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to a concentration of 20 nmol per 109 cells. Numbers indicate the level of
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complementation. For details see Figure 2.
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356
15
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Figure 5. Proposed formation of the E-signal in M. xanthus development. Branched
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chain fatty acid precursor isovaleryl-CoA is synthesized either from leucine or by the
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HMG-CoA pathway. Iso-15:0 fatty acid is used in either lipid biosynthesis leading to
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TG1 (4). Lipid bodies (or lipid vesicles) containing 4 are released into the extracellular
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space by an unknown mechanism or by cell lysis. Subsequent uptake of these lipid
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bodies (or lipid vesicles) containing 4 by the receiving cell (bottom), hydrolysis to 1
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and further modifications then possibly forms the E-signal.
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