Myxobacterium Myxococcus Xanthus Research Articles

Ribonuclease D Processes a Small RNA Regulator of Multicellular Development in Myxobacteria.

By targeting mRNA transcripts, non-coding small RNAs (sRNAs) regulate the expression of genes governing a wide range of bacterial functions. In the social myxobacterium Myxococcus xanthus, the sRNA Pxr serves as a gatekeeper of the regulatory pathway controlling the life-cycle transition from vegetative growth to multicellular fruiting body development. When nutrients are abundant, Pxr prevents the initiation of the developmental program, but Pxr-mediated inhibition is alleviated when cells starve. To identify genes essential for Pxr function, a developmentally defective strain in which Pxr-mediated blockage of development is constitutively active (strain "OC") was transposon-mutagenized to identify suppressor mutations that inactivate or bypass Pxr inhibition and thereby restore development. One of the four loci in which a transposon insertion restored development is rnd, encoding the Ribonuclease D protein (RNase D). RNase D is an exonuclease important for tRNA maturation. Here, we show that disruption of rnd abolishes the accumulation of Pxr-S, the product of Pxr processing from a longer precursor form (Pxr-L) and the active inhibitor of development. Additionally, the decrease in Pxr-S caused by rnd disruption was associated with increased accumulation primarily of a longer novel Pxr-specific transcript (Pxr-XL) rather than of Pxr-L. The introduction of a plasmid expressing rnd reverted cells back to OC-like phenotypes in development and Pxr accumulation, indicating that a lack of RNase D alone suppresses the developmental defect of OC. Moreover, an in vitro Pxr-processing assay demonstrated that RNase D processes Pxr-XL into Pxr-L; this implies that overall, Pxr sRNA maturation requires a sequential two-step processing. Collectively, our results indicate that a housekeeping ribonuclease plays a central role in a model form of microbial aggregative development. To our knowledge, this is the first evidence implicating RNase D in sRNA processing.

Predatory Strategies of Myxococcus xanthus: Prey Susceptibility to OMVs and Moonlighting Enzymes.

Predatory outer membrane vesicles (OMVs) secreted by myxobacteria fuse readily with the outer membranes of Gram-negative bacteria, introducing toxic cargo into their prey. Here we used a strain of the myxobacterium Myxococcus xanthus that produces fluorescent OMVs to assay the uptake of OMVs by a panel of Gram-negative bacteria. M. xanthus strains took up significantly less OMV material than the tested prey strains, suggesting that re-fusion of OMVs with producing organisms is somehow inhibited. The OMV killing activity against different prey correlated strongly with the predatory activity of myxobacterial cells, however, there was no correlation between OMV killing activity and their propensity to fuse with different prey. It has previously been proposed that M. xanthus GAPDH stimulates the predatory activity of OMVs by enhancing OMV fusion with prey cells. Therefore, we expressed and purified active fusion proteins of M. xanthus glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (GAPDH and PGK; moonlighting enzymes with additional activities beyond their roles in glycolysis/gluconeogenesis) to investigate any involvement in OMV-mediated predation. Neither GAPDH nor PGK caused lysis of prey cells or enhanced OMV-mediated lysis of prey cells. However, both enzymes were found to inhibit the growth of Escherichia coli, even in the absence of OMVs. Our results suggest that fusion efficiency is not a determinant of prey killing, but instead resistance to the cargo of OMVs and co-secreted enzymes dictates whether organisms can be preyed upon by myxobacteria.

Cover Feature: Myxococcus xanthus as Host for the Production of Benzoxazoles (ChemBioChem 5/2023)

Benzoxazoles are structural motifs of many active pharmaceutical ingredients. The enzymatic synthesis of these heterocycles is mediated by the concerted action of an ATP-dependent ligase and an amidohydrolase. Expression of the two enzymes in the myxobacterium Myxococcus xanthus enables the recombinant production of benzoxazoles from supplemented aryl carboxylic acids. Cognate substrates as well as enzymes that naturally occur in M. xanthus serve as additional puzzle pieces that allow a diversification of the benzoxazole scaffold. In particular, the endogenous enzyme MxcE participates in the activation and incorporation of substrates, which would otherwise not be accessible for benzoxazole biosynthesis. More information can be found in the Research Article by M. Nett et al.

Plasmalogens and Photooxidative Stress Signaling in Myxobacteria, and How it Unmasked CarF/TMEM189 as the Δ1'-Desaturase PEDS1 for Human Plasmalogen Biosynthesis.

Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond that endows them with unique physical-chemical properties. They have proposed biological roles in membrane organization, fluidity, signaling, and antioxidative functions, and abnormal plasmalogen levels correlate with various human pathologies, including cancer and Alzheimer’s disease. The presence of plasmalogens in animals and in anaerobic bacteria, but not in plants and fungi, is well-documented. However, their occurrence in the obligately aerobic myxobacteria, exceptional among aerobic bacteria, is often overlooked. Tellingly, discovery of the key desaturase indispensable for vinyl ether bond formation, and therefore fundamental in plasmalogen biogenesis, emerged from delving into how the soil myxobacterium Myxococcus xanthus responds to light. A recent pioneering study unmasked myxobacterial CarF and its human ortholog TMEM189 as the long-sought plasmanylethanolamine desaturase (PEDS1), thus opening a crucial door to study plasmalogen biogenesis, functions, and roles in disease. The findings demonstrated the broad evolutionary sweep of the enzyme and also firmly established a specific signaling role for plasmalogens in a photooxidative stress response. Here, we will recount our take on this fascinating story and its implications, and review the current state of knowledge on plasmalogens, their biosynthesis and functions in the aerobic myxobacteria.

Total synthesis of Myxoprincomide, a secondary metabolite from Myxococcus xanthus.

Myxoprincomide, a secondary metabolite of the myxobacterium Myxococcus xanthus DK 1622, is synthesised for the first time. The central, unusual α-ketoamide is generated at the end of the synthesis to avoid side reactions during the synthesis of this rather reactive subunit. Nevertheless, the synthetic natural product is obtained as an isomeric mixture. Detailed analytical investigations show that the identical isomeric mixture is found in the isolated natural product.

Characterization of Constitutive Promoters for the Elicitation of Secondary Metabolites in Myxobacteria.

Genome mining has revealed that myxobacteria contain a myriad of cryptic biosynthetic gene clusters (BGCs). Here, we report the characterization of a panel of myxobacterial promoters with variable strength that are applicable in the engineering of BGCs in myxobacteria. The screened strongest constitutive promoter was used to efficiently enhance the expression of two complex BGCs governing the biosynthesis of myxochromide and DKxanthene in the model myxobacterium Myxococcus xanthus DK1622. We also showcased the combination of promoter engineering and MS2-based spectral networking as an effective strategy to shed light on the previously overlooked chemistry in the family of myxochromide-type lipopeptides. The enriched promoter library substantially expanded the synthetic biology toolkit available for myxobacteria.

Myxobacterial Genomics and Post-Genomics: A Review of Genome Biology, Genome Sequences and Related 'Omics Studies.

Myxobacteria are fascinating and complex microbes. They prey upon other members of the soil microbiome by secreting antimicrobial proteins and metabolites, and will undergo multicellular development if starved. The genome sequence of the model myxobacterium Myxococcus xanthus DK1622 was published in 2006 and 15 years later, 163 myxobacterial genome sequences have now been made public. This explosion in genomic data has enabled comparative genomics analyses to be performed across the taxon, providing important insights into myxobacterial gene conservation and evolution. The availability of myxobacterial genome sequences has allowed system-wide functional genomic investigations into entire classes of genes. It has also enabled post-genomic technologies to be applied to myxobacteria, including transcriptome analyses (microarrays and RNA-seq), proteome studies (gel-based and gel-free), investigations into protein–DNA interactions (ChIP-seq) and metabolism. Here, we review myxobacterial genome sequencing, and summarise the insights into myxobacterial biology that have emerged as a result. We also outline the application of functional genomics and post-genomic approaches in myxobacterial research, highlighting important findings to emerge from seminal studies. The review also provides a comprehensive guide to the genomic datasets available in mid-2021 for myxobacteria (including 24 genomes that we have sequenced and which are described here for the first time).

Functional genomics study of Pseudomonas putida to determine traits associated with avoidance of a myxobacterial predator

Predation contributes to the structure and diversity of microbial communities. Predatory myxobacteria are ubiquitous to a variety of microbial habitats and capably consume a broad diversity of microbial prey. Predator–prey experiments utilizing myxobacteria have provided details into predatory mechanisms and features that facilitate consumption of prey. However, prey resistance to myxobacterial predation remains underexplored, and prey resistances have been observed exclusively from predator–prey experiments that included the model myxobacterium Myxococcus xanthus. Utilizing a predator–prey pairing that instead included the myxobacterium, Cystobacter ferrugineus, with Pseudomonas putida as prey, we observed surviving phenotypes capable of eluding predation. Comparative transcriptomics between P. putida unexposed to C. ferrugineus and the survivor phenotype suggested that increased expression of efflux pumps, genes associated with mucoid conversion, and various membrane features contribute to predator avoidance. Unique features observed from the survivor phenotype when compared to the parent P. putida include small colony variation, efflux-mediated antibiotic resistance, phenazine-1-carboxylic acid production, and increased mucoid conversion. These results demonstrate the utility of myxobacterial predator–prey models and provide insight into prey resistances in response to predatory stress that might contribute to the phenotypic diversity and structure of bacterial communities.

Light-Triggered Carotenogenesis in Myxococcus xanthus: New Paradigms in Photosensory Signaling, Transduction and Gene Regulation.

Myxobacteria are Gram-negative δ-proteobacteria found predominantly in terrestrial habitats and often brightly colored due to the biosynthesis of carotenoids. Carotenoids are lipophilic isoprenoid pigments that protect cells from damage and death by quenching highly reactive and toxic oxidative species, like singlet oxygen, generated upon growth under light. The model myxobacterium Myxococcus xanthus turns from yellow in the dark to red upon exposure to light because of the photoinduction of carotenoid biosynthesis. How light is sensed and transduced to bring about regulated carotenogenesis in order to combat photooxidative stress has been extensively investigated in M. xanthus using genetic, biochemical and high-resolution structural methods. These studies have unearthed new paradigms in bacterial light sensing, signal transduction and gene regulation, and have led to the discovery of prototypical members of widely distributed protein families with novel functions. Major advances have been made over the last decade in elucidating the molecular mechanisms underlying the light-dependent signaling and regulation of the transcriptional response leading to carotenogenesis in M. xanthus. This review aims to provide an up-to-date overview of these findings and their significance.

Supercritical Fluid Extraction Enhances Discovery of Secondary Metabolites from Myxobacteria.

Supercritical fluid extraction (SFE) is widely used for the isolation of natural products from plants, but its application in efforts to identify structurally and physicochemically often dissimilar microbial natural products is limited to date. In this study, we evaluated the impact of SFE on the extractability of myxobacterial secondary metabolites, aiming to improve the prospects of discovering novel natural products. We investigated the influence of different co-solvents on the extraction efficiency of secondary metabolites from three myxobacterial strains and the antimicrobial activity profiles of the corresponding extracts. For each known secondary metabolite, we found extraction conditions using SFE leading to superior yields in the extracts compared to conventional solvent extraction. Compounds with a logP higher than 3 showed the best extraction efficiency using 20% EtOAc as a co-solvent, whereas compounds with logP values lower than 3 were better extractable using more polar co-solvents such as MeOH. Extracts generated with SFE showed increased antimicrobial activities including the presence of activities not explained by known myxobacterial secondary metabolites, highlighting the advantage of SFE for bioactivity-guided isolation. Moreover, non-targeted metabolomics analysis revealed a group of chlorinated metabolites produced by the well-studied model myxobacterium Myxococcus xanthus DK1622, which were not accessible previously due to their low concentration in conventional extracts. The enriched SF extracts were used for isolation and subsequent structure elucidation of chloroxanthic acid A as the founding member of a novel secondary metabolite family. Our findings encourage the increased utilization of SFE as a part of future screening workflows of microbial natural products.

Uncovering the molecular mechanisms of the Pseudomonas aeruginosa PA14 response to myxobacterial predation

Myxobacteria are Gram-negative bacteria, notable for their predatory and antimicrobial activities, which dictate the outcomes of their interactions with neighbouring organisms. They are abundant and widespread in nature, and can significantly affect the microbiome of an environment. We hypothesise that there are underlying molecular mechanisms in prey specieswhich govern the prey’s susceptibility/resistance to the antimicrobial activity of myxobacteria. In this work we attempt to define the mechanisms by which Pseudomonas aeruginosa PA14 resists predation bythe model myxobacterium Myxococcus xanthus. Pseudomonas aeruginosa is an opportunistic pathogen of humans and plants. With the rise in antibiotic resistant organisms, Pseudomonas spp. are categorised as World Health Organisation priority 1 antibiotic-resistant bacteria and are our prey of choice in this study. In collaboration with Dr N. Tucker (Strathclyde), and using a strain of M. xanthus expressing mCherry (courtesy of E. Hoiczyk, Sheffield), we developed 96-well plate assays of predation which measured the optical density of both predator and prey and the florescence of predator at different point intervals. Predation was assayed againsta library of approximately 5700 PA14 mutants to identify strains with increased/decreased susceptibility to predation. Responses of PA14 mutants varied with time and between mutants, allowing us to create a shortlist of candidate genes involved in the prey response to predation. We are currently performing a preliminary analysis of the data using the Integrated Genomic Viewer and Circos plots to assess the genomic organisation of the prey genes that influence susceptibility and resistance to predation.

Dynamics of Solitary Predation by Myxococcus xanthus on Escherichia coli Observed at the Single-Cell Level.

The predatory behavior of Myxococcus xanthus has attracted extensive attention due to its unique social traits and inherent biological activities. In addition to group hunting, individual M. xanthus cells are able to kill and lyse prey cells; however, there is little understanding of the dynamics of solitary predation. In this study, by employing a bacterial tracking technique, we investigated M. xanthus predatory dynamics on Escherichia coli at the single-cell level. The killing and lysis of E. coli by a single M. xanthus cell was monitored in real time by microscopic observation, and the plasmolysis of prey cells was identified at a relatively early stage of solitary predation. After quantitative characterization of their solitary predatory behavior, M. xanthus cells were found to respond more dramatically to direct contact with live E. coli cells than heat-killed or UV-killed cells, showing slower predator motion and faster lysing of prey. Among the three contact-dependent killing modes classified according to the major subareas of M. xanthus cells in contact with prey, leading pole contact was observed most. After killing the prey, approximately 72% of M. xanthus cells were found to leave without thorough degradation of the lysed prey, and this postresidence behavior is described as a lysis-leave pattern, indicating that solitary predation has low efficiency in terms of prey-cell consumption. Our results provide a detailed description of the single-cell level dynamics of M. xanthus solitary predation from both prey and predator perspectives.IMPORTANCE Bacterial predation plays multiple essential roles in bacterial selection and mortality within microbial ecosystems. In addition to its ecological and evolutionary importance, many potential applications of bacterial predation have been proposed. The myxobacterium Myxococcus xanthus is a well-known predatory member of the soil microbial community. Its predation is commonly considered a collective behavior comparable to a wolf pack attack; however, individual M. xanthus cells are also able to competently lead to the lysis of a prey cell. Using a bacterial tracking technique, we are able to observe and analyze solitary predation by M. xanthus on Escherichia coli at the single-cell level and reveal the dynamics of both predator and prey during the process. The present study will not only provide a comprehensive understanding of M. xanthus solitary predation but also help to explain why M. xanthus often displays multicellular characteristic predatory behaviors in nature, while a single cell is capable of predation.

The Predation Strategy of Myxococcus xanthus.

Myxobacteria are ubiquitous in soil environments. They display a complex life cycle: vegetatively growing cells coordinate their motility to form multicellular swarms, which upon starvation aggregate into large fruiting bodies where cells differentiate into spores. In addition to growing as saprophytes, Myxobacteria are predators that actively kill bacteria of other species to consume their biomass. In this review, we summarize research on the predation behavior of the model myxobacterium Myxococcus xanthus, which can access nutrients from a broad spectrum of microorganisms. M. xanthus displays an epibiotic predation strategy, i.e., it induces prey lysis from the outside and feeds on the released biomass. This predatory behavior encompasses various processes: Gliding motility and induced cell reversals allow M. xanthus to encounter prey and to remain within the area to sweep up its biomass, which causes the characteristic “rippling” of preying populations. Antibiotics and secreted bacteriolytic enzymes appear to be important predation factors, which are possibly targeted to prey cells with the aid of outer membrane vesicles. However, certain bacteria protect themselves from M. xanthus predation by forming mechanical barriers, such as biofilms and mucoid colonies, or by secreting antibiotics. Further understanding the molecular mechanisms that mediate myxobacterial predation will offer fascinating insight into the reciprocal relationships of bacteria in complex communities, and might spur application-oriented research on the development of novel antibacterial strategies.

Transcriptional changes when Myxococcus xanthus preys on Escherichia coli suggest myxobacterial predators are constitutively toxic but regulate their feeding.

Predation is a fundamental ecological process, but within most microbial ecosystems the molecular mechanisms of predation remain poorly understood. We investigated transcriptome changes associated with the predation of Escherichia coli by the myxobacterium Myxococcus xanthus using mRNA sequencing. Exposure to pre-killed prey significantly altered expression of 1319 predator genes. However, the transcriptional response to living prey was minimal, with only 12 genes being significantly up-regulated. The genes most induced by prey presence (kdpA and kdpB, members of the kdp regulon) were confirmed by reverse transcriptase quantitative PCR to be regulated by osmotic shock in M. xanthus, suggesting indirect sensing of prey. However, the prey showed extensive transcriptome changes when co-cultured with predator, with 40 % of its genes (1534) showing significant changes in expression. Bacteriolytic M. xanthus culture supernatant and secreted outer membrane vesicles (OMVs) also induced changes in expression of large numbers of prey genes (598 and 461, respectively). Five metabolic pathways were significantly enriched in prey genes up-regulated on exposure to OMVs, supernatant and/or predatory cells, including those for ribosome and lipopolysaccharide production, suggesting that the prey cell wall and protein production are primary targets of the predator’s attack. Our data suggest a model of the myxobacterial predatome (genes and proteins associated with predation) in which the predator constitutively produces secretions which disable its prey whilst simultaneously generating a signal that prey is present. That signal then triggers a regulated feeding response in the predator.

The Myxobacterium Myxococcus xanthus Can Sense and Respond to the Quorum Signals Secreted by Potential Prey Organisms.

The myxobacterium Myxococcus xanthus is a predatory member of the soil microfauna, able to consume bacteria (Gram-negative, Gram-positive), archaea, and fungi. Many potential prey of M. xanthus communicate amongst themselves using acyl homoserine lactones (AHLs) as quorum signals. M. xanthus cannot itself produce AHLs, but could potentially benefit by responding to exogenous AHLs produced during signaling between proximal prey. Four AHLs of different side chain length were tested and all found to delay sporulation of M. xanthus vegetative cells, and to stimulate germination of myxospores, increasing the proportion of predatory vegetative cells in the population. The predatory activity and expansion rates of M. xanthus colonies were also found to be stimulated by AHLs. Thermally inactivated AHLs had no effect on M. xanthus cells, and the response to AHLs depended (non-linearly) on the length of AHL side chain, suggesting that the effect of AHLs was mediated by specific signaling within M. xanthus, rather than being a consequence of the chemical or physical properties of AHLs. Therefore, it seems that the presence of xenic quorum signaling molecules enhances the predatory activity of M. xanthus. AHLs increase the proportion of the population capable of predation, and stimulate the motility and predatory activity of vegetative cells. We therefore propose that in the wild, M. xanthus uses AHLs as markers of nearby prey, potentially eavesdropping on the conversations between prey organisms.

Myxococcus xanthus induces actinorhodin overproduction and aerial mycelium formation by Streptomyces coelicolor

SummaryInteraction of the predatory myxobacterium Myxococcus xanthus with the non‐motile, antibiotic producer Streptomyces coelicolor was examined using a variety of experimental approaches. Myxococcus xanthus cells prey on S. coelicolor, forming streams of ordered cells that lyse the S. coelicolor hyphae in the contact area between the two colonies. The interaction increases actinorhodin production by S. coelicolor up to 20‐fold and triggers aerial mycelium production. Other bacteria are also able to induce these processes in S. coelicolor though to a lesser extent. These studies offer new clues about the expression of genes that remain silent or are expressed at low level in axenic cultures and open the possibility of overproducing compounds of biotechnological interest by using potent inducers synthesized by other bacteria.

Myxococcus xanthus Pph2 Is a Manganese-dependent Protein Phosphatase Involved in Energy Metabolism

The multicellular behavior of the myxobacterium Myxococcus xanthus requires the participation of an elevated number of signal-transduction mechanisms to coordinate the cell movements and the sequential changes in gene expression patterns that lead to the morphogenetic and differentiation events. These signal-transduction mechanisms are mainly based on two-component systems and on the reversible phosphorylation of protein targets mediated by eukaryotic-like protein kinases and phosphatases. Among all these factors, protein phosphatases are the elements that remain less characterized. Hence, we have studied in this work the physiological role and biochemical activity of the protein phosphatase of the family PPP (phosphoprotein phosphatases) designated as Pph2, which is forming part of the same operon as the two-component system phoPR1. We have demonstrated that this operon is induced upon starvation in response to the depletion of the cell energy levels. The increase in the expression of the operon contributes to an efficient use of the scarce energy resources available for developing cells to ensure the completion of the life cycle. In fact, a Deltapph2 mutant is defective in aggregation, sporulation yield, morphology of the myxospores, and germination efficiency. The yeast two-hybrid technology has shown that Pph2 interacts with the gene products of MXAN_1875 and 5630, which encode a hypothetical protein and a glutamine synthetase, respectively. Because Pph2 exhibits Ser/Thr, and to some extent Tyr, Mn(2+)-dependent protein phosphatase activity, it is expected that this function is accomplished by dephosphorylation of the specific protein substrates.

Biosynthesis of 2-Hydroxy andiso-Even Fatty Acids is Connected to Sphingolipid Formation in Myxobacteria

2-Hydroxy fatty acids can be found in several different organisms, including bacteria. In this study, we have studied the biosynthesis of 2-hydroxy fatty acids in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca, resulting in the identification of a family of stereospecific fatty acid alpha-hydroxylases. Although the stereospecificities of the hydroxylases differ between these two species, they share a common function in supporting fatty acid alpha-oxidation; that is, the oxidative shortening of fatty acids. Whereas in S. aurantiaca this process takes place during normal vegetative growth, in M. xanthus it takes place only under developmental conditions. We were also able to identify serine palmitoyltransferase encoding genes involved in sphingolipid biosynthesis as well as sphingolipids themselves in both types of myxobacteria, and were able to show that the alpha-hydroxylation reaction is in fact dependent on the presence of fatty acids bound to sphingolipids.

Cytoprophet: a Cytoscape plug-in for protein and domain interaction networks inference

Cytoprophet is a software tool that allows prediction and visualization of protein and domain interaction networks. It is implemented as a plug-in of Cytoscape, an open source software framework for analysis and visualization of molecular networks. Cytoprophet implements three algorithms that predict new potential physical interactions using the domain composition of proteins and experimental assays. The algorithms for protein and domain interaction inference include maximum likelihood estimation (MLE) using expectation maximization (EM); the set cover approach maximum specificity set cover (MSSC) and the sum-product algorithm (SPA). After accepting an input set of proteins with Uniprot ID/Accession numbers and a selected prediction algorithm, Cytoprophet draws a network of potential interactions with probability scores and GO distances as edge attributes. A network of domain interactions between the domains of the initial protein list can also be generated. Cytoprophet was designed to take advantage of the visual capabilities of Cytoscape and be simple to use. An example of inference in a signaling network of myxobacterium Myxococcus xanthus is presented and available at Cytoprophet's website. http://cytoprophet.cse.nd.edu.

Novel Iso-branched Ether Lipids as Specific Markers of Developmental Sporulation in the Myxobacterium Myxococcus xanthus

Iso-fatty acids (FAs) are the dominant FA family in all myxobacteria analyzed. Furthermore, it was postulated that iso-FAs or compounds derived thereof are involved in fruiting body formation in Myxococcus xanthus, since mutants with a reduced level of iso-FA due to a reduced level of the precursor isovaleryl-CoA, are delayed in aggregation and produce only few myxospores. To elucidate the function of iso-FAs and their corresponding lipids we have analyzed the developmental phenotype of mutants having different levels of iso-FAs resulting in a clear correlation between the amount of iso-FAs and the delay of aggregation and reduction in spore yield. Addition of either isovalerate or 13-methyltetradecanoic acid resulted in restoration of the wild-type FA profile and normal development. Detailed analysis of the fatty acid (FA) profile during fruiting body formation in Myxococcus xanthus wild-type revealed the specific accumulation of 13-methyltetradecanal and 1-O-13-methyltetradecylglycerol which were produced specifically in the myxospores and which are derived from 1-O-(13-methyl-1-Z-tetradecenyl)-2-O-(13-methyltetradecanoyl)-glycero-3-phosphatidylethanolamine (VEPE) and 1,2-di-(13-methyltetradecanoyl)-3-(13-methyltetradecyl)glycerol (TG-1), respectively. The structures of these unusual ether lipids have been determined by spectrometric methods and synthesis (for TG-1). Analysis of several mutants blocked at different stages of development indicated that the biosynthesis of TG-1 is developmentally regulated and that VEPE might be an intermediate in the TG-1 biosynthesis. Finally, addition of TG-1 to mutants blocked in the biosynthesis of isovaleryl-CoA could restore aggregation and sporulation emphasizing the important role of iso-branched lipids for myxobacterial development.