Michael W. Ring

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.

3-Hydroxy-3-Methylglutaryl-Coenzyme A (CoA) Synthase Is Involved in Biosynthesis of Isovaleryl-CoA in the Myxobacterium Myxococcus xanthus during Fruiting Body Formation

Isovaleryl-coenzyme A (IV-CoA) is the starting unit for some secondary metabolites and iso-odd fatty acids in several bacteria. According to textbook biochemistry, IV-CoA is derived from leucine degradation, but recently an alternative pathway that branches from the well-known mevalonate-dependent isoprenoid biosynthesis has been described for myxobacteria. A double mutant was constructed in Myxococcus xanthus by deletion of genes involved in leucine degradation and disruption of mvaS encoding the 3-hydroxy-3-methylglutaryl-coenzyme A synthase. A dramatic decrease of IV-CoA-derived iso-odd fatty acids was observed for the mutant, confirming mvaS to be involved in the alternative pathway. Additional quantitative real-time reverse transcription-PCR experiments indicated that mvaS is transcriptionally regulated by isovalerate. Furthermore, feeding studies employing an intermediate specific for the alternative pathway revealed that this pathway is induced during fruiting body formation, which presumably increases the amount of IV-CoA available when leucine is limited.

Identification of Additional Players in the Alternative Biosynthesis Pathway to Isovaleryl-CoA in the Myxobacterium Myxococcus xanthus

Isovaleryl‐CoA (IV‐CoA) is usually derived from the degradation of leucine by using the Bkd (branched‐chain keto acid dehydrogenase) complex. We have previously identified an alternative pathway for IV‐CoA formation in myxobacteria that branches from the well‐known mevalonate‐dependent isoprenoid biosynthesis pathway. We identified 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) synthase (MvaS) to be involved in this pathway in Myxococcus xanthus, which is induced in mutants with impaired leucine degradation (e.g., bkd−) or during myxobacterial fruiting‐body formation. Here, we show that the proteins required for leucine degradation are also involved in the alternative IV‐CoA biosynthesis pathway through the efficient catalysis of the reverse reactions. Moreover, we conducted a global gene‐expression experiment and compared vegetative wild‐type cells with bkd mutants, and identified a five‐gene operon that is highly up‐regulated in bkd mutants and contains mvaS and other genes that are directly involved in the alternative pathway. Based on our experiments, we assigned roles to the genes required for the formation of IV‐CoA from HMG‐CoA. Additionally, several genes involved in outer‐membrane biosynthesis and a plethora of genes encoding regulatory proteins were decreased in expression levels in the bkd− mutant; this explains the complex phenotype of bkd mutants including a lack of adhesion in developmental submerse culture.

Lipid body formation plays a central role in cell fate determination during developmental differentiation of Myxococcus xanthus

Cell differentiation is widespread during the development of multicellular organisms, but rarely observed in prokaryotes. One example of prokaryotic differentiation is the Gram‐negative bacterium Myxococcus xanthus. In response to starvation, this gliding bacterium initiates a complex developmental programme that results in the formation of spore‐filled fruiting bodies. How the cells metabolically support the necessary complex cellular differentiation from rod‐shaped vegetative cells into spherical spores is unknown. Here, we present evidence that intracellular lipid bodies provide the necessary metabolic fuel for the development of spores. Formed at the onset of starvation, these lipid bodies gradually disappear until they are completely used up by the time the cells have become mature spores. Moreover, it appears that lipid body formation in M. xanthus is an important initial step indicating cell fate during differentiation. Upon starvation, two subpopulations of cells occur: cells that form lipid bodies invariably develop into spores, while cells that do not form lipid bodies end up becoming peripheral rods, which are cells that lack signs of morphological differentiation and stay in a vegetative‐like state. These data indicate that lipid bodies not only fuel cellular differentiation but that their formation represents the first known morphological sign indicating cell fate during differentiation.

Straight-Chain Fatty Acids Are Dispensable in the Myxobacterium Myxococcus xanthus for Vegetative Growth and Fruiting Body Formation

Inactivation of the MXAN_0853 gene blocked the production in Myxococcus xanthus of straight-chain fatty acids which otherwise represent 30% of total fatty acids. Despite this drastic change in the fatty acid profile, no change in phenotype could be observed, which contrasts with previous interpretations of the role of straight-chain fatty acids in the organisms development.

Phaselicystis flava gen. nov., sp. nov., an arachidonic acid-containing soil myxobacterium, and the description of Phaselicystidaceae fam. nov.

A bacterial strain designated SBKo001(T) was isolated from a forest soil sample from Mt Makiling in Laguna, Philippines. It shows the general characteristics associated with myxobacteria, such as swarming of Gram-negative, rod-shaped vegetative cells, fruiting body formation and bacteriolytic activity. The strain is mesophilic, strictly aerobic and chemoheterotrophic and also exhibits resistance to various antibiotics. Major fatty acids are iso-C(15 : 0), C(17 : 1) 2-OH and C(20 : 4) (arachidonic acid). The G+C content of the genomic DNA is 69.2 mol%. A reference strain, NOSO-1 (=DSM 53757), isolated from the Etosha Basin in Namibia, shares nearly the same characteristics with SBKo001(T). The identical 16S rRNA gene sequences of the two strains show 94 % identity to strains of the cellulose-degrading Byssovorax and Sorangium species. Phylogenetic analysis reveals a novel branch diverging from the Polyangiaceae, Sorangiineae, Myxococcales. Their uniqueness in morphological growth stages, unusual fatty acid profile, broad-spectrum antibiotic resistance and branch divergence from the Polyangiaceae imply that strains SBKo001(T) and NOSO-1 not only represent a novel genus and species, proposed here as Phaselicystis flava gen. nov., sp. nov., but also belong to a new family, Phaselicystidaceae fam. nov. The type strain of Phaselicystis flava is SBKo001(T) (=DSM 21295(T) =NCCB 100230(T)).

Biosynthesis of 2‐Hydroxy and iso‐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 α‐hydroxylases. Although the stereospecificities of the hydroxylases differ between these two species, they share a common function in supporting fatty acid α‐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 α‐hydroxylation reaction is in fact dependent on the presence of fatty acids bound to sphingolipids.

Isoprenoids Are Essential for Fruiting Body Formation in Myxococcus xanthus

It was recently shown that Myxococcus xanthus harbors an alternative and reversible biosynthetic pathway to isovaleryl coenzyme A (CoA) branching from 3-hydroxy-3-methylglutaryl-CoA. Analyses of various mutants in these pathways for fatty acid profiles and fruiting body formation revealed for the first time the importance of isoprenoids for myxobacterial development.

Functional analysis of desaturases from the myxobacterium Myxococcus xanthus

The fatty acid (FA) profiles of myxobacteria contain FA species with double bonds at the Delta(5) and Delta(11) positions, the latter being rather unusual among bacteria. Despite this knowledge, the mechanism for introduction of these double bonds has never been described before in myxobacteria. Searches for candidate genes in the genome of the model organism Myxococcus xanthus revealed 16 genes, which have been annotated as FA desaturases. However, due to redundant substrate specificity, functional analyses of these enzymes by construction of inactivation mutants did not lead to the identification of their function or substrate specificity. Therefore, we elucidated the regioselectivity of the desaturation reactions by heterologous expression of eight desaturases from M. xanthus in Pseudomonas putida and thus could prove five of them to be indeed active as desaturases, with three (MXAN_1742, MXAN_3495 and MXAN_5461) and two (MXAN_0317 and MXAN_6306) acting as Delta(5) and Delta(11) desaturases, respectively. This is the first report about the heterologous expression and regioselectivity of FA desaturases in myxobacteria.