Gertrud Schwär

A Type II Polyketide Synthase is Responsible for Anthraquinone Biosynthesis in Photorhabdus luminescens

Type II polyketide synthases are involved in the biosynthesis of numerous clinically relevant secondary metabolites with potent antibiotic or anticancer activity. Until recently the only known producers of type II PKSs were members of the Gram‐positive actimomycetes, well‐known producers of secondary metabolites in general. Here we present the second example of a type II PKS from Gram‐negative bacteria. We have identified the biosynthesis gene cluster responsible for the production of anthraquinones (AQs) from the entomopathogenic bacterium Photorhabdus luminescens. This is the first example of AQ production in Gram‐negative bacteria, and their heptaketide origin was confirmed by feeding experiments. Deletion of a cyclase/aromatase involved in AQ biosynthesis resulted in accumulation of mutactin and dehydromutactin, which have been described as shunt products of typical octaketide compounds from streptomycetes, and a pathway for AQ formation from octaketide intermediates is discussed.

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.

Bendigoles D–F, bioactive sterols from the marine sponge-derived Actinomadura sp. SBMs009

Marine derived actinomycetes have become an important source of bioactive natural products. Here we report the structure and bioactivity of the bendigoles D-F (1-3), 3-keto sterols isolated from the new marine sponge derived bacterium, Actinomadura sp. SBMs009. The isolation of these compounds was guided by a novel high-content screen for NF-κB and glucocorticoid receptor (GR) activity, and cytotoxicity assays. The structures of 1-3 were determined by detailed analysis of NMR, MS, and single crystal X-ray diffraction data. Interestingly, 1 displayed cytotoxicity against the L929 (mouse fibroblast) cell line with an IC(50) approximated to 30 μM and was the most active inhibitor of GR-translocation, while 3 was the most effective inhibitor of NF-κB nuclear translocation with an IC(50) of 71 μM.

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.

Mutasynthesis-derived myxalamids and origin of the isobutyryl-CoA starter unit of myxalamid B.

Myxalamids are potent inhibitors of the eukaryotic electron transport chain produced by different myxobacteria. Here, we describe the identification of the myxalamid biosynthesis gene cluster from Myxococcus xanthus. Additionally, new myxalamids (5–13) have been obtained by mutasynthesis from bkd mutants of M. xanthus and Stigmatella aurantiaca. Moreover, as these bkd mutants are still able to produce myxalamid B (2), the origin of the isobutyryl‐CoA (IB‐CoA) starter unit required for its biosynthesis has been determined. In a M. xanthus bkd mutant, IB‐CoA originates from valine, but in S. aurantiaca this starter unit is derived from α‐oxidation of iso‐odd fatty acids, thereby connecting primary and secondary metabolism.

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.

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.

A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+]i (between 122 and 334 nm) and [Ca2+]o (between 23 and 625 μm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down‐regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin‐dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+]o. We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination.