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Biomedical subjects

Rolf Müller

Publications and source records attributed to Rolf Müller.

At least 19 recordsLinked to original sources

The unique DKxanthene secondary metabolite family from the myxobacterium Myxococcus xanthus is required for developmental sporulation.

Under starvation conditions myxobacteria form multicellular fruiting bodies in which vegetative cells differentiate into heat- and desiccation-resistant myxospores. Myxobacteria in general are a rich source of secondary metabolites that often exhibit biological activities rarely found in nature. Although the involvement of a yellow compound in sporulation and fruiting body formation of Myxococcus xanthus was described almost 30 years ago, the chemical principle of the pigment remained elusive. This work presents the isolation and structure elucidation of a unique class of pigments that were named DKxanthenes (DKX). The corresponding biosynthetic gene cluster was identified, and DKX-negative mutants were constructed to investigate the physiological role of DKX during development. In these mutants, fruiting body formation was delayed. Moreover, severely reduced amounts of viable spores were observed after 120 h of starvation, whereas no viable spores were formed at all after 72 h. The addition of purified DKX to the mutants resulted in the formation of viable spores after 72 h. Even though an antioxidative activity could be assigned to DKX, the true biochemical mechanism underlying the complementation remains to be elucidated.

Antioxidants↗

Filamin-regulated F-actin assembly is essential for morphogenesis and controls phototaxis in Dictyostelium.

Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.

Actins↗

Noseleaf furrows in a horseshoe bat act as resonance cavities shaping the biosonar beam.

Horseshoe bats emit their ultrasonic biosonar pulses through nostrils surrounded by intricately shaped protuberances (noseleaves). While these noseleaves have been hypothesized to affect the sonar beam, their physical function has never been analyzed. Using numerical methods, we show that conspicuous furrows in the noseleaf act as resonance cavities shaping the sonar beam. This demonstrates that (a) animals can use resonances in external, half-open cavities to direct sound emissions, (b) structural detail in the faces of bats can have acoustic effects even if it is not adjacent to the emission sites, and (c) specializations in the biosonar system of horseshoe bats allow for differential processing of subbands of the pulse in the acoustic domain.

Acoustics↗

On the biosynthetic origin of methoxymalonyl-acyl carrier protein, the substrate for incorporation of "glycolate" units into ansamitocin and soraphen A.

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.

Acyl Carrier Protein↗

Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus.

Although many secondary metabolites exhibiting important pharmaceutical and agrochemical activities have been isolated from myxobacteria, most of these microorganisms remain difficult to handle genetically. To utilize their metabolic potential, heterologous expression methodologies are currently being developed. Here, the Red/ET recombination technology was used to perform all required gene cluster engineering steps in Escherichia coli prior to the transfer into the chromosome of the heterologous host. We describe the integration of the complete 57-kbp myxothiazol biosynthetic gene cluster reconstituted from two cosmids from a cosmid library of the myxobacterium Stigmatella aurantiaca DW4-3/1 into the chromosome of the thus far best-characterized myxobacterium, Myxococcus xanthus, in one step. The successful integration and expression of the myxothiazol biosynthetic genes in M. xanthus results in the production of myxothiazol in yields comparable to the natural producer strain.

Bacterial Proteins↗

The annexins of Dictyostelium.

Annexins are a highly conserved ubiquitous family of Ca2+- and phospholipid-binding proteins present in nearly all eukaryotic cells. Analysis of the Dictyostelium genome revealed the presence of two annexin genes, the annexin C1 gene (nxnA) giving rise to two isoforms of 47 and 51 kDa (previously synexin), and the annexin C2 gene (nxnB) coding for a 56-kDa protein with 33% sequence identity to annexin C1. Annexin C2 is expressed at very low and constant levels throughout development. Quantification by real-time PCR indicated that it is present in about 35-fold lower amounts compared to annexin C1. We have used a GFP-tagged annexin C2 to study its cellular distribution and dynamics. In cell fractionation studies, annexin C2 cofractionates with annexin C1 and is enriched in the 100,000 g pellet. Like annexin C1, GFP-AnxC2 stains the plasma membrane. In addition it is present in the perinuclear region and overlaps to some degree with the Golgi apparatus, whereas annexin C1 is present on intracellular membranes resembling endosomal membranes and in the nucleus. Annexin C2 is not observed in the nucleus. An annexin C1 mutant (SYN-) which shows a defect during multicellular development can be rescued by full-length annexin C1, whereas overexpression of GFP-AnxC2 did not rescue the developmental defect The data support the concept that annexins, although having a highly conserved structure, participate in different functions in a cell.

Amino Acid Sequence↗

Analysis of myxobacterial secondary metabolism goes molecular.

During the last 20 years myxobacteria have made their way from highly exotic organisms to one of the major sources of microbial secondary metabolites besides actinomycetes and fungi. The pharmaceutical interest in these peculiar prokaryotes lies in their ability to produce a variety of structurally unique compounds and/or metabolites with rare biological activities. This review deals with the recent progress toward a better understanding of the biology, the genetics, the biochemistry and the regulation of secondary metabolite biosynthesis in myxobacteria. These research efforts paved the way to sophisticated in vitro studies and to the heterologous expression of complete biosynthetic pathways in conjunction with their targeted manipulation. The progress made is a prerequisite for using the vast resource of myxobacterial diversity regarding secondary metabolism more efficiently in the future.

Gene Expression Regulation, Bacterial↗

Development of simple media which allow investigations into the global regulation of chivosazol biosynthesis with Sorangium cellulosum So ce56.

Media, completely different with respect to their complexity, were investigated for growth and secondary metabolism with Sorangium cellulosum strain So ce56. While technical substrates are best for metabolite production, these media are not suitable to study gene expressions. Free amino acids as present in soluble media based on peptones were found to inhibit polyketide biosynthesis. Therefore synthetic growth and production media were developed. Different nitrogen- and carbon sources as well as trace elements were studied with respect to growth and regulation of chivosazol biosynthesis. A simple defined medium with asparagine as nitrogen source, glucose as carbon source and zinc ions as essential trace element is proposed for future studies into secondary metabolism using transcriptomics and proteomics.

Culture Media↗

Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads.

Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be "silent" under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.

Acyltransferases↗

A unique mechanism for methyl ester formation via an amide intermediate found in myxobacteria.

Secondary metabolism involves a broad diversity of biochemical reactions that result in a wide variety of biologically active compounds. Terminal amide formation during the biosynthesis of the myxobacterial electron-transport inhibitor, myxothiazol, was analyzed by heterologous expression of the unique nonribosomal-peptide synthetase, MtaG, and incubation with a synthesized substrate mimic. These experiments provide evidence that the terminal amide is formed from a carrier protein-bound myxothiazol acid that is thioesterified to MtaF. This intermediate is transformed to an amide by extension with glycine and subsequent oxidative cleavage by MtaG. The final steps of melithiazol assembly involve a highly similar protein-bound intermediate (attached to MelF, a homologue of MtaF), which is transformed to an amide by MelG (homologue of MtaG). In this study, we also show that the amide moiety of myxothiazol A can be hydrolyzed in vivo to the formerly unknown free myxothiazol acid by heterologous expression of melJ in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The methyltransferase MelK can finally methylate the acid to give rise to the methyl ester, which is produced as the final product in the melithiazol A biosynthetic pathway. These experiments clarify the role of MelJ and MelK during melithiazol assembly.

Acrylates↗

Myxovirescin A biosynthesis is directed by hybrid polyketide synthases/nonribosomal peptide synthetase, 3-hydroxy-3-methylglutaryl-CoA synthases, and trans-acting acyltransferases.

Myxococcus xanthus DK1622 is shown to be a producer of myxovirescin (antibiotic TA) antibiotics. The myxovirescin biosynthetic gene cluster spans at least 21 open reading frames (ORFs) and covers a chromosomal region of approximately 83 kb. In silico analysis of myxovirescin ORFs in conjunction with genetic studies suggests the involvement of four type I polyketide synthases (PKSs; TaI, TaL, TaO, and TaP), one major hybrid PKS/NRPS (Ta-1), and a number of monofunctional enzymes similar to the ones involved in type II fatty-acid biosynthesis (FAB). Whereas deletion of either taI or taL causes a dramatic drop in myxovirescin production, deletion of both genes (DeltataIL) leads to the complete loss of myxovirescin production. These results suggest that both TaI and TaL PKSs might act in conjunction with a methyltransferase, reductases, and a monooxygenase to produce the 2-hydroxyvaleryl-S-ACP starter that is proposed to act as the biosynthetic primer in the initial condensation reaction with glycine. Polymerization of the remaining 11 acetates required for lactone formation is directed by 12 modules of Ta-1, TaO, and TaP megasynthetases. All modules, except for the first module of TaL, lack cognate acyltransferase (AT) domains. Furthermore, deletion of a discrete tandem AT-encoded by taV-blocks myxovirescin production; this suggests an "in trans" mode of action. To embellish the macrocycle with methyl and ethyl moieties, assembly of the myxovirescin scaffold is proposed to switch twice from PKS to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)-like biochemistry during biosynthesis. Disruption of the S-adenosylmethionine (SAM)-dependent methyltransferase, TaQ, shifts production toward two novel myxovirescin analogues, designated myxovirescin Q(a) and myxovirescin Q(c). NMR analysis of purified myxovirescin Q(a) revealed the loss of the methoxy carbon atom. This novel analogue lacks bioactivity against E. coli.

Acyltransferases↗

Molecular and biochemical studies of chondramide formation-highly cytotoxic natural products from Chondromyces crocatus Cm c5.

The jaspamide/chondramide family of depsipeptides are mixed PKS/NRPS natural products isolated from marine sponges and a terrestrial myxobacterium that potently affect the function of the actin cytoskeleton. As a first step to improve production in heterologous host cells and permit genetic approaches to novel analogs, we have cloned and characterized the chondramide biosynthetic genes from the myxobacterium Chondromyces crocatus Cm c5. In addition to the expected PKS and NRPS genes, the cluster encodes a rare tyrosine aminomutase for beta-tyrosine formation and a previously unknown tryptophan-2-halogenase. Conditions for gene transfer into C. crocatus Cm c5 were developed, and inactivation of several genes corroborated their proposed function and served to define the boundaries of the cluster. Biochemical characterization of the final NRPS adenylation domain confirmed the direct activation of beta-tyrosine, and fluorinated chondramides were produced through precursor-directed biosynthesis.

Adenine↗

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation.

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.

Acyl Coenzyme A↗

Proteome analysis of Myxococcus xanthus by off-line two-dimensional chromatographic separation using monolithic poly-(styrene-divinylbenzene) columns combined with ion-trap tandem mass spectrometry.

Myxobacteria are potent producers of secondary metabolites exhibiting diverse biological activities and pharmacological potential. The proteome of Myxococcus xanthus DK1622 was characterized by two-dimensional chromatographic separation of tryptic peptides from a lysate followed by tandem mass spectrometric identification. The high degree of orthogonality of the separation system employing polymer-based strong cation-exchange and monolithic reversed-phase stationary phases was clearly demonstrated. Upon automated database searching, 1312 unique peptides were identified, which were associated with 631 unique proteins. High-molecular polyketide synthetases and nonribosomal peptide synthetases, known to be involved in the biosynthesis of various secondary metabolites, were readily detected. Besides the identification of gene products associated with the production of known secondary metabolites, proteins could also be identified for six gene clusters, for which no biosynthetic product has been known so far.

Amino Acid Sequence↗

Induction of PPARbeta and prostacyclin (PGI2) synthesis by Raf signaling: failure of PGI2 to activate PPARbeta.

A role for the nuclear receptor peroxisome proliferator-activated receptor-beta (PPARbeta) in oncogenesis has been suggested by a number of observations but its precise role remains elusive. Prostaglandin I2 (PGI2, prostacyclin), a major arachidonic acid (AA) derived cyclooxygenase (Cox) product, has been proposed as a PPARbeta agonist. Here, we show that the 4-hydroxytamoxifen (4-OHT) mediated activation of a C-Raf-estrogen receptor fusion protein leads to the induction of both the PPARbeta and Cox-2 genes, concomitant with a dramatic increase in PGI2 synthesis. Surprisingly, however, 4-OHT failed to activate PPARbeta transcriptional activity, indicating that PGI2 is insufficient for PPARbeta activation. In agreement with this conclusion, the overexpression of ectopic Cox-2 and PGI2 synthase (PGIS) resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPARbeta. Conversely, inhibition of PGIS blocked PGI2 synthesis but did not affect the AA mediated activation of PPARbeta. Our data obtained with four different cell types and different experimental strategies do not support the prevailing opinion that PGI2 plays a significant role in the regulation of PPARbeta.

Animals↗

A helical biosonar scanning pattern in the Chinese noctule, Nyctalus plancyi.

Directivity and sound diffraction of the pinna of the Chinese Noctule (Nyctalus plancyi) have been studied numerically. The pinna was found capable of generating a periodic helical scanning pattern over frequency, if the tragus and the thickened lower ledge of the pinna rim were in an appropriate position. During the helical scan, a directivity pattern with a strong mainlobe alternated with a pattern dominated by a conical sleeve of sidelobes. This alternation was present, even when an unfavorable arrangement of the pinna disrupted the overall helical scanning pattern. In the fully formed helical scan, the orientation of main and sidelobes for different frequencies revealed a spatial ordering which extends volume coverage. Five different pinna parts have been removed from the digital pinna-shape representations in turn to assess their influence on the directivity. Of these parts, the tragus stem and the thickened lower ledge of the pinna rim were found to have the largest overall impact. The anatomical prominence of these structures was hence in agreement with their acoustic functionality. In the near-field, tragus stem and lower ledge were seen to act primarily through large shifts in the wavefield phase in both directions.

Acoustics↗

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.

Acyl Coenzyme A↗