• 2-Hydroxysorangiadenosine: Structure and Biosynthesis of a Myxobacterial Sesquiterpene-Nucleoside.

      Okoth, Dorothy A; Hug, Joachim J; Garcia, Ronald; Spröer, Cathrin; Overmann, Jörg; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-06-09)
      Myxobacteria represent an under-investigated source for biologically active natural products featuring intriguing structural moieties with potential applications, e.g., in the pharmaceutical industry. Sorangiadenosine and the here-discovered 2-hydroxysorangiadenosine are myxobacterial sesquiterpene-nucleosides with an unusual structural moiety, a bicyclic eudesmane-type sesquiterpene. As the biosynthesis of these rare terpene-nucleoside hybrid natural products remains elusive, we investigated secondary metabolomes and genomes of several 2-hydroxysorangiadenosine-producing myxobacteria. We report the isolation and full structure elucidation of 2-hydroxysorangiadenosine and its cytotoxic and antibiotic activities and propose a biosynthetic pathway in the myxobacterium Vitiosangium cumulatum MCy10943T.
    • Biocompatible bacteria-derived vesicles show inherent antimicrobial activity.

      Schulz, Eilien; Goes, Adriely; Garcia, Ronald; Panter, Fabian; Koch, Marcus; Müller, Rolf; Fuhrmann, Kathrin; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2018-11-28)
      Up to 25,000 people die each year from resistant infections in Europe alone, with increasing incidence. It is estimated that a continued rise in bacterial resistance by 2050 would lead up to 10 million annual deaths worldwide, exceeding the incidence of cancer deaths. Although the design of new antibiotics is still one way to tackle the problem, pharmaceutical companies investigate far less into new drugs than 30 years ago. Incorporation of antibiotics into nanoparticle drug carriers ("nanoantibiotics") is currently investigated as a promising strategy to make existing antibiotics regain antimicrobial strength and overcome certain types of microbial drug resistance. Many of these synthetic systems enhance the antimicrobial effect of drugs by protecting antibiotics from degradation and reducing their side effects. Nevertheless, they often cannot selectively target pathogenic bacteria and - due to their synthetic origin - may induce side-effects themselves. In this work, we present the characterisation of naturally derived outer membrane vesicles (OMVs) as biocompatible and inherently antibiotic drug carriers. We isolated OMVs from two representative strains of myxobacteria, Cystobacter velatus Cbv34 and Sorangiineae species strain SBSr073, a bacterial order with the ability of lysing other bacterial strains and currently investigated as sources of new secondary metabolites. We investigated the myxobacterias' inherent antibacterial properties after isolation by differential centrifugation and purification by size-exclusion chromatography. OMVs have an average size range of 145-194 nm. We characterised their morphology by electron cryomicroscopy and found that OMVs are biocompatible with epithelial cells and differentiated macrophages. They showed a low endotoxin activity comparable to those of control samples, indicating a low acute inflammatory potential. In addition, OMVs showed inherent stability under different storage conditions, including 4 °C, -20 °C, -80 °C and freeze-drying. OMV uptake in Gram-negative model bacterium Escherichia coli (E. coli) showed similar to better incorporation than liposome controls, indicating the OMVs may interact with model bacteria via membrane fusion. Bacterial uptake correlated with antimicrobial activity of OMVs as measured by growth inhibition of E. coli. OMVs from Cbv34 inhibited growth of E. coli to a comparable extent as the clinically established antibiotic gentamicin. Liquid-chromatography coupled mass spectrometry analyses revealed the presence of cystobactamids in OMVs, inhibitors of bacterial topoisomerase currently studied to treat different Gram-negative and Gram-positive pathogens. This work, may serve as an important basis for further evaluation of OMVs derived from myxobacteria as novel therapeutic delivery systems against bacterial infections.
    • Correlating chemical diversity with taxonomic distance for discovery of natural products in myxobacteria.

      Hoffmann, Thomas; Krug, Daniel; Bozkurt, Nisa; Duddela, Srikanth; Jansen, Rolf; Garcia, Ronald; Gerth, Klaus; Steinmetz, Heinrich; Müller, Rolf; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-02-23)
      Some bacterial clades are important sources of novel bioactive natural products. Estimating the magnitude of chemical diversity available from such a resource is complicated by issues including cultivability, isolation bias and limited analytical data sets. Here we perform a systematic metabolite survey of ~2300 bacterial strains of the order Myxococcales, a well-established source of natural products, using mass spectrometry. Our analysis encompasses both known and previously unidentified metabolites detected under laboratory cultivation conditions, thereby enabling large-scale comparison of production profiles in relation to myxobacterial taxonomy. We find a correlation between taxonomic distance and the production of distinct secondary metabolite families, further supporting the idea that the chances of discovering novel metabolites are greater by examining strains from new genera rather than additional representatives within the same genus. In addition, we report the discovery and structure elucidation of rowithocin, a myxobacterial secondary metabolite featuring an uncommon phosphorylated polyketide scaffold.
    • Fatty acid-related phylogeny of myxobacteria as an approach to discover polyunsaturated omega-3/6 Fatty acids.

      Garcia, Ronald; Pistorius, Dominik; Stadler, Marc; Müller, Rolf (2011-04)
      In an analysis of 47 aerobic myxobacterial strains, representing 19 genera in suborders Cystobacterineae, Nannocystineae, Sorangiineae, and a novel isolate, "Aetherobacter" SBSr008, an enormously diverse array of fatty acids (FAs) was found. The distribution of straight-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) supports the reported clustering of strains in the phylogenetic tree based on 16S rRNA genes. This finding additionally allows the prediction and assignment of the novel isolate SBSr008 into its corresponding taxon. Sorangiineae predominantly contains larger amounts of SCFA (57 to 84%) than BCFA. On the other hand, Cystobacterineae exhibit significant BCFA content (53 to 90%), with the exception of the genus Stigmatella. In Nannocystineae, the ratio of BCFA and SCFA seems dependent on the taxonomic clade. Myxobacteria could also be identified and classified by using their specific and predominant FAs as biomarkers. Nannocystineae is remarkably unique among the suborders for its absence of hydroxy FAs. After the identification of arachidonic (AA) FA in Phaselicystidaceae, eight additional polyunsaturated fatty acids (PUFAs) belonging to the omega-6 and omega-3 families were discovered. Here we present a comprehensive report of FAs found in aerobic myxobacteria. Gliding bacteria belonging to Flexibacter and Herpetosiphon were chosen for comparative analysis to determine their FA profiles in relation to the myxobacteria.
    • Future Directions of Marine Myxobacterial Natural Product Discovery Inferred from Metagenomics.

      Garcia, Ronald; La Clair, James J; Müller, Rolf; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-08-29)
      Over the last two decades, halophilic (organisms that thrive at high salt concentrations) and halotolerant (organisms that have adapted to high salt concentrations) myxobacteria emerged as an important source of structurally diverse secondary metabolites from the marine environment. This review explores the advance of metagenomics analysis and 16S rRNA gene phylogeny of the cultured and uncultured myxobacteria from marine and other salt-environments up to July 2018. The diversity of novel groups of myxobacteria in these environments appears unprecedented, especially in the
    • Metabolic and Biosynthetic Diversity in Marine Myxobacteria.

      Gemperlein, Katja; Zaburannyi, Nestor; Garcia, Ronald; La Clair, James J; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (2018-09-05)
      Prior to 2005, the vast majority of characterized myxobacteria were obtained from terrestrial habitats. Since then, several species of halotolerant and even obligate marine myxobacteria have been described. Chemical analyses of extracts from these organisms have confirmed their ability to produce secondary metabolites with unique chemical scaffolds. Indeed, new genera of marine-derived myxobacteria, particularly Enhygromyxa, have been shown to produce novel chemical scaffolds that differ from those observed in soil myxobacteria. Further studies have shown that marine sponges and terrestrial myxobacteria are capable of producing similar or even identical secondary metabolites, suggesting that myxobacterial symbionts may have been the true producers. Recent in silico analysis of the genome sequences available from six marine myxobacteria disclosed a remarkably versatile biosynthetic potential. With access to ever-advancing tools for small molecule and genetic evaluation, these studies suggest a bright future for expeditions into this yet untapped resource for secondary metabolites
    • Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections.

      Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K; et al. (MDPI, 2020-01-12)
      In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
    • Myxobakterielle Naturstofffabriken

      Krug, Daniel; Garcia, Ronald; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2020-02-01)
      Myxococcus xanthus is a prime example of soil-living myxobacteria featuring a complex lifestyle, including coordinated movement through swarming, predatory feeding on other microorganisms, and the formation of multicellular fruiting bodies. Due to its biosynthetic capabilities for secondary metabolite production and its applicability as biotechno-logical chassis organism for heterologous expression, Myxococcus stands out as a biochemical factory for bioactive molecules with future applications, not only in human therap
    • Structure and biosynthesis of sorangipyranone - a new γ-dihydropyrone from the myxobacterial strain MSr12020.

      Okoth, Dorothy A; Hug, Joachim J; Mándi, Attila; Kurtán, Tibor; Garcia, Ronald; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Oxford Academic, 2021-05-18)
      Sorangipyranone was isolated as a novel natural product featuring a unique 2,3-dihydro-γ-4H-pyrone scaffold from cultures of the myxobacterial strain MSr12020. We report here the full structure elucidation of sorangipyranone by spectroscopic techniques including 2D NMR and high-resolution mass spectrometry together with the analysis of the biosynthetic pathway. Determination of the absolute configuration was performed by time-dependent density functional theory-electronic circular dichroism calculations and determination of the applicability of the Snatzke's helicity rule, to correlate the high-wavelength n→π* electronic circular dichroism (ECD) transition and the absolute configuration of the 2,3-dihydro-4H-γ-pyrone, was done by the analysis of low-energy conformers and the Kohn-Sham orbitals. Sorangipyranone outlines a new class of a γ-dihydropyrone-containing natural product comprised of malonyl-CoA-derived building blocks and features a unique polyketide scaffold. In silico analysis of the genome sequence of the myxobacterial strain MSr12020 complemented with feeding experiments employing stable isotope-labeled precursors allowed the identification and annotation of a candidate biosynthetic gene cluster that encodes a modular polyketide synthase assembly line. A model for the biosynthetic pathway leading to the formation of the γ-dihydropyrone scaffold is presented in this study.
    • Vitiosangium cumulatum gen. nov., sp. nov. and Vitiosangium subalbum sp. nov., novel soil myxobacteria from Nepal , and emended descriptions of genus Archangium and Angiococcus, and of Cystobacteraceae family.

      Awal, Ram Prasad; Garcia, Ronald; Gemperlein, Katja; Wink, Joachim; Kunwar, Bikram; Parajuli, Niranjan; Müller, Rolf; Helmholtz-Institue für Pharmazeutische Forschung Saarland (HIPS), Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-01-28)
      Bacterial strains designated MCy10943T and MCy10944T were isolated in 2014 from dried Nepalese soil samples collected in 2013 from Phukot, Kalikot, Western Nepal and Godawari, Lalitpur, Central Nepal. The novel organisms showed typical myxobacterial growth characteristics which include swarming colony and fruiting body formation on solid surfaces, and a predatory ability to lyse microorganisms. The strains were aerobic, mesophilic, chemoheterotrophic and showed resistance to various antibiotics. The major cellular fatty acids common to both organisms were C17:0 2-OH, iso-C15:0, C16:1 and iso-C17:0. The G + C content of the genomic DNA was 72-75 mol %. Phylogenetic analysis showed that the strains belong to the family Cystobacteraceae, suborder Cystobacterineae, order Myxococcales. The 16S rRNA gene sequences of both strains showed 97-98 % similarity to Archangium gephyra DSM 2261T, Cystobacter violaceus DSM 14727T, and 96.7-97 % to Cystobacter fuscus DSM 2262T and Angiococcus disciformis DSM 52716T. Polyphasic taxonomic characterisation suggested that strains MCy10943T and MCy10944T represent two distinct species of a novel genus, for which the names Vitiosangium cumulatum and Vitiosangium subalbum are proposed. The type strain of Vitiosangium cumulatum is MCy10943T (=DSM 102952T =NCCB 100600T) while for Vitiosangium subalbum is MCy10944T (=DSM 102953T =NCCB 100601T). In addition, the genera Archangium and Angiococcus, and the family Cystobacteraceae is herewith emended.