• Acetyl-CoA carboxylase 1 regulates endothelial cell migration by shifting the phospholipid composition.

      Glatzel, Daniel K; Koeberle, Andreas; Pein, Helmut; Löser, Konstantin; Stark, Anna; Keksel, Nelli; Werz, Oliver; Müller, Rolf; Bischoff, Iris; Fürst, Robert; HIPS, Helmholtz-Institute für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2018-02)
      The enzyme acetyl-CoA carboxylase (ACC) plays a crucial role in fatty acid metabolism. In recent years, ACC has been recognized as a promising drug target for treating different diseases. However, the role of ACC in vascular endothelial cells (ECs) has been neglected so far. To characterize the role of ACC, we used the ACC inhibitor, soraphen A, as a chemical tool, and also a gene silencing approach. We found that ACC1 was the predominant isoform in human umbilical vein ECs as well as in human microvascular ECs and that soraphen A reduced the levels of malonyl-CoA. We revealed that ACC inhibition shifted the lipid composition of EC membranes. Accordingly, membrane fluidity, filopodia formation, and migratory capacity were reduced. The antimigratory action of soraphen A depended on an increase in the cellular proportion of PUFAs and, most importantly, on a decreased level of phosphatidylglycerol. Our study provides a causal link between ACC, membrane lipid composition, and cell migration in ECs. Soraphen A represents a useful chemical tool to investigate the role of fatty acid metabolism in ECs and ACC inhibition offers a new and valuable therapeutic perspective for the treatment of EC migration-related diseases.
    • The actin targeting compound Chondramide inhibits breast cancer metastasis via reduction of cellular contractility.

      Menhofer, Magdalena H; Kubisch, Rebekka; Schreiner, Laura; Zorn, Matthias; Foerster, Florian; Mueller, Rolf; Raedler, Joachim O; Wagner, Ernst; Vollmar, Angelika M; Zahler, Stefan; Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Saarbru ¨ cken, Germany. (2014)
      A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent.
    • Activity-guided screening of bioactive natural compounds implementing a new glucocorticoid-receptor-translocation assay and detection of new anti-inflammatory steroids from bacteria.

      Kaufmann, Katrin; Simmons, Luke; Herrmann, Jennifer; Schwär, Gertrud; Luniak, Nora; Müller, Rolf; Department of Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany. kaufm@uni-trier.de (2013-01)
      Using an in vitro cell-based assay in a flow-design, we have applied activity-guided screening to search for new bioactive compounds isolated from microorganisms. A first assay employs the stable expression of nuclear factor kappa B (NF-κB) while a second assay utilizes the glucocorticoid receptor (GR) coupled to green fluorescent protein. A specialized assay was implemented for both the translocation of NF-κB and to inhibit the translocation of cytokine-mediated NF-κB. In addition, we developed in a wide palette of cell lines used for a highly specialized GR-translocation assay to detect anti-inflammatory effects. This approach demonstrates the straight-forward combination of cell-based assays arranged with an automated fluorescence microscope. This allows for the direct sorting of extracts which are acting in a pharmaceutically interesting way. Initial results using this technique have led to the detection of new anti-inflammatory steroids from bacterial crude extracts.
    • The AibR-isovaleryl coenzyme A regulator and its DNA binding site - a model for the regulation of alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus.

      Bock, Tobias; Volz, Carsten; Hering, Vanessa; Scrima, Andrea; Müller, Rolf; Blankenfeldt, Wulf; Hel,holtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-12-09)
      Isovaleryl coenzyme A (IV-CoA) is an important building block of iso-fatty acids. In myxobacteria, IV-CoA is essential for the formation of signaling molecules involved in fruiting body formation. Leucine degradation is the common source of IV-CoA, but a second, de novo biosynthetic route to IV-CoA termed AIB (alternative IV-CoA biosynthesis) was recently discovered in M. xanthus The AIB-operon contains the TetR-like transcriptional regulator AibR, which we characterize in this study. We demonstrate that IV-CoA binds AibR with micromolar affinity and show by gelshift experiments that AibR interacts with the promoter region of the AIB-operon once IV-CoA is present. We identify an 18-bp near-perfect palindromic repeat as containing the AibR operator and provide evidence that AibR also controls an additional genomic locus coding for a putative acetyl-CoA acetyltransferase. To elucidate atomic details, we determined crystal structures of AibR in the apo, the IV-CoA- and the IV-CoA-DNA-bound state to 1.7 Å, 2.35 Å and 2.92 Å, respectively. IV-CoA induces partial unfolding of an α-helix, which allows sequence-specific interactions between AibR and its operator. This study provides insights into AibR-mediated regulation and shows that AibR functions in an unusual TetR-like manner by blocking transcription not in the ligand-free but in the effector-bound state.
    • An Unprecedented Octahydro-3H-oxeto[2,3,4-ij]isochromene Ring System Formed by a Trichloromethyl-Anion-Induced Reaction Cascade

      Schmidt, Witali; Jones, Peter; Herrmann, Jennifer; Müller, Rolf; Schulz, Stefan; Helmholtz-Institut für Pharmaceutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany.; Institute of Organic Chemistry, Technische Universität Braunschweig; Institute of Inorganic and Analytical Chemistry, Technische Universität Braunschweig; Department Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Saarland University; Department Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Saarland University; Institute of Organic Chemistry, Technische Universität Braunschweig (2016-11-18)
    • Analytics of the therapeutic peptide aviptadil by sheathless CE-MS and comparison with nanoRP-HPLC-MS.

      Gross, Peter C; Burkart, Sonja C; Müller, Rolf; Biotech Processes and Analytics Department, PharmBioTec GmbH, D-66123 Saarbrücken, Germany. Electronic address: p.gross@pharmbiotec.de. (2014-01)
      Purification and quality control of therapeutic peptides is often performed by one single method, RP-HPLC. As usage of an orthogonal technique is highly advisable for quality assurance, capillary electrophoresis (CE) employing a coated capillary coupled via a sheathless interface to a mass spectrometer was applied in parallel. The basic therapeutic peptide aviptadil served as a model substance to study the impurity profiles revealing 15 detectable impurities using CE-MS, two were detected by an appropriate nanoRP-HPLC-MS method. None of the impurities detected by CE were observed in LC and vice versa. The LOD in CE-MS was determined in the base peak electropherogram at ∼1fmol, a value 2500 times smaller than the LOD found in nanoRP-HPLC-MS (3pmol). In nanoRP-HPLC-MS only 0.2% of the extrapolated CE-MS signal for a 25ng aviptadil load was observed. We conclude that both, the LOD as well as the impurity profile of aviptadil, as analyzed by nanoRP-HPLC are influenced by both, the ligand-derivatized silica matrix and the flow-rate. Peptides may disappear completely and their variable emergence may lead to the determination of incorrect ratios as present in the sample.
    • Anti-leukemic effects of the V-ATPase inhibitor Archazolid A.

      Zhang, Siwei; Schneider, Lina S; Vick, Binje; Grunert, Michaela; Jeremias, Irmela; Menche, Dirk; Müller, Rolf; Vollmar, Angelika M; Liebl, Johanna; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2015-12-22)
      Prognosis for patients suffering from T-ALL is still very poor and new strategies for T-ALL treatment are urgently needed. Our study shows potent anti-leukemic effects of the myxobacterial V-ATPase inhibitor Archazolid A. Archazolid A reduced growth and potently induced death of leukemic cell lines and human leukemic samples. By inhibiting lysosomal acidification, Archazolid A blocked activation of the Notch pathway, however, this was not the mechanism of V-ATPase inhibition relevant for cell death induction. In fact, V-ATPase inhibition by Archazolid A decreased the anti-apoptotic protein survivin. As underlying mode of action, this work is in line with recent studies from our group demonstrating that Archazolid A induced S-phase cell cycle arrest by interfering with the iron metabolism in leukemic cells. Our study provides evidence for V-ATPase inhibition as a potential new therapeutic option for T-ALL.
    • Antimalarial activity of the myxobacterial macrolide chlorotonil a.

      Held, Jana; Gebru, Tamirat; Kalesse, Markus; Jansen, Rolf; Gerth, Klaus; Müller, Rolf; Mordmüller, Benjamin (2014-11)
      Myxobacteria are Gram-negative soil-dwelling bacteria belonging to the phylum Proteobacteria. They are a rich source of promising compounds for clinical application, such as epothilones for cancer therapy and several new antibiotics. In the course of a bioactivity screening program of secondary metabolites produced by Sorangium cellulosum strains, the macrolide chlorotonil A was found to exhibit promising antimalarial activity. Subsequently, we evaluated chlorotonil A against Plasmodium falciparum laboratory strains and clinical isolates from Gabon. Chlorotonil A was highly active, with a 50% inhibitory concentration between 4 and 32 nM; additionally, no correlations between the activities of chlorotonil A and artesunate (rho, 0.208) or chloroquine (rho, -0.046) were observed. Per os treatment of Plasmodium berghei-infected mice with four doses of as little as 36 mg of chlorotonil A per kg of body weight led to the suppression of parasitemia with no obvious signs of toxicity. Chlorotonil A acts against all stages of intraerythrocytic parasite development, including ring-stage parasites and stage IV to V gametocytes, and it requires only a very short exposure to the parasite to exert its antimalarial action. Conclusively, chlorotonil A has an exceptional and unprecedented profile of action and represents an urgently required novel antimalarial chemical scaffold. Therefore, we propose it as a lead structure for further development as an antimalarial chemotherapeutic.
    • antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters.

      Weber, Tilmann; Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A; Müller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko; Medema, Marnix H; Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Saarland University, Saarbrucken, Germany. (2015-05-06)
      Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.
    • Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents.

      Castoldi, Arianna; Empting, Martin; De Rossi, Chiara; Mayr, Karsten; Dersch, Petra; Hartmann, Rolf; Müller, Rolf; Gordon, Sarah; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2018-12-05)
      The objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections. Aspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri. InvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration. InvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy.
    • Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment.

      Hauf, Samuel; Herrmann, Jennifer; Miethke, Marcus; Gibhardt, Johannes; Commichau, Fabian M; Müller, Rolf; Fuchs, Stephan; Halbedel, Sven; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-Blackwell, 2019-01-15)
      Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multi drug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild type cells were not known. Using RNA-Seq and promoter-lacZ fusions we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil. This article is protected by copyright. All rights reserved.
    • 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.
    • The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of Escherichia coli.

      Donner, Jannik; Reck, Michael; Bunk, Boyke; Jarek, Michael; App, Constantin Benjamin; Meier-Kolthoff, Jan P; Overmann, Jörg; Müller, Rolf; Kirschning, Andreas; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr, 7,38124 Braunschweig, Germany. (2017-11-01)
      The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.
    • Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme.

      Wang, Yuanyou; Schnell, Bastien; Baumann, Sascha; Müller, Rolf; Begley, Tadhg P; HIPS, Helmholtz-Institut füt Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (2017-02-08)
      The biosynthesis of branched alkoxy groups, such as the unique t-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metabolites contain an isopropyl group proposed to be formed using CysS, a cobalamin-dependent radical S-adenosylmethionine (SAM) methyltransferase. Here, we reconstitute the CysS-catalyzed reaction, on p-aminobenzoate thioester substrates, and demonstrate that it not only catalyzes sequential methylations of a methyl group to form ethyl and isopropyl groups but remarkably also sec-butyl and t-butyl groups. To our knowledge, this is the first in vitro reconstitution of a cobalamin-dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t-butyl group.
    • Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.

      Kolinko, Isabel; Lohße, Anna; Borg, Sarah; Raschdorf, Oliver; Jogler, Christian; Tu, Qiang; Pósfai, Mihály; Tompa, Eva; Plitzko, Jürgen M; Brachmann, Andreas; Wanner, Gerhard; Müller, Rolf; Zhang, Youming; Schüler, Dirk; Ludwig-Maximilians-Universität München, Department of Biology I, Großhaderner Straße 2-4, 82152 Martinsried, Germany. (2014-03)
      The synthetic production of monodisperse single magnetic domain nanoparticles at ambient temperature is challenging. In nature, magnetosomes--membrane-bound magnetic nanocrystals with unprecedented magnetic properties--can be biomineralized by magnetotactic bacteria. However, these microbes are difficult to handle. Expression of the underlying biosynthetic pathway from these fastidious microorganisms within other organisms could therefore greatly expand their nanotechnological and biomedical applications. So far, this has been hindered by the structural and genetic complexity of the magnetosome organelle and insufficient knowledge of the biosynthetic functions involved. Here, we show that the ability to biomineralize highly ordered magnetic nanostructures can be transferred to a foreign recipient. Expression of a minimal set of genes from the magnetotactic bacterium Magnetospirillum gryphiswaldense resulted in magnetosome biosynthesis within the photosynthetic model organism Rhodospirillum rubrum. Our findings will enable the sustainable production of tailored magnetic nanostructures in biotechnologically relevant hosts and represent a step towards the endogenous magnetization of various organisms by synthetic biology.
    • Biosynthesis of methyl-proline containing griselimycins, natural products with anti-tuberculosis activity.

      Lukat, Peer; Katsuyama, Yohei; Wenzel, Silke; Binz, Tina; König, Claudia; Blankenfeldt, Wulf; Brönstrup, Mark; Müller, Rolf; Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-11-01)
      Griselimycins (GMs) are depsidecapeptides with superb anti-tuberculosis activity. They contain up to three (2S,4R)-4-methyl-prolines (4-MePro), of which one blocks oxidative degradation and increases metabolic stability in animal models. The natural congener with this substitution is only a minor component in fermentation cultures. We showed that this product can be significantly increased by feeding the reaction with 4-MePro and we investigated the molecular basis of 4-MePro biosynthesis and incorporation. We identified the GM biosynthetic gene cluster as encoding a nonribosomal peptide synthetase and a sub-operon for 4-MePro formation. Using heterologous expression, gene inactivation, and in vitro experiments, we showed that 4-MePro is generated by leucine hydroxylation, oxidation to an aldehyde, and ring closure with subsequent reduction. The crystal structures of the leucine hydroxylase GriE have been determined in complex with substrates and products, providing insight into the stereospecificity of the reaction.
    • Biosynthesis of Oxytetracycline by Streptomyces rimosus:
Past, Present and Future Directions in the Development
of Tetracycline Antibiotics.

      Petković, Hrvoje; Lukežič, Tadeja; Šušković, Jagoda; Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-03)
      Natural tetracycline (TC) antibiotics were the first major class of therapeutics to earn the distinction of 'broad-spectrum antibiotics' and they have been used since the 1940s against a wide range of both Gram-positive and Gram-negative pathogens, mycoplasmas, intracellular chlamydiae, rickettsiae and protozoan parasites. The second generation of semisynthetic tetracyclines, such as minocycline and doxycycline, with improved antimicrobial potency, were introduced during the 1960s. Despite emerging resistance to TCs erupting during the 1980s, it was not until 2006, more than four decades later, that a third--generation TC, named tigecycline, was launched. In addition, two TC analogues, omadacycline and eravacycline, developed via (semi)synthetic and fully synthetic routes, respectively, are at present under clinical evaluation. Interestingly, despite very productive early work on the isolation of a Streptomyces aureofaciens mutant strain that produced 6-demethyl-7-chlortetracycline, the key intermediate in the production of second- and third-generation TCs, biosynthetic approaches in TC development have not been productive for more than 50 years. Relatively slow and tedious molecular biology approaches for the genetic manipulation of TC-producing actinobacteria, as well as an insufficient understanding of the enzymatic mechanisms involved in TC biosynthesis have significantly contributed to the low success of such biosynthetic engineering efforts. However, new opportunities in TC drug development have arisen thanks to a significant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology approaches, and, importantly, to a much deeper understanding of TC biosynthesis, mostly gained over the last two decades.
    • Concepts and Methods to Access Novel Antibiotics from Actinomycetes.

      Hug, Joachim J; Bader, Chantal D; Remškar, Maja; Cirnski, Katarina; Müller, Rolf (MPDI, 2018-05-22)
      Actinomycetes have been proven to be an excellent source of secondary metabolites for more than half a century. Exhibiting various bioactivities, they provide valuable approved drugs in clinical use. Most microorganisms are still untapped in terms of their capacity to produce secondary metabolites, since only a small fraction can be cultured in the laboratory. Thus, improving cultivation techniques to extend the range of secondary metabolite producers accessible under laboratory conditions is an important first step in prospecting underexplored sources for the isolation of novel antibiotics. Currently uncultured actinobacteria can be made available by bioprospecting extreme or simply habitats other than soil. Furthermore, bioinformatic analysis of genomes reveals most producers to harbour many more biosynthetic gene clusters than compounds identified from any single strain, which translates into a silent biosynthetic potential of the microbial world for the production of yet unknown natural products. This review covers discovery strategies and innovative methods recently employed to access the untapped reservoir of natural products. The focus is the order of actinomycetes although most approaches are similarly applicable to other microbes. Advanced cultivation methods, genomics- and metagenomics-based approaches, as well as modern metabolomics-inspired methods are highlighted to emphasise the interplay of different disciplines to improve access to novel natural products.
    • 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.
    • Covalent Lectin Inhibition and Application in Bacterial Biofilm Imaging.

      Wagner, Stefanie; Hauck, Dirk; Hoffmann, Michael; Sommer, Roman; Joachim, Ines; Müller, Rolf; Imberty, Anne; Varrot, Annabelle; Titz, Alexander; HIPS, Helmholtz-Institut für pharmazeutische Forchung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-09-28)
      Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing "pathoblockers" preventing biofilm formation and virulence. A covalent lectin inhibitor specific to a carbohydrate binding site is described for the first time. Its application in the LecA-specific in vitro imaging of biofilms formed by P. aeruginosa is also reported.