• Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

      Hennessen, Fabienne; Miethke, Marcus; Zaburannyi, Nestor; Loose, Maria; Lukežič, Tadeja; Bernecker, Steffen; Hüttel, Stephan; Jansen, Rolf; Schmiedel, Judith; Fritzenwanker, Moritz; et al. (MDPI, 2020-09-18)
      The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.
    • Draft Genome Sequence and Annotation of the Obligate Bacterial Endosymbiont Caedibacter taeniospiralis, Causative Agent of the Killer Phenotype in Paramecium tetraurelia.

      Zaburannyi, Nestor; Grosser, Katrin; Gasparoni, Gilles; Müller, Rolf; Schrallhammer, Martina; Simon, Martin; Helmholtz Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2018-01-18)
      Caedibacter taeniospiralis is an obligate endosymbiont living in the cytoplasm of Paramecium tetraureliaC. taeniospiralis causes the so-called killer trait, eliminating intraspecific competitors of its host when released into the medium by the concerted action of the unusual protein structure R-body (refractile body) in addition to an as-yet-unknown toxin.
    • Dual-Seq reveals genome and transcriptome of Caedibacter taeniospiralis, obligate endosymbiont of Paramecium.

      Pirritano, Marcello; Zaburannyi, Nestor; Grosser, Katrin; Gasparoni, Gilles; Müller, Rolf; Simon, Martin; Schrallhammer, Martina; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (NPG, 2020-06-16)
      Interest in host-symbiont interactions is continuously increasing, not only due to the growing recognition of the importance of microbiomes. Starting with the detection and description of novel symbionts, attention moves to the molecular consequences and innovations of symbioses. However, molecular analysis requires genomic data which is difficult to obtain from obligate intracellular and uncultivated bacteria. We report the identification of the Caedibacter genome, an obligate symbiont of the ciliate Paramecium. The infection does not only confer the host with the ability to kill other cells but also renders them immune against this effect. We obtained the C. taeniospiralis genome and transcriptome by dual-Seq of DNA and RNA from infected paramecia. Comparison of codon usage and expression level indicates that genes necessary for a specific trait of this symbiosis, i.e. the delivery of an unknown toxin, result from horizontal gene transfer hinting to the relevance of DNA transfer for acquiring new characters. Prediction of secreted proteins of Caedibacter as major agents of contact with the host implies, next to several toxin candidates, a rather uncharacterized secretome which appears to be highly adapted to this symbiosis. Our data provides new insights into the molecular establishment and evolution of this obligate symbiosis and for the pathway characterization of toxicity and immunity.
    • Genome Analysis of the Fruiting Body-Forming Myxobacterium Chondromyces crocatus Reveals High Potential for Natural Product Biosynthesis.

      Zaburannyi, Nestor; Bunk, Boyke; Maier, Josef; Overmann, Jörg; Müller, Rolf; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany. (2016)
      Here, we report the complete genome sequence of the type strain of the myxobacterial genus Chondromyces, Chondromyces crocatus Cm c5. It presents one of the largest prokaryotic genomes featuring a single circular chromosome and no plasmids. Analysis revealed an enlarged set of tRNA genes, along with reduced pressure on preferred codon usage compared to that of other bacterial genomes. The large coding capacity and the plethora of encoded secondary metabolite biosynthetic gene clusters are in line with the capability of Cm c5 to produce an arsenal of antibacterial, antifungal, and cytotoxic compounds. Known pathways of the ajudazol, chondramide, chondrochloren, crocacin, crocapeptin, and thuggacin compound families are complemented by many more natural compound biosynthetic gene clusters in the chromosome. Whole-genome comparison of the fruiting-body-forming type strain (Cm c5, DSM 14714) to an accustomed laboratory strain which has lost this ability (nonfruiting phenotype, Cm c5 fr-) revealed genetic changes in three loci. In addition to the low synteny found with the closest sequenced representative of the same family, Sorangium cellulosum, extensive genetic information duplication and broad application of eukaryotic-type signal transduction systems are hallmarks of this 11.3-Mbp prokaryotic genome.
    • Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis.

      Lukežič, Tadeja; Pikl, Špela; Zaburannyi, Nestor; Remškar, Maja; Petković, Hrvoje; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (BMC (part of Springer), 2020-12-19)
      Background: Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today’s antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. Results: We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. Conclusions: We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains allgene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action.
    • Kibdelosporangium persicum sp. nov., a new member of the Actinomycetes from a hot desert in Iran.

      Safaei, Nasim; Nouioui, Imen; Mast, Yvonne; Zaburannyi, Nestor; Rohde, Manfred; Schumann, Peter; Müller, Rolf; Wink, Joachim; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.;HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Microbiology Society, 2021-01-11)
      Isolate 4NS15T was isolated from a neglected arid habitat in Kerman, Iran. The strain showed 16S rRNA gene sequence similarity values of 98.9 % to the type strains of Kibdelosporangium aridum subsp. aridum, Kibdelosporangium phytohabitans and Kibdelosporangium philippinense and 98.6 % to the type strain K. aridum subsp. largum, respectively. Genome-based phylogenetic analysis revealed that isolate 4NS15T is closely related to Kibdelosporangium aridum subsp. aridum DSM 43828T. The digital DNA-DNA hybridization value between the genome sequences of 4NS15T and strain DSM 43828T is 29.8 %. Strain 4NS15T produces long chains of spores without a sporangium-like structure which can be distinguished from other Kibdelosporangium species. Isolate 4NS15T has a genome size of 10.35 Mbp with a G+C content of 68.1 mol%. Whole-cell hydrolysates of isolate 4NS15T are rich in meso-diaminopimelic acid and cell-wall sugars such as arabinose, galactose, glucose and ribose. Major fatty acids (>10 %) are C16 : 0, iso-C16 : 0 and iso-C15 : 0. The phospholipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylhydroxyethanolamine, aminolipid and glycoaminolipid. The predominant menaquinone is MK-9(H4). Based on its phenotypic and genotypic characteristics, isolate 4NS15T (NCCB 100701=CIP 111705=DSM 110728) merits recognition as representing a novel species of the genus Kibdelosporangium, for which the name Kibdelosporangium persicum sp. nov. is proposed.
    • 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
    • Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

      Knapp, Andreas; Voget, Sonja; Gao, Rong; Zaburannyi, Nestor; Krysciak, Dagmar; Breuer, Michael; Hauer, Bernhard; Streit, Wolfgang R; Müller, Rolf; Daniel, Rolf; et al. (2015-10-17)
      Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.
    • The natural product carolacton inhibits folate-dependent C1 metabolism by targeting FolD/MTHFD.

      Fu, Chengzhang; Sikandar, Asfandyar; Donner, Jannik; Zaburannyi, Nestor; Herrmann, Jennifer; Reck, Michael; Wagner-Döbler, Irene; Koehnke, Jesko; Müller, Rolf; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124Braunschweig, Germany. (2017-11-16)
      The natural product carolacton is a macrolide keto-carboxylic acid produced by the myxobacterium Sorangium cellulosum, and was originally described as an antibacterial compound. Here we show that carolacton targets FolD, a key enzyme from the folate-dependent C1 metabolism. We characterize the interaction between bacterial FolD and carolacton biophysically, structurally and biochemically. Carolacton binds FolD with nanomolar affinity, and the crystal structure of the FolD-carolacton complex reveals the mode of binding. We show that the human FolD orthologs, MTHFD1 and MTHFD2, are also inhibited in the low nM range, and that micromolar concentrations of carolacton inhibit the growth of cancer cell lines. As mitochondrial MTHFD2 is known to be upregulated in cancer cells, it may be possible to use carolacton as an inhibitor tool compound to assess MTHFD2 as an anti-cancer target.
    • Synthetic biology approaches and combinatorial biosynthesis towards heterologous lipopeptide production.

      Yan, Fu; Burgard, Christian; Popoff, Alexander; Zaburannyi, Nestor; Zipf, Gregor; Maier, Josef; Bernauer, Hubert S; Wenzel, Silke C; Müller, Rolf; HIPS, Helmholtz-Institut füt Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (2018-10-14)
      Synthetic biology techniques coupled with heterologous secondary metabolite production offer opportunities for the discovery and optimisation of natural products. Here we developed a new assembly strategy based on type IIS endonucleases and elaborate synthetic DNA platforms, which could be used to seamlessly assemble and engineer biosynthetic gene clusters (BGCs). By applying this versatile tool, we designed and assembled more than thirty different artificial myxochromide BGCs, each around 30 kb in size, and established heterologous expression platforms using a derivative of Myxococcus xanthus DK1622 as a host. In addition to the five native types of myxochromides (A, B, C, D and S), novel lipopeptide structures were produced by combinatorial exchange of nonribosomal peptide synthetase (NRPS) encoding genes from different myxochromide BGCs. Inspired by the evolutionary diversification of the native myxochromide megasynthetases, the ancestral A-type NRPS was engineered by inactivation, deletion, or duplication of catalytic domains and successfully converted into functional B-, C- and D-type megasynthetases. The constructional design approach applied in this study enables combinatorial engineering of complex synthetic BGCs and has great potential for the exploitation of other natural product biosynthetic pathways.