Table of Contents I

Identification and characterization of selected secondary metabolite biosynthetic pathways from Xenorhabdus nematophila Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biowissenschaften (15) der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Daniela Reimer aus Offenbach am Main Frankfurt 2013 (D 30) vom Fachbereich Biowissenschaften (15) der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekan: Prof. Dr. Starzinski-Powitz Gutachter: Prof. Dr. Helge B. Bode, Prof. Dr. Eckhard Boles Datum der Disputation: 22.08.2013 Meinen Eltern "Two roads diverged in a yellow wood, And sorry I could not travel both And be one traveler, long I stood And looked down one as far as I could To where it bent in the undergrowth; Then took the other, as just as fair, And having perhaps the better claim, Because it was grassy and wanted wear; Though as for that the passing there Had worn them really about the same, And both that morning equally lay In leaves no step had trodden black. Oh, I kept the first for another day! Yet knowing how way leads on to way, I doubted if I should ever come back. I shall be telling this with a sigh Somewhere ages and ages hence: Two roads diverged in a wood, and I - I took the one less traveled by, And that has made all the difference."* * Robert Frost, poem "The road not taken", published in the collection Mountain Interval, Henry Holt and Company, New York, 1916. Table of contents i Table of contents Abstract.................................................................................................................................... 1 Zusammenfassung................................................................................................................... 5 Introduction............................................................................................................................. 13 Mutualistic and entomopathogenic bacteria as a source for natural products 15 The genera Xenorhabdus and Photorhabdus and their associated symbiotic partners 17 Taxonomy of entomopathogenic nematodes and bacteria 17 The life cycle of the entomopathogenic nematodes Steinernema and Heterorhabditis 18 Genus Xenorhabdus 20 Genus Photorhabdus 23 Secondary metabolites of Xenorhabdus and Photorhabdus 24 Nonribosomal peptide synthetases and polyketide synthases 30 Reference list 36 Chapter 1.................................................................................................................................. 51 A new type of pyrrolidine biosynthesis is involved in the late steps of xenocoumacin production in Xenorhabdus nematophila 53 Chapter 2.................................................................................................................................. 71 Genetic analysis of xenocoumacin antibiotic production in the mutualistic bacterium Xenorhabdus nematophila 73 Chapter 3.................................................................................................................................. 99 A natural prodrug activation mechanism in nonribosomal peptide synthesis 101 Chapter 4.................................................................................................................................. 137 Determination of the absolute configuration of peptide natural products by using stable isotope labeling and mass spectrometry 139 ii Table of contents Chapter 5.................................................................................................................................. 165 Rhabdopeptides from entomopathogenic bacteria as examples for insect-associated secondary metabolites 167 Concluding Remarks................................................................................................................ 201 A widespread natural prodrug activation mechanism in xenocoumacin biosynthesis 203 Rapid structure elucidation of linear and cyclic nonribosomally produced peptides by stable isotope labeling and mass spectrometry 213 Distribution of insect-associated highly N-methylated rhabdopeptide derivatives in Xenorhabdus and Photorhabdus 216 Regulation of secondary metabolite biosynthesis in Xenorhabdus nematophila 219 Reference list 221 Acknowledgment................................................................................................................... 229 Curriculum Vitae.................................................................................................................... 233 Eidesstattliche Erklärung....................................................................................................... 237 Abstract 1 Abstract 2 Abstract Abstract 3 Bacteria of the genera Xenorhabdus and Photorhabdus are entomopathogenic bacteria symbiotically associated with entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis, respectively. Detailed studies for the understanding of the regulation system in the tripartial mutualism-pathogenesis relationship between the bacteria, the nematode and the infected host have shown that secondary metabolites produced by the bacteria are either involved in the pathogenesis against numerous insect larvae or play an important role in the symbiosis towards the nematode. Several classes of structurally diverse secondary metabolites with a broad spectrum of bioactivities (e.g. antibacterial, insecticidal, antifungal) are known from different Xenorhabdus and Photorhabdus strains and are produced by nonribosomal peptide synthetases (NRPS) and the fatty acid synthase (FAS)-related polyketide synthases (PKS) or even hybrids thereof. During this work, xenocoumacin 1 (XCN 1) and 2 (XCN 2), the major antimicrobial compounds produced by Xenorhabdus nematophila and their corresponding biosynthetic gene cluster were identified and studied in detail. Although both compounds show antibiotic activity against Gram- positive bacteria, XCN 1 is much more active and additionally shows good activity against different fungi. Xenocoumacins are synthesized via a non colinear hybrid PKS/NRPS multienzyme (xcnA-N), consisting of six transcriptional units identified by real time PCR. The biosynthesis can be divided into enzymes responsible for the biosynthesis of the core structure (XcnAFHIJKL), including the hydroxymalonyl-ACP (XcnBCDE), in proteins involved in an interesting drug activation mechanism (XcnAG) and for a resistance conferring inactivation pathway (XcnMN). Five different prexenocoumacins are formed by the xenocoumacin biosynthetic machinery as inactive prodrugs inside the cytoplasm. XcnG, a bifunctional protein with a periplasmic peptidase domain and three additional transmembrane helices cleaves the acylated D-asparagine residue from all prexenocoumacin derivatives to form the bioactive XCN 1 as sole compound. Furthermore, XCN 1 is secreted by an ABC transporter TolC-like protein complex and is thought to be involved in killing microbes living inside the insect gut or other bacterial food competitors during the infection cycle and the nematode development. As XCN 1 is also toxic to the producing strain, this compound is taken up by X. nematophila and a detoxification by XcnMN via a conversion of XCN 1 into the less active XCN 2 occurs due to a new type of pyrrolidine ring formation. A desaturase (XcnN) and a saccharopine dehydrogenase-like enzyme (XcnM) are essential for this unusual transformation via two new identified intermediates and the catalytic reaction is regulated by the response regulator OmpR. OmpR was identified as a negative regulator of xcnA-L required for the biosynthesis of XCN 1 and as a positive regulator responsible for the self-resistance mechanism. The differential expression may therefore be part of a response to balance the necessary level between XCN 1 and XCN 2 to avoid self-toxicity and as a result to optimize the fitness of the strain. Astonishingly, homologues of the membrane-bound and D-asparagine-specific peptidase (XcnG) and the encoding NRPS for the starting module (XcnA) for the acylated D-asparagine residue 4 Abstract were identified in many different bacterial genera. Thus indicating a widespread and important mechanism for the activation of secondary metabolites as it was earlier only known from ribosomal biosynthesis and should be considered especially during the in silico analysis of secondary metabolite biosynthetic gene clusters and their predicted products during large-scale genome mining approaches. Moreover, six novel linear peptides named rhabdopeptides (RDPs) have been identified after the identification of the corresponding rdp gene cluster using a promoter trap strategy (IVET) for the detection of insect inducible genes. Detailed analysis revealed that these compounds participate in virulence towards insects and are produced upon bacterial infection of a suitable insect host. As rhabdopeptide production is initially upregulated upon infections but rdp mutant strains display no severe virulence defect, rhabdopeptides are suggested to function during the insect bioconversion and nematode reproduction phases of the Xenorhabdus life cycle due to an abundant production after the insect death. The structures of the highly N-methylated nonribosomally derived rhabdopeptides were deduced exclusively from stable isotope labeling experiments combined with detailed mass spectrometric analysis and represent a new class of N-methylated peptides carrying a decarboxylated amino acid. Besides rhabdopeptides, a new xenortide derivative from X. nematophila and the cyclic GameXPeptides

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