Identification and characterization of selected secondary metabolite biosynthetic pathways from Xenorhabdus nematophila
Frankfurt (Main), Univ., Diss., 2013
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Tag hinzufügen | Zusammenfassung: | Frankfurt (Main), Univ., Diss., 2013 Bacteria of the genera Xenorhabdus and Photorhabdus are entomopathogenic bacteria symbiotically associated with 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 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. The XCN biosynthesis can be divided into enzymes responsible for the biosynthesis of the core structure, including the hydroxymalonyl-ACP, in proteins involved in an interesting drug activation mechanism and for a resistance conferring inactivation pathway. 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 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, which 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. Homologues of the membrane-bound and D-asparagine-specific peptidase and the encoding NRPS for the starting module for the acylated D-asparagine residue 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. Moreover, six novel linear peptides named rhabdopeptides have been identified after the identification of the corresponding rdp gene cluster using a promoter trap strategy 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, the cyclic GameXPeptides from P. luminescens were identified and their structures were elucidated. The combination of labeling experiments and mass spectrometry enables a rapid identification of building blocks. In particular it allows to distinguish between isobar amino acids such as leucine and isoleucine in nonribosomally produced peptides. Furthermore, stable isotope labeling can be used as a method to determine the absolute amino acid configuration of compounds directly in the producer strain without derivatization reagents. Hence, in this work the absolute configuration of the GameXPeptides was successfully determined. |
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| Beschreibung: | 1 CD-R 12 cm Beil. ([1] Bl.) |