Kleinschmidt, Katrin (2012) Isolierung und Charakterisierung von Bakterien aus Meeressedimenten und ihr Potential zur Produktion von antimikrobiellen Peptiden und Polyketiden . (PhD/ Doctoral thesis), Christian-Albrechts-Universität Kiel, Kiel, Germany, 148 pp.

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Abstract

As microbial resistence against known antibiotics increases, the search for new chemical structures becomes more important. Especially marine bacteria are of particular research interest as natural product producer. To further asses the potential marine bacteria from different sediments were isolated, cultivated and analyzed with chemical and biomolecular methods. To bypass the limitations of existing methods for natural product discovery the search for alternative strategies e.g. genome-based strategies, is necessary. For example PCR-based screening with special primers is a fast and efficient possibility to assess the potential of a species to produce natural products. It can therefore be used to select isolates for further tests. Over 50% of the investigated isolates (especially Actinobacteria and Firmicutes) contained gene clusters for non-ribosomal peptidsynthetases (NRPS) or polyketidsynthases (PKS) and/or show antimicrobial activity against some test organisms. Of these isolates the representatives of Bacillus amyloliqufaciens were chosen and their natural product production was compared to each other. The investigation of equal taxa from different habitats is not common. Nine (out of ten) Bacillus amyloliquefaciens isolates had secondary metabolite gene clusters and showed differences in the production of natural products. A location-independent production of different antimicrobial non-ribosomal lipopeptides (substance classes of surfactin, iturin and fengycin) and twelve unknown substances could be shown. This demonstrates that bacteria of the same taxon (16sRNA similarity ≥99,5 %) are a viable source for the search of new substances. Especially the taxa Bacillus and Streptomyces are known as good natural product producer. Two isolates were chosen (gtP20b Bacillus sp. and C42 Streptomyces sp.) and the metabolites and genomes were analyzed. The crude extract from isolate gtP20b inhibited the test organisms Xanthomonas campestris and Candida glabrata and it could detected gene clusters for NRPS with PCR. It produces non-ribosomal lipopeptides (substance classes iturine, surfactine and fengycine) as well as six unknown substances in culture. The genome sequencing revealed known NRPS gene clusters as well as unknown gene clusters for PKS and NRPS/PKS hybrid. The detected lipopeptides of isolate gtP20b were assigned to annotated NRPS gene clusters. The other putative gene cluster of PKS and hybridsystems contain a potential for novel natural products. The identification of these unknown gene cluster is still in progress. The isolate C42 (Streptomyces sp.) was selected due to inhibiting to Candida glabrata and NRPS and PKS gene clusters detected during PCR. Two sphingolipids and two new unknown non-ribosomal peptides were identified with chemical and chromatographic methods. With HPLS/MS and GC/MS the identity and configuration of the eight amino acids of peptide m/z 898.9 was detected. The peptid m/z 898.9 contain s D-alanin, Dphenylalanin, L-valin, D-valin, D-leucin, L- (allo) isoleucin and D- (allo) isoleucin. Amino acids with the D-configuration can only be incorporated into peptides with specially NRPS domains. This proves that the new peptide m/z 898.9 is synthesized with non-ribosomal synthetases. Approximately 60 % of the genome from C42 have been sequenced. The genome included a number of known and unknown NRPS and PKS gene cluster with putative products that could not be produced in the lab. This work shows that the combination of cultivation and genome-based methods allows to take advantages of biosynthetic potential of marine bacteria. New isolates of known bacteria taxa from marine sediments show strain specific distinctions in the natural product production and are good producers for natural products.

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