Isolierung und Charakterisierung von Bakterien aus Meeressedimenten und ihr Potential zur Produktion von antimikrobiellen Peptiden und Polyketiden

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

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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

Document Type: Thesis (Doctoral thesis/PhD)
Thesis Advisors: Imhoff, Johannes F. and Schönheit, Peter
Keywords: Marine Bacteria; Polyketide; AMP; Microbiology; natural products
Research affiliation: OceanRep > GEOMAR > FB3 Marine Ecology > FB3-MI Marine Microbiology
OceanRep > GEOMAR > Applied R&D > Centre for Marine Substances (KiWiZ)
Projects: KIWIZ
Date Deposited: 27 Sep 2012 08:34
Last Modified: 12 Feb 2013 08:40

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