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Evolutionary Genomics of Bacillus thuringiensis

dc.contributor.advisorLiesegang, Heiko Dr.
dc.contributor.authorHollensteiner, Jacqueline
dc.date.accessioned2017-11-07T09:07:24Z
dc.date.available2017-11-07T09:07:24Z
dc.date.issued2017-11-07
dc.identifier.urihttp://hdl.handle.net/11858/00-1735-0000-0023-3F58-3
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-6567
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc570de
dc.titleEvolutionary Genomics of Bacillus thuringiensisde
dc.typedoctoralThesisde
dc.contributor.refereeDaniel, Rolf Prof. Dr.
dc.date.examination2017-02-21
dc.description.abstractengNowadays, many evolutionary processes and involved key factors are known but how they influence natural genetic variation and phenotypic traits in bacteria is under debate. However, the mechanism of evolution as well as the adaptive potential of B. thuringiensis as a species is poorly understood. Especially, strains highly specific to various ecological niches also with regard to their specific hosts, the complex lifestyle switches and the selection of different virulence factors provided makes B. thuringiensis to a good model for the study of evolution. A glimpse is known about single evolutionary mechanisms which partially contribute to “rapid evolution”, and there is a lack of information about how a given selection regime determines the various opportunities of B. thuringiensis as a species. The aim of this study was to investigate the different genomic mechanisms to gain insights in the emerging of successful new strains from the complex and divers evolutionary puzzle of the species B. thuringiensis. In this thesis, evolutionary mechanisms of B. thuringiensis were investigated by genomic approaches. In particular, whole genomes of nematicidal B. thuringiensis strains were sequenced and analyzed with a focus on virulence factors, fitness factors, methylation pattern, metabolic properties and mobile elements such as bacteriophages, IS elements, transposases, which could contribute to a successful infection and show new insights into how B. thuringiensis is able to evolve, adapt and survive in various ecological niches and where the broad host range has its origin. The genomes of B. thuringiensis MYBT18246, MYBT18247 and B. thuringiensis MYBT18679 were sequenced and they contained approximately 15% to 20% genetic material which encodes genomic elements assigned to genome plasticity. Moreover, all investigated strains encode a various number of different Cry-toxins which was specific for their selection regimes and were encoded on either plasmids or in the chromosome. Additionally, it was shown that prophages act as likely candidates for the mobilization of chromosomally encoded cry-toxins in B. thuringiensis MYBT18246. Host-parasite evolution experiments were performed to evaluate the speed of evolution of nematicidal B. thuringiensis strains in a continuous arms race with their host organism Caenorhabditis elegans by looking at the general genetic trait mechanism under selection, including adaptive changes in real time using large-scale phenotyping, population next generation sequencing, and genetic analysis of the identified candidate genes. We showed that high virulence was specifically favored during pathogen–host coevolution rather than pathogen one-sided adaptation to a non changing host or to an environment without host and that high virulence was specifically favored during pathogen–host coevolution rather than pathogen one-sided adaptation to a non changing host or to an environment without host. Moreover, the Cry toxins of B. thuringiensis MYBT 18679 with high virulence specifically swept to fixation in all of the independent replicate populations under coevolution which was high correlated with elevated copy numbers of the plasmid containing the nematicidal toxin genes. The effect of specific nematicidal cry toxin genes (Cry14Aa and Cry21Aa) and their impact on virulence has been confirmed by molecular reconstruction in mutant strains and tested against the susceptible host Caenorhabditis elegans. Taken together we could show that the evolution of B. thuringiensis seems to be a multi-mechanism process influenced by variety of different parameters such as genomic plasticity, natural conditions and interaction partners including hosts, competitors or commensals. Besides the B. thuringiensis species specific experiments, we investigated how the approach of how NGS methods can be successfully implemented not only in in depth bacteriophage research. Furthermore we determined how phages can impact biotechnology and can be used in biotechnological relevant strains (B. licheniformis DSM13) where strain optimization is crucial for a sufficient production. In total, seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Induction experiments using mitymycin C followed by a phage particle analysis from the wild-type strain and prophage deletion mutant strains revealed differences in activity of the prophage regions. Three showed activity and active particle production which were backtracked via transmission electron microscopy (TEM). The ability of prophage BLi_Pp6 to generate particles was confirmed by sequencing of particle- protected DNA mapping to prophage locus BLi_Pp6. This NGS approach increases the sensitivity of prophage activity analysis. A second focus in this thesis was set on the biocontrol-potential of B. thuringiensis. It is well known as biocontrol agent against a broad spectrum of insects, nematodes, mites and protozoa depending on their armory of toxins (Cry, Cyt, Vip and Sip toxins). However, the interaction structures such as competition, amensalism, expoitation, neutralism, commensalism, mutualism, or symbiosis in complex environments between animals, plants, fungi and B. thuringiensis has been rarely studied in detail. This includes also the question of the antifungal potential of B. thuringiensis and their potential as control agent against other pests such as phytopathogenic fungi. Therefore we investigated the anti-fungal potential of host-plant associated Bcsl group species, especially B. thuringiensis, against wilt causing phytopathogenic Verticillia. In particular, the natural occurrence of wildtype B. thuringiensis and other Bcsl group species members sampled from Solanum lycopersicum (tomato) with the plant as primary ecological niche were investigated by 16S amplicon sequencing. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum. In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities. This indicates that chitinases contribute to antifungal activities.de
dc.contributor.coRefereeHoppert, Michael PD Dr.
dc.subject.engB. thuringiensis, Genomicsde
dc.identifier.urnurn:nbn:de:gbv:7-11858/00-1735-0000-0023-3F58-3-2
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de
dc.identifier.ppn1003198988


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