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Assessing the impact of transposon activity and megaplasmid characteristics on the genetic stability of the model organism shewanella oneidensis
Citation Link: https://doi.org/10.15480/882.17327
Publikationstyp
Doctoral Thesis
Date Issued
2026
Sprache
English
Author(s)
Fritz, Benjamin
Advisor
Referee
Title Granting Institution
PrƤsidium der TUHH
Place of Title Granting Institution
Hamburg
Examination Date
2025-10-08
Institute
TORE-DOI
Citation
Technische UniverstiƤt Hamburg (2026)
The facultative anaerobe Shewanella oneidensis is a model organism widely studied for its extracellular electron transfer capabilities, making it a promising candidate for bioelectrochemical systems (BES). However, its presumed genetic instability presents a key challenge, particularly for long-term industrial applications. This study investigates two major factors influencing genetic robustness in S. oneidensis: transposon activity and the presence of the megaplasmid (MP).
Transposable elements (TEs) play a crucial role in genome plasticity. Their high activity can lead to disruptive mutations, compromising strain stability. To assess transposase mobility under TE-activating conditions, transcriptomic analysis and stress-induced transposition experiments were combined with whole-genome sequencing. The results indicate that certain insertion sequences (IS), particularly ISSOD1, ISSOD2 and ISSOD9, exhibit high transposition rates, contributing to genetic instability. To mitigate these effects, CRISPR-Cas-deaminase-based genome editing was employed to introduce premature stop codons into active transposase genes. This deactivation successfully reduced transposase-related mutations emanating from the ISSOD2 family, demonstrating a viable strategy for stabilizing the S. oneidensis genome. ISSOD9 activity appeared to be strongly linked to the presence of plasmids in transformed strains, suggesting its transposition mechanism is associated with horizontal gene transfer. This finding has significant implications for studies utilizing external plasmids, as it underscores the potential for unintended genomic alterations.
In parallel, the role of the megaplasmid in genetic stability was examined, which harbors numerous TEs and contains mutation-prone regions, increasing TE motility potential. Using an adaptive evolutionary approach, it was attempted to delete the MP while circumventing post-segregational killing caused by toxin-antitoxin pairs that contribute to stabilized vertical plasmid transfer. Although complete megaplasmid loss was not achieved, approximately 35 % of its sequence was deleted, including nine TEs and an identified hotspot for integration. Notably, the unexpected loss of the region containing the origin of replication suggests a metastable state for the MP.
By integrating genome-wide analyses with targeted genetic modifications, this study provides valuable insights into the mechanisms governing genetic stability in S. oneidensis. These findings contribute to optimizing S. oneidensis as a robust production strain for BES applications, with broader implications for microbial engineering and industrial biotechnology.
Transposable elements (TEs) play a crucial role in genome plasticity. Their high activity can lead to disruptive mutations, compromising strain stability. To assess transposase mobility under TE-activating conditions, transcriptomic analysis and stress-induced transposition experiments were combined with whole-genome sequencing. The results indicate that certain insertion sequences (IS), particularly ISSOD1, ISSOD2 and ISSOD9, exhibit high transposition rates, contributing to genetic instability. To mitigate these effects, CRISPR-Cas-deaminase-based genome editing was employed to introduce premature stop codons into active transposase genes. This deactivation successfully reduced transposase-related mutations emanating from the ISSOD2 family, demonstrating a viable strategy for stabilizing the S. oneidensis genome. ISSOD9 activity appeared to be strongly linked to the presence of plasmids in transformed strains, suggesting its transposition mechanism is associated with horizontal gene transfer. This finding has significant implications for studies utilizing external plasmids, as it underscores the potential for unintended genomic alterations.
In parallel, the role of the megaplasmid in genetic stability was examined, which harbors numerous TEs and contains mutation-prone regions, increasing TE motility potential. Using an adaptive evolutionary approach, it was attempted to delete the MP while circumventing post-segregational killing caused by toxin-antitoxin pairs that contribute to stabilized vertical plasmid transfer. Although complete megaplasmid loss was not achieved, approximately 35 % of its sequence was deleted, including nine TEs and an identified hotspot for integration. Notably, the unexpected loss of the region containing the origin of replication suggests a metastable state for the MP.
By integrating genome-wide analyses with targeted genetic modifications, this study provides valuable insights into the mechanisms governing genetic stability in S. oneidensis. These findings contribute to optimizing S. oneidensis as a robust production strain for BES applications, with broader implications for microbial engineering and industrial biotechnology.
Subjects
S
oneidensis
CRISPR-CAS
Transposases
genetic engineering
genetics
DDC Class
579: Microorganisms, Fungi and Algae
660.6: Biotechnology
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Dissertation_Benjamin_Fritz_2025.pdf
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