Etablierung und Untersuchung artifizieller Multi- Replikon Systeme im Alphaproteobakterium Sinorhizobium meliloti
Loading...
Files
Date
Publisher
Philipps-Universität Marburg
Supervisors
Abstract
The present work describes studies on the pABC vector family as well as artificial genome fusions, from
which future hybrid genome configurations in the α-proteobacterium Sinorhizobium meliloti
(Ensifer meliloti) can be derived. These contribute to the understanding of genomic organization
principles and replication mechanisms, as well as the advancement of methods for genome editing in
S. meliloti and related organisms within the α-proteobacteria. The novel pABC vector system was
developed in previous work for S. meliloti. It was found that the pABC vector family can be used as a
functional extension of the tripartite S. meliloti genome. In the context of this work, the stability of this
modular replicon system was investigated under metabolic stress through the expression of the Lux-like
regulator ExpR. Similarly, compatibility of the pABC vector principle beyond S. meliloti was
demonstrated by developing pABC derivatives specifically for Methylorubrum extorquens AM1, which
serves as a platform for a future C1 bioeconomy. Further optimization of the pABC replicon system was
achieved by removing type IIS restriction endonuclease recognition sequences in several vector
modules, allowing the efficient use of modular cloning strategies (MoClo). The addition of vectors with
additional functional modules for site-specific recombination or new assembly possibilities expanded
the applications of the pABC system for genome editing. Finally, it was shown that pABC-based genetic
circuits in S. meliloti can be a powerful alternative to their genomic integration.
Another part of this work involved the construction and characterization of different fusions of the
tripartite S. meliloti genome. Physiological studies showed that morphology, growth and the ability to
enter into a functional symbiosis with Medicago sativa were hardly affected in the genome fusion
variants. Differences compared to the wild-type strain were only observed under special stress
conditions or with an unfavorable replichor composition. Using a modified fluorescence labeling
system, the spatio-temporal organization and coordination of the segregation of the fusion genomes
during the cell cycle was characterized. In this context, it was observed that key elements of genome
partitioning were largely conserved. In an S. meliloti strain carrying a fusion product of the chromosome,
pSymA, and pSymB, it was observed that both the replication origins of the secondary replicons and the
partitioning apparatus of pSymB were no longer essential. A triple-fusion derivative without the
replication origins of the secondary replicons exhibited significantly asymmetrical replicore ratios as
well as abnormal localization and segregation patterns of the chromosomal replication origin. A
missense mutation in the gene for the histidine kinase CckA and a partial inversion of the fused replicon
were identified as suppressors of the unfavorable replicon composition by sequencing the respective
genomes.
Review
Metadata
Contributors
Supervisor:
Dates
Created: 2024Issued: 2025-03-13Updated: 2025-03-13
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
Synthetic BiologyGenome Editing
DFG-subjects
BiotechnologieGenom-EditierungSynthetische Biologie
DDC-Numbers
570
show more
Wagner, Marcel (0009-0005-1963-3072): Etablierung und Untersuchung artifizieller Multi- Replikon Systeme im Alphaproteobakterium Sinorhizobium meliloti. : Philipps-Universität Marburg 2025-03-13. DOI: https://doi.org/10.17192/z2024.0225.
License
Except where otherwised noted, this item's license is described as Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 - CC BY NC ND