A streamlined Workflow for Transposon Mutagenesis in Corynebacterium glutamicum Based on Third-Generation Sequencing and its Application for Advanced Genetic Screens

This work comprises the establishment and utilization of the prophage- and insertion sequence(IS)-free *Corynebacterium glutamicum* platform strain CR101 for an efficient integration system, a transposon mutagenesis system based on the endogenous IS*Cg1* transposase and a streamlined workflow for transposon mutant library preparation and sequencing using Oxford Nanopore Technologies (ONT) nanopore Sequencing. Mobile genetic elements (MGEs) destabilize the genetic material of their hosts. While this can contribute to evolutionary plasticity in nature, it is often detrimental in a research and industrial context. Therefore, this work presents and builds upon the ”zero-background” strain CR101 derived from the industrial amino acid producer *Corynebacterium glutamicum*.<br /> Furthermore, untargeted genetic screens using transposon mutagenesis need to be comprehensive using tenth of thousands of individual insertions to achieve transposition into each gene with high level of confidence. This prohibits a single mutant-based approach and requires screening of the transposon library as a whole. In this respect, third generation nanopore sequencing offers the possibility to identify all mutations in a library and to compare libraries made in different mutant strains or collectively grown under different conditions.<br /> In the first part of this work, the genesis of CR101 is described, with systematic removal of all known full and partial IS-elements, starting from the prophage-free strain MB001. This is followed by developing a transposon mutagenesis system using the removed IS*Cg1* transposase gene to insert a selectable marker at random positions into the genome. IS*Cg1* was found to be active in *Escherichia coli*, the common host organism for cloning and propagation of plasmids. On the one hand, this indicates the potential for the IS*Cg1* transposon mutagenesis system to be adapted for other hosts. On the other hand, it impedes easy propagation of a plasmid carrying transposase and transposon of IS*Cg1*. Three plasmid-based approaches were pursued to circumvent this issue: One separating the transposase and the transposon on two plasmids to be transformed consecutively, one using *Saccharomyces cerevisiae* as host for assembly and propagation of a suicide plasmid in *C. glutamicum* and finally a plasmid assembled and propagated in *E. coli* but with a synthetic biology switch for tight repression of IS*Cg1*. The latter, named pML10, proved to be the most fruitful approach. The switch utilizes the Bxb1 phage integrase from *Mycobacterium smegmatis*. The integrase targets a synthetic promoter on pML10 flanked by the corresponding *attB* and *attP* sites. Recombination results in the hybrid *attL* and *attR* sites inverting the promoter in between, which renders transcription of the downstream IS*Cg1* active. For this, *bxbI* was integrated into the genome of CR101, resulting in the strain ML102.<br /> Surprisingly, transformation of pML10 into ML102 resulted in an integration of the plasmid into a newly discovered partially conserved *attB* site in *groEL1*, one of two *groEL* genes in *C. glutamicum*. The gene where integration occurred is disrupted by IS*Cg1c* in the ATCC wild type. This discovery denotes an efficient integration system in ML102 and indicates possible utility of the BxbI integrase in other related bacteria. Removal of *groEL1* resulted in ML103, in which random transposition of IS*Cg1* across the genome could be demonstrated.<br /> A streamlined workflow for preparation of precisely enriched transposition site libraries for nanopore sequencing was developed and combined with the use of the alignment tool *crossalign*, specifically designed for Tn-seq application.<br /> In the second part, the workflow was applied to generate transposon mutant libraries for CR101 and C1* using Tn5 for increased transformability. C1* is a genome-reduced strain derived from a common progenitor of CR101, CR099. The idea was to identify genes that might have become essential in C1* due to removed redundancies that were classified as non-essential. Additionally, the mutant libraries were cultivated in complex and minimal medium and samples were taken for sequencing over three time points. These samples were processed according to the established workflow followed by a *DESeq2* differential analysis of gene-specific mutation frequencies. While direct comparison between the two strains proved difficult due to library size differences, the data provided interesting information, enabling categorization of genes beyond essential and non-essential.<br /> Lastly, the workflow, combined with Tn5 transposome mutant libraries with different genetic screens was utilized to identify *ftsK* as a candidate for compensating the loss of function in a *C. glutamicum* MB001 *∆smc* mutant. Structural Maintenance of Chromosomes (SMC) proteins are crucial in chromosome segregation. Deletion of *smc* however, does not affect the growth of *C. glutamicum*, which presented a fascinating use-case for the developed workflow. For this, Tn5 transposon mutant libraries in MB001 and *∆smc* were produced and prepared, sequenced and mapped accordingly, followed by a differential analysis of gene-specific mutation frequencies using *DESeq2*. Apart from determining *ftsK* as essential in the *∆smc* background, the data was used to generate an extensive transposition map, which can be helpful to derive essentiality of genes in MB001.<br /> In summary, this dissertation documents the development and application of the prophage- and IS-free *Corynebacterium glutamicum* platform strain CR101, featuring an efficient integration system, a transposon mutagenesis system utilizing the endogenous IS*Cg1* transposase, and a streamlined workflow for transposon mutant library preparation and sequencing via Oxford Nanopore Technologies, that is transferable to other transposon mutagenesis systems and enables advanced genetic screens.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2025

Author dc:creator

• Linder, Marten

Repository record source_url

https://pub.uni-bielefeld.de/record/3003809

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3003809