Abstract
Mobile genetic elements (MGEs) contribute to instability of the host genome and plasmids. Previously, removal of the prophages in the industrial amino acid producer Corynebacterium glutamicum ATCC 13 032 resulted in strain MB001 which showed better survival under stress conditions and increased transformability. Still, eight families of Insertion Sequence (IS) elements with 27 potentially active members remain in MB001, two of which were demonstrated to be detrimental in biotechnological processes. In this study, systematical deletion of all complete IS elements in MB001 resulted in the MGE-free strain CR101. CR101 shows growth characteristics identical to the wildtype and the increased transformability of MB001. Due to its improved genome stability, we consider this strain to be an optimal host for basic research and biotechnology. As a “zero-background” host, it is also an ideal basis to study C. glutamicum IS elements. Re-sequencing of CR101 revealed that only five spontaneous point mutations had occurred during the construction process, highlighting the low mutation rate of C. glutamicum on the nucleotide level. In a second step, we developed an easily applicable ISCg1-based transposon mutagenesis system to randomly transpose a selectable marker. For optimal plasmid stability during cloning in Escherichia coli, the system utilizes a genetic switch based on the phage integrase Bxb1. Use of this integrase revealed the presence of a functional attB site in the C. glutamicum genome. To avoid cross-talk with our system and increase ease-of-use, we removed the attB site and also inserted the Bxb1 encoding gene into the chromosome of CR101. Successful insertion of single markers was verified by sequencing randomly selected mutants. Sequencing pooled mutant libraries revealed only a weak target site specificity, seemingly random distribution of insertion sites and no general strand bias. The resulting strain, ML103, together with plasmid pML10 provides a easily customizable system for random mutagenesis in an otherwise genomically stable C. glutamicum. Taken together, the MGE-free C. glutamicum strain CR101, the derivative ML103, and the plasmid pML10 provide a useful set of tools to study C. glutamicum in the future.
Highlights
Corynebacterium glutamicum is a GRAS-classified (Generally Recognized as Safe), Gram-positive bacterium which is known to be non-pathogenic, non-motile, and non-sporulating
“genome healing” approach: First, using BLAST we searched for the sequence of the ISCg elements with 2,500 bp flanks in the genome sequences of 3 C. glutamicum strains available at our lab ATCC 869, “Brevibacterium flavum” ATCC 067, and “Corynebacterium crenatum” AS1.452) to identify the orthologous regions in the three genomes
In those cases where 1) there was a syntenous region present, but 2) without the ISCg element found in MB001, we compared the number of differences between the target sequence and that of MB001 in the 750 bp directly up- and downstream of the missing region
Summary
Corynebacterium glutamicum is a GRAS-classified (Generally Recognized as Safe), Gram-positive bacterium which is known to be non-pathogenic, non-motile, and non-sporulating. While many C. glutamicum strains have been isolated, to our knowledge just two are widely used in basic research and biotechnology: strain ATCC 13 032 and strain R This might in part be due to the presence of the paracrystalline protein surface (S) layer in many other C. glutamicum strains (Hansmeier et al, 2004) that was shown to interfere with DNA transfer by electroporation in Caulobatcer species (Gilchrist and Smit, 1991) and in part due to the early availability of complete genome sequences for the two strains (Ikeda and Nakagawa, 2003; Kalinowski et al, 2003; Yukawa et al, 2007).
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