Biomedical subjects

Yota Tsuge

Publications and source records attributed to Yota Tsuge.

8 recordsLinked to original sources

PubMed2007

Random segment deletion based on IS31831 and Cre/loxP excision system in Corynebacterium glutamicum.

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2-186 kb). A total of 393.6 kb (11.9% of C. glutamicum R genome) coding for 331 genes was confirmed to be nonessential under standard laboratory conditions. The deletion strains, generated using only two vectors, varied not only in their lengths but also the location of the deletion along the C. glutamicum R genome. By comparing and analyzing the generated deletion strains, identification of nonessential genes, the roles of genes of hitherto unknown function, and gene-gene interactions can be easily and efficiently determined.

Blotting, Southern↗

PubMed2006

High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library.

A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

Chromosome Mapping↗

PubMed2005

Multiple large segment deletion method for Corynebacterium glutamicum.

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.

Chromosome Deletion↗

PubMed2005

A new insertion sequence, IS14999, from Corynebacterium glutamicum.

A new insertion sequence from Corynebacterium glutamicum ATCC 14999 was isolated and characterized. This IS element, designated IS14999, comprised a 1149 bp nucleotide sequence with 22 bp imperfect terminal inverted repeats. IS14999 carries a single open reading frame of 345 amino acids encoding a putative transposase that appears to have partial homology to IS642, an IS630/Tc1 superfamily element, at the C-terminal region in the amino acid sequence. This indicated that IS14999 belonged to the IS630/Tc1 superfamily, which was first identified in C. glutamicum. IS14999 has a unique distance of 38 amino acid residues between the second and third amino acids in the DDE motif, which is well known as the catalytic centre of transposase. This suggested that IS14999 constituted a new subfamily of the IS630/Tc1 superfamily. A phylogenetic tree constructed on the basis of amino acid sequences of transposases revealed that this new transposable element was more similar to eukaryotic Tc1/mariner family elements than to prokaryotic IS630 family elements. Added to the fact that IS14999 was present in only a few C. glutamicum strains, this implies that IS14999 was probably acquired by a recent lateral transfer event from eukaryotic cells. Analysis of the insertion site in C. glutamicum R revealed that IS14999 appeared to transpose at random and always caused a target duplication of a 5'-TA-3' dinucleotide upon insertion, like the other IS630/Tc1 family elements. These findings indicated that IS14999 could be a powerful tool for genetic manipulation of corynebacteria and related species.

Amino Acid Sequence↗

PubMed2005

Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA.

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

Bacterial Proteins↗

PubMed2005

New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence.

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.

Base Sequence↗

PubMed2005

Large-scale engineering of the Corynebacterium glutamicum genome.

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.

Corynebacterium glutamicum↗

PubMed2004

Cre/loxP-mediated deletion system for large genome rearrangements in Corynebacterium glutamicum.

Genome rearrangement is an increasingly important technique to facilitate the understanding of genome functions. A Cre/loxP-mediated deletion system for large-scale genome rearrangements in Corynebacterium glutamicum was developed. By comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes, distinct 14.5-kb and 56-kb regions not essential for cell survival were identified and targeted for deletion. By homologous recombination, loxP sites were integrated at each end of the target region. Deletions between the two chromosomal loxP sites in the presence of Cre recombinase were highly efficient. Accurate deletion was observed in all 96 Cre-expressing strains tested. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of 11 and 58 predicted ORF(s), respectively, upon the deletion of the 14.5-kb and 56-kb regions, the cells still exhibited normal growth under standard laboratory conditions. Based on the precision of its deletion, the Cre/loxP system provides a new, efficient genome rearrangement technique for studying C. glutamicum.

Corynebacterium glutamicum↗