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Applied and Environmental Microbiology, May 2006, p. 3750-3755, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3750-3755.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

High-Throughput Transposon Mutagenesis of Corynebacterium glutamicum and Construction of a Single-Gene Disruptant Mutant Library{dagger}

Nobuaki Suzuki,1 Naoko Okai,1 Hiroshi Nonaka,1 Yota Tsuge,1,2 Masayuki Inui,1 and Hideaki Yukawa1,2*

Microbiology Research Group, Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizu-Cho, Soraku-Gun, Kyoto 619-0292, Japan,1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan2

Received 15 November 2005/ Accepted 17 February 2006

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.

* Corresponding author. Mailing address: Microbiology Research Group, Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizu-Cho, Soraku-Gun, Kyoto 619-0292, Japan. Phone: 81-774-75-2308. Fax: 81-774-75-2321. E-mail: mmg-lab{at}rite.or.jp.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, May 2006, p. 3750-3755, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3750-3755.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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