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Journal of Bacteriology, June 2003, p. 3333-3343, Vol. 185, No. 11
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.11.3333-3343.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Distribution and Organization of Auxotrophic Genes on the Multichromosomal Genome of Burkholderia multivorans ATCC 17616

Harunobu Komatsu, Yoshiyuki Imura, Akira Ohori, Yuji Nagata, and Masataka Tsuda*

Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan

Received 4 December 2002/ Accepted 12 March 2003

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this ß-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions. Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome. More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome. Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome. The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.

* Corresponding author. Mailing address: Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan. Phone and fax: 81-22-217-5699. E-mail: mtsuda{at}ige.tohoku.ac.jp.

Journal of Bacteriology, June 2003, p. 3333-3343, Vol. 185, No. 11
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.11.3333-3343.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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