Journal Article

Ordered Cosmid Library of the <i>Mesorhizobium loti</i> MAFF303099 Genome for Systematic Gene Disruption and Complementation Analysis

Yoshiyuki Hattori, Hirofumi Omori, Masaki Hanyu, Noriko Kaseda, Elina Mishima, Takakazu Kaneko, Satoshi Tabata and Kazuhiko Saeki

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 12, pages 1542-1557
Published in print December 2002 | ISSN: 0032-0781
Published online December 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf175
Ordered Cosmid Library of the Mesorhizobium loti MAFF303099 Genome for Systematic Gene Disruption and Complementation Analysis

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  • Biochemistry
  • Molecular and Cell Biology
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For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively. The genome of M. loti consists of a single chromosome and two plasmids. The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E. coli. The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps. We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones. As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome. All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930–4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor. The results supported the effectiveness of the approach to detect gene functions.

Keywords: Keywords: Exopolysaccharide — Genome library — Mesorhizobium loti — Nitrogen fixation — Symbiosis.; Abbreviation: ORF, open reading frame.

Journal Article.  7417 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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