Journal Article

Construction of Signature-tagged Mutant Library in <i>Mesorhizobium loti</i> as a Powerful Tool for Functional Genomics

Yoshikazu Shimoda, Hisayuki Mitsui, Hiroko Kamimatsuse, Kiwamu Minamisawa, Eri Nishiyama, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda, Sayaka Shinpo, Akiko Watanabe, Mitsuyo Kohara, Manabu Yamada, Yasukazu Nakamura, Satoshi Tabata and Shusei Sato

in DNA Research

Published on behalf of Kazusa DNA Research Institute

Volume 15, issue 5, pages 297-308
Published in print October 2008 | ISSN: 1340-2838
Published online July 2008 | e-ISSN: 1756-1663 | DOI: http://dx.doi.org/10.1093/dnares/dsn017

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Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.

Keywords: Mesorhizobium loti; signature-tagged mutagenesis; mutant library; reverse genetics

Journal Article.  6455 words.  Illustrated.

Subjects: Genetics and Genomics

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