Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a fla
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ORIGINAL PAPER
Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a Xagellar mutant Luc F. M. Rouws · Jean L. Simões-Araújo · Adriana S. Hemerly · José I. Baldani
Received: 17 July 2007 / Revised: 19 October 2007 / Accepted: 17 November 2007 / Published online: 5 December 2007 © Springer-Verlag 2007
Abstract Gluconacetobacter diazotrophicus is a nitrogen-Wxing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an eYcient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5™Tnp Transposome™ (Epicentre). Up to 1 £ 106 mutants per microgram of transposome were generated in a single electroporation experiment. Insertionsite Xanking sequences were ampliWed by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion Xanks could be identiWed, conWrming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a
homolog of the Xagellar gene XgA, and was therefore predicted to be aVected in Xagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked Xagella and was non-motile on soft agar. Interestingly, it was also strongly aVected in the ability to form bioWlm on glass wool. Keywords Nitrogen-Wxing bacteria · Endophytes · Transposome · Inverse PCR · Flagella · Motility · BioWlm Abbreviations Tz Transposon-like DNA element from EZ::Tn5™ Tnp Transposome™ IPCR Inverse PCR
Introduction
Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (doi:10.1007/s00203-007-0330-x) contains supplementary material, which is available to authorized users. L. F. M. Rouws · J. L. Simões-Araújo · J. I. Baldani (&) CNPAB/EMBRAPA, BR465, Km7, Seropédica, RJ 23890-000, Brazil e-mail: [email protected] L. F. M. Rouws · A. S. Hemerly Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, RJ 21941-590, Brazil A. S. Hemerly Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 915, Rio de Janeiro, RJ 22460-030, Brazil
Gluconacetobacter diazotrophicus is a Gram-negative diazotrophic bacterium that was originally isolated from apoplastic Xuids of Brazilian sugarcane (Cavalcante and Döbereiner 1988; Gillis et al. 1989), which may obtain up to 60% of its nitrogen from biological nitrogen Wxation (Lima et al. 1987; Boddey et al. 2003). Gluconacetobacter diazotrophicus appears to be highly adapted to life inside the sugarcane plant since its optimal growth occurs at 10% sucrose and pH 5.5; conditions found inside this plant (Baldani and Baldani 2005). Additionally, G. diazotrophicus is assumed to play an important role in biological nitrogen Wxation in
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