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Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant

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An Erratum to this article was published on 11 January 2008

Abstract

Gluconacetobacter diazotrophicus is a nitrogen-fixing 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 efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5™<KAN-2>Tnp Transposome™ (Epicentre). Up to 1 × 106 mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming 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 flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.

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Abbreviations

Tz:

Transposon-like DNA element from EZ::Tn5™<KAN-2>Tnp Transposome™

IPCR:

Inverse PCR

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Acknowledgments

We would like to thank Geraldo B. da Cruz for his help with electron microscopy and Kátia R.S. Teixeira and Marcia S. Vidal for helpful suggestions. We are grateful to Robert M. Boddey for English corrections and to Stefan Schwab for helpful suggestions and critically reading the manuscript. This study was financially supported by Pronex/Faperj (project number E-26/171.208/2003), CNPq (project number 50.6355/2004-7) and Instituto Milênio (process number 420274/2005-7). The first author received a CNPq fellowship.

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Authors and Affiliations

  1. CNPAB/EMBRAPA, BR465, Km7, Seropédica, RJ, 23890-000, Brazil

    Luc F. M. Rouws, Jean L. Simões-Araújo & José I. Baldani

  2. Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, RJ, 21941-590, Brazil

    Luc F. M. Rouws & Adriana S. Hemerly

  3. 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

    Adriana S. Hemerly

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  1. Luc F. M. Rouws
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  4. José I. Baldani
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Correspondence to José I. Baldani.

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Communicated by Erko Stackebrandt.

An erratum to this article can be found at http://dx.doi.org/10.1007/s00203-007-0344-4

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Rouws, L.F.M., Simões-Araújo, J.L., Hemerly, A.S. et al. Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant. Arch Microbiol 189, 397–405 (2008). https://doi.org/10.1007/s00203-007-0330-x

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