01936naa a2200193 a 450000100080000000500110000800800410001910000200006024501390008026000090021950000830022852012260031165000370153765300260157470000270160070000190162770000190164677300770166516267922015-03-16       2008    bl uuuu     u00u1 u    #d1 aROUWS, L. F. M.  aValidation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant.  c2008  aParceria: UFRRJ; Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro.  aGluconacetobacter 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?&ltKAN-2&gtTnp 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.  aGluconacetobacter diazotrophicus  aBactéria endofítica1 aSIMÕES ARAÚJO, J. L.1 aHEMERLY, A. S.1 aBALDANI, J. I.  tArchives of Microbiology, New Yorkgv. 189, n. 4, p. 397-405, apr. 2008.