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  • Title: Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant.
    Author: Rouws LF, Simões-Araújo JL, Hemerly AS, Baldani JI.
    Journal: Arch Microbiol; 2008 Apr; 189(4):397-405. PubMed ID: 18060666.
    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 x 10(6) 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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