Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant.
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.
CNPAB/EMBRAPA, BR465, Km7, Seropédica, RJ, 23890-000, Brazil.
Luc F M
LF
Rouws
Jean L
JL
Simões-Araújo
Adriana S
AS
Hemerly
José I
JI
Baldani
D016947:Acetobacteraceae / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development; D002876:Chromosomes, Bacterial / Q000235:genetics; D004251:DNA Transposable Elements; D018274:Electroporation; D005407:Flagella / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D004251:DNA Transposable Elements