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

Arch Microbiol. 2008 Apr;189(4):397-405. doi: 10.1007/s00203-007-0330-x. Epub 2007 Dec 5.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Acetobacteraceae / genetics*
  • Acetobacteraceae / physiology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biofilms / growth & development
  • Chromosomes, Bacterial / genetics
  • DNA Transposable Elements*
  • Electroporation
  • Flagella / genetics*
  • Flagella / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Insertional*
  • Phenotype
  • Polymerase Chain Reaction

Substances

  • Bacterial Proteins
  • DNA Transposable Elements