title: A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa.
abstract: Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.
  
  
  
  To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.
  
  
  
  We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.
authors.affiliation: Department of Forest Mycology and Plant Pathology, Uppsala BioCenter, SLU, Uppsala, Sweden.
authors.forename: Seong-Bin
authors.initials: SB
authors.lastname: Kim
authors.forename: Salme
authors.initials: S
authors.lastname: Timmusk
mesh_terms: D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors; D059765:Homologous Recombination; D009154:Mutation; D000070057:Paenibacillus polymyxa / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics; D001614:beta-Amylase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000516:alpha-Amylases; D001614:beta-Amylase