title: Construction of a mariner-based transposon vector for use in insertion sequence mutagenesis in selected members of the Rhizobiaceae.
abstract: BACKGROUND
  The Rhizobiaceae family of Gram-negative bacteria often engage in symbiosis with plants of economic importance. Historically, genetic studies to identify the function of individual genes, and characterize the biology of these bacteria have relied on the use of classical transposon mutagenesis. To increase the rate of scientific discovery in the Rhizobiaceae there is a need to adapt high-throughput genetic screens like insertion sequencing for use in this family of bacteria. Here we describe a Rhizobiaceae compatible MmeI-adapted mariner transposon that can be used with insertion sequencing for high-throughput genetic screening.
  
  
  RESULTS
  The newly constructed mariner transposon pSAM_Rl mutagenized R. leguminosarum, S. meliloti, and A. tumefaciens at a high frequency. In R. leguminosarum, mutant pools were generated that saturated 88% of potential mariner insertions sites in the genome. Analysis of the R. leguminosarum transposon insertion sequencing data with a previously described hidden Markov model-based method resulted in assignment of the contribution of all annotated genes in the R. leguminosarum 3841 genome for growth on a complex medium. Good concordance was observed between genes observed to be required for growth on the complex medium, and previous studies.
  
  
  CONCLUSIONS
  The newly described Rhizobiaceaee compatible mariner transposon insertion sequencing vector pSAM_Rl has been shown to mutagenize at a high frequency and to be an effective tool for use in high-throughput genetic screening. The construction and validation of this transposon insertion sequencing tool for use in the Rhizobiziaceae will provide an opportunity for researchers in the Rhizobiaceae community to use high-throughput genetic screening, allowing for significant increase in the rate of genetic discovery, particularly given the recent release of genome sequences from many Rhizobiaceae strains.
authors.affiliation: Department of Biology, University of Regina, 3737 Wascana Parkway, Regina, SK, S4S 0A2, Canada. benjamin.j.perry@gmail.com.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Perry
authors.affiliation: Department of Biology, University of Regina, 3737 Wascana Parkway, Regina, SK, S4S 0A2, Canada. chris.yost@uregina.ca.
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Yost
mesh_terms: D004251:DNA Transposable Elements; D005820:Genetic Testing / Q000379:methods; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D012230:Rhizobiaceae / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements