Transposon mutagenesis of PGPR Bacillus strains B9601-Y2 and FZB42 for detection of genes important for plant growth promotion and biocontrol. Development and application of a screening procedure permitting identification of transposants with altered plant growth promoting properties, antifungal and antibacterial properties.
Responsible: Dr. He, Kunming and Dr. Borriss, Berlin
Both partners are in charge to develop suitable screening systems allowing high throughout tests of transposon mutants generated in WP4. After identification and final proof of those mutants, sequence determination will be performed.
The nonreplicative class1 transposon pIC333 containing a mini Tn10 construct is used for random mutagenesis of the PGPR Bacillus subtilis strain B9601-Y2 previously shown to display a general antifungal action due to proteins secreted in the environment (He, personal communication). In a similar way the mariner transposon TnYLB-1(pMarA,B) will be used to mutagenize Bacillus amyloliquefaciens FZB42. Mariner transposons insert more randomly as do the bacterial transposons (Tn10 or Tn917). To identify the Bacillus genes disrupted by the insertions, as well as to further characterize the insertion sites, chromosomal DNA will be extracted from selected clones exhibiting an apparently altered phenotype and three randomly selected clones. These DNA will be used in an inverse PCR protocol that amplifies the chromosomal DNA abutting the transposons´ ITR. The genome of FZB42 is now completely sequenced, greatly facilitating identification of gene sequences from short sequence tags obtained from regions flanking the transposon insertions.