We start with a stepr F- strain and mix with E.coli that is strepr and resistant to neomycinr (neor is on Tn5 transposon on F-plasmid).
conjugate salmonella with tn5 transposon, plate on neomycin & get neomycin resistant colonies & these have tranposon in genome & then assay for those mutants with defective virulence. We need to use the LD50 assay.
Mutant screening, transposon mutagenesis where transposon integrated randomly and find which do not exhibit Nod formation.
- WT Rhizobia (SmR, NeoS, F-)
- Mix with E. coli (SmS, NeoR ((presence of Tn5 transposon, transposons oftentimes carry antibiotic resistance markers)), F+)
- Remember that Tn5 transposon not capable of independent replication
- Confugation such that Tn5 must integrate into CMSM.
- Select rhizobia: SmR + NeoR 6000 Tn5 insertion mutants identified.
To select for rhizobia with Tn5 integrated we select for streprneor cells and 6,000 strains identified. These are tn5 insertion mutants. We screen for mutants performing nodulation. Assay for that is to screen for nodulation is root hair curling assay. We will also screen for nitrogen fixation. Screen for ability to convert acetylene to ethylene. This is done on all 6,000 mutants several times. Out of this, at least 50 different genes identified for nodule formation and nitrogen fixation. The first group is mutants completely incapable of forming nodules. The second group can form nodules but not nitrogen fixation (nod+fix-). Nod A, B, C and D were isolated. Rhizobia can be free-living and symbiotic. NodA not present when cells grown free-living, but add plant root secretions and you will get nodA expression. reporter gene fusion to test activity of nodA promoter.