Stability of mutations in a Sphingomonas strain

Sphingomonas sp. strain RW1 is able to mineralise dibenzofuran and dibenzo-p-dioxin. Three mutants were constructed that could not use dibenzofuran or dibenzo-p-dioxin as a carbon source but were able to grow with the succeeding metabolites of the pathway. Two different mutagenic agents were applied, a chemical treatment with 1-methyl-3-nitro-1-nitrosoguanidine, resulting in mutants RW1-N6 and RW1-N7, and a biological insertion mutagenesis with the mini-Tn5 transposon pBSL118, resulting in mutant RW1-M3. Southern blot analysis and PCR experiments confirmed a single insertion of the mini-Tn5 into one of the genes coding for the oxygenase component of the dibenzofuran 4,4a-dioxygenase system. The genetic stability of these mutants was examined after growth with complex medium under nonselective conditions. All three mutants failed to revert to wild-type metabolic functions.