SUMMARY: Three Py2 mutants (M3, M8 and M10) lacking epoxyalkane-degrading activity were isolated and characterized. All mutants were able to grow on acetone, the degradation product of 1,2-epoxypropane conversions. Furthermore, they contained the unidentified “low molecular mass fraction” (LMF) necessary for epoxyalkane-degrading activity. Three cosmids from a gene bank complemented the mutation in M10 and M8 but not in mutant M3. Epoxyalkane-degrading activity in crude extracts of 1,2-epoxypropane-grown complemented mutants was similar to the wild-type activity. Surprisingly, M10 transformed with complementing cosmid pEP9 showed a constitutively expressed epoxyalkane-degrading activity, which was not observed in the wild-type strain. The cosmid pEP9 was conjugated into GJ10, which is not able to degrade 1,2-epoxypropane. In crude extracts of . autotrophicus GJ10(pEP9), epoxyalkane-degrading activity was demonstrated, but only after the addition of the LMF from Py2. Hybridization experiments demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9. Subcloning revealed a 4.8 kb RI-dlll fragment to be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found.


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