COMPLEMENTATION OF XANTHOBACTER-PY2 MUTANTS DEFECTIVE IN EPOXYALKANE DEGRADATION, AND EXPRESSION AND NUCLEOTIDE-SEQUENCE OF THE COMPLEMENTING DNA FRAGMENT

Three Xanthobacter Py2 mutants (M3, M8 and M10) lacking epoxyalkane-de grading activity were isolated and characterized. All mutants were abl e to grow on acetone, the degradation product of 1,2-epoxypropane conv ersions. Furthermore, they contained the unidentified 'low molecular m ass fraction' (LMF) necessary for epoxyalkane-degrading activity. Thre e 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-t ype 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 c onjugated into Xanthobacter autotrophicus GJ10, which is not able to d egrade 1,2-epoxypropane. In crude extracts of X. autotrophicus GJ10(pE P9), epoxyalkane-degrading activity was demonstrated, but only after t he addition of the LMF from Xanthobacter Py2. Hybridization experiment s demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9 . Subcloning revealed a 4.8 kb EcoRI-HindIII fragment to be necessary for complementing the mutant M10. In the sequence of this fragment fou r different ORFs were found.