Complementation of Xanthobacter Py2 mutants defective in epoxyalkane degradation, and expression and nucleotide sequence of the complementing DNA fragment

Division of Industrial Microbiology, Department of Food Science, Agricultural University, PO Box 8129, 6700 EV Wageningen, The Netherlands ABSTRACT SUMMARY: Three Xanthobacter 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 Xanthobacter autotrophicus GJ10, which is not able to degrade 1,2-epoxypropane. In crude extracts of X . autotrophicus GJ10(pEP9), epoxyalkane-degrading activity was demonstrated, but only after the addition of the LMF from Xanthobacter Py2. Hybridization experiments demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9. Subcloning revealed a 4.8 kb Eco RI- Hin dlll fragment to be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found. Author for correspondence: Jelto Swaving. Tel: +31 8370 83393. Fax: +31 8370 84978.e-mail: Jelto.Swaving@algemeen.im.wau.nl Keywords: Xanthobacter Py2, epoxyalkane degradation, complementation