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-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 EcoRI-HindIII fragment to be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found.
Swaving
J
J
Department of Food Science, Agricultural University, Wageningen, The Netherlands.
Weijers
C A
CA
van Ooyen
A J
AJ
de Bont
J A
JA
D000473:Alkanes / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004852:Epoxy Compounds / Q000378:metabolism*; D005816:Genetic Complementation Test; D006088:Gram-Negative Aerobic Bacteria / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames / Q000235:genetics*; D015183:Restriction Mapping; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
D000473:Alkanes; D004852:Epoxy Compounds; C009068:propylene oxide