1887

Abstract

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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-141-2-477
1995-02-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/141/2/mic-141-2-477.html?itemId=/content/journal/micro/10.1099/13500872-141-2-477&mimeType=html&fmt=ahah

References

  1. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli. . J Mol Biol 41:459–472
    [Google Scholar]
  2. De Bont J. A. M. 1993; Biotransformation of optically pure epoxides Tetrahedron Asymmetry . 4:1331–1340
    [Google Scholar]
  3. Figurski D. H., Helinski D. R. 1979; Replication of an origin- containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA . 76:1648–1652
    [Google Scholar]
  4. Janssen D. B., Scheper A., Dijkhuizen L., Witholt B. 1985; Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10. . Appl Environ Microbiol 49:673–677
    [Google Scholar]
  5. Janssen D. B., Pries F., Van Der Ploeg J., Kazemier B., Terpstra P., Witholt B. 1989; Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene. . J Bact 171:6791–6799
    [Google Scholar]
  6. Keen N. T., Tamaki S., Kobayahi D., Trollinger D. 1988; Improved broad-host-range plasmids for DNA cloning in Gram- negative bacteria.. Gene 70:191–197
    [Google Scholar]
  7. Leak D. J., Aikens P. J., Seyed-Mahmoudian M. 1992; The microbial production of epoxides. . Trends Biotechnol 10:256–261
    [Google Scholar]
  8. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  9. Sanger F., Nicklen S., Coulsen A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
    [Google Scholar]
  10. Thijssen H. H. W., Janssen Y. P. G., Vervoort L. T. M. 1994; Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents.. Biochem J 297:277–280
    [Google Scholar]
  11. Van Ginkel C. G., De Bont J. A. M. 1986; Isolation and characterization of alkene-utilizing Xanthobacter spp. . Arch Microbiol 145:403–407
    [Google Scholar]
  12. Weijers C. A. G. M., De Haan A., De Bont J. A. M. 1988a; Chiral resolution of 2,3-epoxyalkanes by Xanthobacter Py2. . Appl Microbiol Biotechnol 27:337–340
    [Google Scholar]
  13. Weijers C. A. G. M., De Haan A., De Bont J. A. M. 1988b; Microbial production and metabolism of epoxides. . Microbiol Sci 5:156–159
    [Google Scholar]
  14. Weijers C. A. G. M., Jongejan H., Franssen M. C. R., De Groot Ae., De Bont J. A. M. 1994; Dithiol- and NAD-dependent degradation of epoxyalkanes by Xanthobacter Py2. . Appl Microbiol Biotechnol (in press)
    [Google Scholar]
  15. Wiegant W. M., De Bont J. A. M. 1980; A new route for ethylene glycol metabolism in Mycobacterium E44. . J Gen Microbiol 120:325–331
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-141-2-477
Loading
/content/journal/micro/10.1099/13500872-141-2-477
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error