Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Nov;83(22):8467–8471. doi: 10.1073/pnas.83.22.8467

Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria.

J L Ramos, A Stolz, W Reineke, K N Timmis
PMCID: PMC386951  PMID: 3022293

Abstract

Stimulation of transcription from positively regulated promoters involves regulatory proteins that have been activated, generally as a consequence of binding low molecular weight effector molecules. To define essential structural features of effectors for one positively acting gene regulator, the xylS-encoded protein, which activates the TOL plasmid meta-cleavage pathway operon promoters, effector activities of a wide range of benzoate derivatives have been systematically analyzed, and mutant xylS-encoded proteins exhibiting altered effector specificities have been generated and characterized. Cloned mutant xylS genes were trans dominant in partial diploids containing the wild-type xylS allele and could therefore be used to effect expansion of the range of aromatic compounds completely or partially degraded by Pseudomonas bacteria. The method developed to isolate mutant xylS-encoded proteins has general applicability and could in principle be used to isolate gene regulator specificity mutants of any inducible regulatory system.

Full text

PDF
8467

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bochner B. R., Huang H. C., Schieven G. L., Ames B. N. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. doi: 10.1128/jb.143.2.926-933.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dorn E., Hellwig M., Reineke W., Knackmuss H. J. Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad. Arch Microbiol. 1974;99(1):61–70. doi: 10.1007/BF00696222. [DOI] [PubMed] [Google Scholar]
  4. Franklin F. C., Lehrbach P. R., Lurz R., Rueckert B., Bagdasarian M., Timmis K. N. Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway. J Bacteriol. 1983 May;154(2):676–685. doi: 10.1128/jb.154.2.676-685.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Harayama S., Lehrbach P. R., Timmis K. N. Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2. J Bacteriol. 1984 Oct;160(1):251–255. doi: 10.1128/jb.160.1.251-255.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harayama S., Leppik R. A., Rekik M., Mermod N., Lehrbach P. R., Reineke W., Timmis K. N. Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product. J Bacteriol. 1986 Aug;167(2):455–461. doi: 10.1128/jb.167.2.455-461.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harayama S., Rekik M., Timmis K. N. Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida. Mol Gen Genet. 1986 Feb;202(2):226–234. doi: 10.1007/BF00331641. [DOI] [PubMed] [Google Scholar]
  8. Inouye S., Nakazawa A., Nakazawa T. Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS. J Bacteriol. 1981 Nov;148(2):413–418. doi: 10.1128/jb.148.2.413-418.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lehrbach P. R., Zeyer J., Reineke W., Knackmuss H. J., Timmis K. N. Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13. J Bacteriol. 1984 Jun;158(3):1025–1032. doi: 10.1128/jb.158.3.1025-1032.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mermod N., Lehrbach P. R., Reineke W., Timmis K. N. Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters. EMBO J. 1984 Nov;3(11):2461–2466. doi: 10.1002/j.1460-2075.1984.tb02156.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mermod N., Ramos J. L., Lehrbach P. R., Timmis K. N. Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria. J Bacteriol. 1986 Aug;167(2):447–454. doi: 10.1128/jb.167.2.447-454.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Raibaud O., Schwartz M. Positive control of transcription initiation in bacteria. Annu Rev Genet. 1984;18:173–206. doi: 10.1146/annurev.ge.18.120184.001133. [DOI] [PubMed] [Google Scholar]
  14. Reineke W., Knackmuss H. J. Chemical structure and biodegradability of halogenate aromatic compounds. Substituent effects on 1,2-dioxygenation of benzoic acid. Biochim Biophys Acta. 1978 Sep 6;542(3):412–423. doi: 10.1016/0304-4165(78)90372-0. [DOI] [PubMed] [Google Scholar]
  15. Sala-Trepat J. M., Evans W. C. The meta cleavage of catechol by Azotobacter species. 4-Oxalocrotonate pathway. Eur J Biochem. 1971 Jun 11;20(3):400–413. doi: 10.1111/j.1432-1033.1971.tb01406.x. [DOI] [PubMed] [Google Scholar]
  16. Worsey M. J., Franklin F. C., Williams P. A. Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2. J Bacteriol. 1978 Jun;134(3):757–764. doi: 10.1128/jb.134.3.757-764.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES