Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Mar;172(3):1556–1561. doi: 10.1128/jb.172.3.1556-1561.1990

Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene.

S Pille 1, J C Chuat 1, A M Breton 1, J D Clément-Métral 1, F Galibert 1
PMCID: PMC208632  PMID: 2137818

Abstract

Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R. sphaeroides thioredoxin. An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.

Full text

PDF
1556

Images in this article

1558FIG. 1
on p.1558

Selected References

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

  1. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  2. Clement-Metral J. D. Activation of ALA synthetase by reduced thioredoxin in Rhodopseudomonas spheroides Y. FEBS Lett. 1979 May 1;101(1):116–120. doi: 10.1016/0014-5793(79)81307-1. [DOI] [PubMed] [Google Scholar]
  3. Clement-Metral J. D., Holmgren A., Cambillau C., Jörnvall H., Eklund H., Thomas D., Lederer F. Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y. Eur J Biochem. 1988 Mar 1;172(2):413–419. doi: 10.1111/j.1432-1033.1988.tb13902.x. [DOI] [PubMed] [Google Scholar]
  4. Clement-Metral J. D., Hög J. O., Holmgren A. Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y. Eur J Biochem. 1986 Nov 17;161(1):119–126. doi: 10.1111/j.1432-1033.1986.tb10131.x. [DOI] [PubMed] [Google Scholar]
  5. Donohue T. J., McEwan A. G., Kaplan S. Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides cytochrome c2 gene. J Bacteriol. 1986 Nov;168(2):962–972. doi: 10.1128/jb.168.2.962-972.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eklund H., Cambillau C., Sjöberg B. M., Holmgren A., Jörnvall H., Hög J. O., Brändén C. I. Conformational and functional similarities between glutaredoxin and thioredoxins. EMBO J. 1984 Jul;3(7):1443–1449. doi: 10.1002/j.1460-2075.1984.tb01994.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gleason F. K., Holmgren A. Thioredoxin and related proteins in procaryotes. FEMS Microbiol Rev. 1988 Dec;4(4):271–297. doi: 10.1111/j.1574-6968.1988.tb02747.x. [DOI] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  9. Holmgren A. Thioredoxin. Annu Rev Biochem. 1985;54:237–271. doi: 10.1146/annurev.bi.54.070185.001321. [DOI] [PubMed] [Google Scholar]
  10. Huber H. E., Russel M., Model P., Richardson C. C. Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity. J Biol Chem. 1986 Nov 15;261(32):15006–15012. [PubMed] [Google Scholar]
  11. Kiley P. J., Donohue T. J., Havelka W. A., Kaplan S. DNA sequence and in vitro expression of the B875 light-harvesting polypeptides of Rhodobacter sphaeroides. J Bacteriol. 1987 Feb;169(2):742–750. doi: 10.1128/jb.169.2.742-750.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kiley P. J., Kaplan S. Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides light-harvesting B800-850-alpha and B800-850-beta genes. J Bacteriol. 1987 Jul;169(7):3268–3275. doi: 10.1128/jb.169.7.3268-3275.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LAURENT T. C., MOORE E. C., REICHARD P. ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEOTIDES. IV. ISOLATION AND CHARACTERIZATION OF THIOREDOXIN, THE HYDROGEN DONOR FROM ESCHERICHIA COLI B. J Biol Chem. 1964 Oct;239:3436–3444. [PubMed] [Google Scholar]
  15. Lim C. J., Fuchs J. A., McFarlan S. C., Hogenkamp H. P. Cloning, expression, and nucleotide sequence of a gene encoding a second thioredoxin from Corynebacterium nephridii. J Biol Chem. 1987 Sep 5;262(25):12114–12119. [PubMed] [Google Scholar]
  16. Lim C. J., Geraghty D., Fuchs J. A. Cloning and nucleotide sequence of the trxA gene of Escherichia coli K-12. J Bacteriol. 1985 Jul;163(1):311–316. doi: 10.1128/jb.163.1.311-316.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lim C. J., Gleason F. K., Fuchs J. A. Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli. J Bacteriol. 1986 Dec;168(3):1258–1264. doi: 10.1128/jb.168.3.1258-1264.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lim C. J., Haller B., Fuchs J. A. Thioredoxin is the bacterial protein encoded by fip that is required for filamentous bacteriophage f1 assembly. J Bacteriol. 1985 Feb;161(2):799–802. doi: 10.1128/jb.161.2.799-802.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mark D. F., Richardson C. C. Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1976 Mar;73(3):780–784. doi: 10.1073/pnas.73.3.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Muller E. D., Chory J., Kaplan S. Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides. J Bacteriol. 1985 Jan;161(1):469–472. doi: 10.1128/jb.161.1.469-472.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Russel M., Model P. The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins. J Biol Chem. 1986 Nov 15;261(32):14997–15005. [PubMed] [Google Scholar]
  23. Russel M., Model P. Thioredoxin is required for filamentous phage assembly. Proc Natl Acad Sci U S A. 1985 Jan;82(1):29–33. doi: 10.1073/pnas.82.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  25. Sistrom W. R. Transfer of chromosomal genes mediated by plasmid r68.45 in Rhodopseudomonas sphaeroides. J Bacteriol. 1977 Aug;131(2):526–532. doi: 10.1128/jb.131.2.526-532.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tabor S., Huber H. E., Richardson C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed] [Google Scholar]
  27. Wallace B. J., Kushner S. R. Genetic and physical analysis of the thioredoxin (trxA) gene of Escherichia coli K-12. Gene. 1984 Dec;32(3):399–408. doi: 10.1016/0378-1119(84)90015-5. [DOI] [PubMed] [Google Scholar]
  28. Wollman E. E., d'Auriol L., Rimsky L., Shaw A., Jacquot J. P., Wingfield P., Graber P., Dessarps F., Robin P., Galibert F. Cloning and expression of a cDNA for human thioredoxin. J Biol Chem. 1988 Oct 25;263(30):15506–15512. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES