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. 1993 Apr 15;90(8):3486–3490. doi: 10.1073/pnas.90.8.3486

Synthesis of a gene for sensory rhodopsin I and its functional expression in Halobacterium halobium.

M P Krebs 1, E N Spudich 1, H G Khorana 1, J L Spudich 1
PMCID: PMC46325  PMID: 8475097

Abstract

We have designed, synthesized, and expressed in Halobacterium halobium a gene encoding sensory rhodopsin I (SR-I). The gene has been optimized for cassette mutagenesis by incorporating 30 unique restriction sites with uniform spacing throughout the 720-bp coding region. For expression, the coding region was placed downstream of the promoter and translation initiation region of the bacterioopsin gene on a selectable vector. This construct encodes SR-I with an extended N terminus that includes the 13-amino acid leader sequence and the 8-amino acid N terminus of bacterioopsin. To obtain a SR-I- H. halobium strain for expressing the synthetic gene, we used homologous recombination to delete the chromosomal gene encoding SR-I, sopI. The deletion strain was transformed with the synthetic sopI expression vector. Using antibody directed against the C-terminal region of SR-I, we detected in transformant membranes a protein with the electrophoretic mobility expected for SR-I with a processed N-terminal extension. The synthetic gene product was functionally identical to SR-I. Its flash-induced absorption difference spectrum and photochemical reaction cycle in membrane envelope vesicles were characteristic of SR-I. The protein fully restored phototaxis responses in the deletion strain.

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Selected References

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

  1. Alam M., Hazelbauer G. L. Structural features of methyl-accepting taxis proteins conserved between archaebacteria and eubacteria revealed by antigenic cross-reaction. J Bacteriol. 1991 Sep;173(18):5837–5842. doi: 10.1128/jb.173.18.5837-5842.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blanck A., Oesterhelt D., Ferrando E., Schegk E. S., Lottspeich F. Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor. EMBO J. 1989 Dec 20;8(13):3963–3971. doi: 10.1002/j.1460-2075.1989.tb08579.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bogomolni R. A., Spudich J. L. Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6250–6254. doi: 10.1073/pnas.79.20.6250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dunn R., McCoy J., Simsek M., Majumdar A., Chang S. H., Rajbhandary U. L., Khorana H. G. The bacteriorhodopsin gene. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6744–6748. doi: 10.1073/pnas.78.11.6744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ferretti L., Karnik S. S., Khorana H. G., Nassal M., Oprian D. D. Total synthesis of a gene for bovine rhodopsin. Proc Natl Acad Sci U S A. 1986 Feb;83(3):599–603. doi: 10.1073/pnas.83.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Khorana H. G. Bacteriorhodopsin, a membrane protein that uses light to translocate protons. J Biol Chem. 1988 Jun 5;263(16):7439–7442. [PubMed] [Google Scholar]
  7. Krebs M. P., Hauss T., Heyn M. P., RajBhandary U. L., Khorana H. G. Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):859–863. doi: 10.1073/pnas.88.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Krebs M. P., Mollaaghababa R., Khorana H. G. Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1987–1991. doi: 10.1073/pnas.90.5.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lam W. L., Doolittle W. F. Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5478–5482. doi: 10.1073/pnas.86.14.5478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lanyi J. K., MacDonald R. E. Light-induced transport in Halobacterium halobium. Methods Enzymol. 1979;56:398–407. doi: 10.1016/0076-6879(79)56038-8. [DOI] [PubMed] [Google Scholar]
  11. Nassal M., Mogi T., Karnik S. S., Khorana H. G. Structure-function studies on bacteriorhodopsin. III. Total synthesis of a gene for bacterio-opsin and its expression in Escherichia coli. J Biol Chem. 1987 Jul 5;262(19):9264–9270. [PubMed] [Google Scholar]
  12. Ni B. F., Chang M., Duschl A., Lanyi J., Needleman R. An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium. Gene. 1990 May 31;90(1):169–172. doi: 10.1016/0378-1119(90)90456-2. [DOI] [PubMed] [Google Scholar]
  13. Spudich E. N., Hasselbacher C. A., Spudich J. L. Methyl-accepting protein associated with bacterial sensory rhodopsin I. J Bacteriol. 1988 Sep;170(9):4280–4285. doi: 10.1128/jb.170.9.4280-4285.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Spudich E. N., Sundberg S. A., Manor D., Spudich J. L. Properties of a second sensory receptor protein in Halobacterium halobium phototaxis. Proteins. 1986 Nov;1(3):239–246. doi: 10.1002/prot.340010306. [DOI] [PubMed] [Google Scholar]
  15. Spudich E. N., Takahashi T., Spudich J. L. Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7746–7750. doi: 10.1073/pnas.86.20.7746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Spudich J. L., Bogomolni R. A. Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature. 1984 Dec 6;312(5994):509–513. doi: 10.1038/312509a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Spudich J. L., Bogomolni R. A. Sensory rhodopsins of halobacteria. Annu Rev Biophys Biophys Chem. 1988;17:193–215. doi: 10.1146/annurev.bb.17.060188.001205. [DOI] [PubMed] [Google Scholar]
  18. Sundberg S. A., Alam M., Spudich J. L. Excitation signal processing times in Halobacterium halobium phototaxis. Biophys J. 1986 Nov;50(5):895–900. doi: 10.1016/S0006-3495(86)83530-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sundberg S. A., Bogomolni R. A., Spudich J. L. Selection and properties of phototaxis-deficient mutants of Halobacterium halobium. J Bacteriol. 1985 Oct;164(1):282–287. doi: 10.1128/jb.164.1.282-287.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wölfer U., Dencher N. A., Büldt G., Wrede P. Bacteriorhodopsin precursor is processed in two steps. Eur J Biochem. 1988 May 16;174(1):51–57. doi: 10.1111/j.1432-1033.1988.tb14061.x. [DOI] [PubMed] [Google Scholar]
  21. Yan B., Nakanishi K., Spudich J. L. Mechanism of activation of sensory rhodopsin I: evidence for a steric trigger. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9412–9416. doi: 10.1073/pnas.88.21.9412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yan B., Takahashi T., Johnson R., Derguini F., Nakanishi K., Spudich J. L. All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in Halobacterium halobium. Biophys J. 1990 Apr;57(4):807–814. doi: 10.1016/S0006-3495(90)82600-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yao V. J., Spudich J. L. Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11915–11919. doi: 10.1073/pnas.89.24.11915. [DOI] [PMC free article] [PubMed] [Google Scholar]

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