Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Apr;177(8):2064–2073. doi: 10.1128/jb.177.8.2064-2073.1995

Complete DNA sequence, specific Tn5 insertion map, and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides.

H P Lang 1, R J Cogdell 1, S Takaichi 1, C N Hunter 1
PMCID: PMC176850  PMID: 7721699

Abstract

The carotenoid biosynthesis genes form a cluster within the genome of Rhodobacter sphaeroides, lying in the middle of a larger cluster and 45 kb in length, which contains genes for bacteriochlorophyll biosynthesis and for the reaction center and light-harvesting apoproteins. The positions and approximate limits of the carotenoid genes were determined previously by localized transposon Tn5 mutagenesis and by comparison with the closely related Rhodobacter capsulatus carotenoid gene cluster. In this report, analysis of the DNA and deduced amino acid sequences of the carotenoid genes in R. sphaeroides are presented. Twenty-five Tn5 insertion mutants were used to produce a base-specific Tn5 insertion map of this region, and carotenoid gene assignment was supported by spectroscopic, ultrastructural, and high-pressure liquid chromatography analyses of these mutants. A region in the 3' end of crtD which affects bacteriochlorophyll biosynthesis was discovered, and CrtA was found to possess a proline-rich C-terminal region containing a repeated (Ala-Pro)n motif. CrtF also showed a high degree of sequence conservation with eukaryotic O-methyltransferases. This study provides gene sequences and assignments based upon a comprehensive structural, spectroscopic, and biochemical analysis of a range of carotenoid biosynthetic mutants; in each mutation, the point of Tn5 insertion is determined accurate to 1 bp on the gene cluster.

Full Text

The Full Text of this article is available as a PDF (360.3 KB).

Selected References

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

  1. Abillon E., Bremier L., Cardinaud R. Conformational calculations on the Ala14-Pro27 LC1 segment of rabbit skeletal myosin. Biochim Biophys Acta. 1990 Mar 1;1037(3):394–400. doi: 10.1016/0167-4838(90)90042-e. [DOI] [PubMed] [Google Scholar]
  2. Allen J. P., Feher G., Yeates T. O., Komiya H., Rees D. C. Structure of the reaction center from Rhodobacter sphaeroides R-26: the protein subunits. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6162–6166. doi: 10.1073/pnas.84.17.6162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Armstrong G. A., Alberti M., Leach F., Hearst J. E. Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet. 1989 Apr;216(2-3):254–268. doi: 10.1007/BF00334364. [DOI] [PubMed] [Google Scholar]
  4. Armstrong G. A., Cook D. N., Ma D., Alberti M., Burke D. H., Hearst J. E. Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation. J Gen Microbiol. 1993 May;139(5):897–906. doi: 10.1099/00221287-139-5-897. [DOI] [PubMed] [Google Scholar]
  5. Baker M. E., Fanestil D. D. Mammalian peripheral-type benzodiazepine receptor is homologous to CrtK protein of rhodobacter capsulatus, a photosynthetic bacterium. Cell. 1991 May 31;65(5):721–722. doi: 10.1016/0092-8674(91)90379-d. [DOI] [PubMed] [Google Scholar]
  6. Bauer C. E., Young D. A., Marrs B. L. Analysis of the Rhodobacter capsulatus puf operon. Location of the oxygen-regulated promoter region and the identification of an additional puf-encoded gene. J Biol Chem. 1988 Apr 5;263(10):4820–4827. [PubMed] [Google Scholar]
  7. Bhandari D. G., Levine B. A., Trayer I. P., Yeadon M. E. 1H-NMR study of mobility and conformational constraints within the proline-rich N-terminal of the LC1 alkali light chain of skeletal myosin. Correlation with similar segments in other protein systems. Eur J Biochem. 1986 Oct 15;160(2):349–356. doi: 10.1111/j.1432-1033.1986.tb09978.x. [DOI] [PubMed] [Google Scholar]
  8. Brown A. E., Eiserling F. A., Lascelles J. Bacteriochlorophyll Synthesis and the Ultrastructure of Wild Type and Mutant Strains of Rhodopseudomonas spheroides. Plant Physiol. 1972 Dec;50(6):743–746. doi: 10.1104/pp.50.6.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chamovitz D., Misawa N., Sandmann G., Hirschberg J. Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthesis enzyme. FEBS Lett. 1992 Jan 27;296(3):305–310. doi: 10.1016/0014-5793(92)80310-d. [DOI] [PubMed] [Google Scholar]
  10. Chang C. H., el-Kabbani O., Tiede D., Norris J., Schiffer M. Structure of the membrane-bound protein photosynthetic reaction center from Rhodobacter sphaeroides. Biochemistry. 1991 Jun 4;30(22):5352–5360. doi: 10.1021/bi00236a005. [DOI] [PubMed] [Google Scholar]
  11. Cheng X., Kumar S., Posfai J., Pflugrath J. W., Roberts R. J. Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine. Cell. 1993 Jul 30;74(2):299–307. doi: 10.1016/0092-8674(93)90421-l. [DOI] [PubMed] [Google Scholar]
  12. Coomber S. A., Chaudhri M., Connor A., Britton G., Hunter C. N. Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides. Mol Microbiol. 1990 Jun;4(6):977–989. doi: 10.1111/j.1365-2958.1990.tb00670.x. [DOI] [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dogbo O., Laferriére A., D'Harlingue A., Camara B. Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7054–7058. doi: 10.1073/pnas.85.19.7054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dryden S. C., Kaplan S. Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides. Nucleic Acids Res. 1990 Dec 25;18(24):7267–7277. doi: 10.1093/nar/18.24.7267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fraley R. T., Lueking D. R., Kaplan S. Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Polypeptide insertion into growing membrane. J Biol Chem. 1978 Jan 25;253(2):458–464. [PubMed] [Google Scholar]
  17. Garí E., Toledo J. C., Gibert I., Barbé J. Nucleotide sequence of the methoxyneurosporene dehydrogenase gene from Rhodobacter sphaeroides: comparison with other bacterial carotenoid dehydrogenases. FEMS Microbiol Lett. 1992 May 15;72(1):103–108. doi: 10.1016/0378-1097(92)90497-c. [DOI] [PubMed] [Google Scholar]
  18. Giuliano G., Pollock D., Scolnik P. A. The gene crtI mediates the conversion of phytoene into colored carotenoids in Rhodopseudomonas capsulata. J Biol Chem. 1986 Oct 5;261(28):12925–12929. [PubMed] [Google Scholar]
  19. Hunter C. N., Hundle B. S., Hearst J. E., Lang H. P., Gardiner A. T., Takaichi S., Cogdell R. J. Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides. J Bacteriol. 1994 Jun;176(12):3692–3697. doi: 10.1128/jb.176.12.3692-3697.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hunter C. N., McGlynn P., Ashby M. K., Burgess J. G., Olsen J. D. DNA sequencing and complementation/deletion analysis of the bchA-puf operon region of Rhodobacter sphaeroides: in vivo mapping of the oxygen-regulated puf promoter. Mol Microbiol. 1991 Nov;5(11):2649–2661. doi: 10.1111/j.1365-2958.1991.tb01974.x. [DOI] [PubMed] [Google Scholar]
  21. Hunter C. N., van Grondelle R., Olsen J. D. Photosynthetic antenna proteins: 100 ps before photochemistry starts. Trends Biochem Sci. 1989 Feb;14(2):72–76. doi: 10.1016/0968-0004(89)90047-9. [DOI] [PubMed] [Google Scholar]
  22. Kagan R. M., Clarke S. Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch Biochem Biophys. 1994 May 1;310(2):417–427. doi: 10.1006/abbi.1994.1187. [DOI] [PubMed] [Google Scholar]
  23. Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  24. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  25. Lang H. P., Cogdell R. J., Gardiner A. T., Hunter C. N. Early steps in carotenoid biosynthesis: sequences and transcriptional analysis of the crtI and crtB genes of Rhodobacter sphaeroides and overexpression and reactivation of crtI in Escherichia coli and R. sphaeroides. J Bacteriol. 1994 Jul;176(13):3859–3869. doi: 10.1128/jb.176.13.3859-3869.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lang H. P., Hunter C. N. The relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex in Rhodobacter sphaeroides. Biochem J. 1994 Feb 15;298(Pt 1):197–205. doi: 10.1042/bj2980197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Math S. K., Hearst J. E., Poulter C. D. The crtE gene in Erwinia herbicola encodes geranylgeranyl diphosphate synthase. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6761–6764. doi: 10.1073/pnas.89.15.6761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McGlynn P., Hunter C. N. Genetic analysis of the bchC and bchA genes of Rhodobacter sphaeroides. Mol Gen Genet. 1993 Jan;236(2-3):227–234. doi: 10.1007/BF00277117. [DOI] [PubMed] [Google Scholar]
  29. Michalowski C. B., Löffelhardt W., Bohnert H. J. An ORF323 with homology to crtE, specifying prephytoene pyrophosphate dehydrogenase, is encoded by cyanelle DNA in the eukaryotic alga Cyanophora paradoxa. J Biol Chem. 1991 Jun 25;266(18):11866–11870. [PubMed] [Google Scholar]
  30. Olsen J. D., Hunter C. N. Protein structure modelling of the bacterial light-harvesting complex. Photochem Photobiol. 1994 Dec;60(6):521–535. doi: 10.1111/j.1751-1097.1994.tb05144.x. [DOI] [PubMed] [Google Scholar]
  31. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  32. Sandmann G., Misawa N. New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species. FEMS Microbiol Lett. 1992 Jan 15;69(3):253–257. doi: 10.1016/0378-1097(92)90656-9. [DOI] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scolnik P. A., Walker M. A., Marrs B. L. Biosynthesis of carotenoids derived from neurosporene in Rhodopseudomonas capsulata. J Biol Chem. 1980 Mar 25;255(6):2427–2432. [PubMed] [Google Scholar]
  35. Singh R. K., Britton G., Goodwin T. W. Carotenoid biosynthesis in Rhodopseudomonas spheroides. S-adenosylmethionine as the methylating agent in the biosynthesis of spheroidene and spheroidenone. Biochem J. 1973 Oct;136(2):413–419. doi: 10.1042/bj1360413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smith H. O., Annau T. M., Chandrasegaran S. Finding sequence motifs in groups of functionally related proteins. Proc Natl Acad Sci U S A. 1990 Jan;87(2):826–830. doi: 10.1073/pnas.87.2.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Taylor D. P., Cohen S. N., Clark W. G., Marrs B. L. Alignment of genetic and restriction maps of the photosynthesis region of the Rhodopseudomonas capsulata chromosome by a conjugation-mediated marker rescue technique. J Bacteriol. 1983 May;154(2):580–590. doi: 10.1128/jb.154.2.580-590.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  39. Turner S. L., Russell G. C., Williamson M. P., Guest J. R. Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli. Protein Eng. 1993 Jan;6(1):101–108. doi: 10.1093/protein/6.1.101. [DOI] [PubMed] [Google Scholar]
  40. Wellington C. L., Beatty J. T. Overlapping mRNA transcripts of photosynthesis gene operons in Rhodobacter capsulatus. J Bacteriol. 1991 Feb;173(4):1432–1443. doi: 10.1128/jb.173.4.1432-1443.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wellington C. L., Taggart A. K., Beatty J. T. Functional significance of overlapping transcripts of crtEF, bchCA, and puf photosynthesis gene operons in Rhodobacter capsulatus. J Bacteriol. 1991 May;173(9):2954–2961. doi: 10.1128/jb.173.9.2954-2961.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Williamson M. P. The structure and function of proline-rich regions in proteins. Biochem J. 1994 Jan 15;297(Pt 2):249–260. doi: 10.1042/bj2970249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  45. Yen H. C., Marrs B. Map of genes for carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata. J Bacteriol. 1976 May;126(2):619–629. doi: 10.1128/jb.126.2.619-629.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Young D. A., Bauer C. E., Williams J. C., Marrs B. L. Genetic evidence for superoperonal organization of genes for photosynthetic pigments and pigment-binding proteins in Rhodobacter capsulatus. Mol Gen Genet. 1989 Jul;218(1):1–12. doi: 10.1007/BF00330558. [DOI] [PubMed] [Google Scholar]
  47. Young D. A., Rudzik M. B., Marrs B. L. An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus. FEMS Microbiol Lett. 1992 Aug 15;74(2-3):213–218. doi: 10.1016/0378-1097(92)90431-m. [DOI] [PubMed] [Google Scholar]
  48. Zsebo K. M., Hearst J. E. Genetic-physical mapping of a photosynthetic gene cluster from R. capsulata. Cell. 1984 Jul;37(3):937–947. doi: 10.1016/0092-8674(84)90428-8. [DOI] [PubMed] [Google Scholar]
  49. de Bont J. A., Scholten A., Hansen T. A. Dna-Dna hybridization of Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains. Arch Microbiol. 1981 Jan;128(3):271–274. doi: 10.1007/BF00422528. [DOI] [PubMed] [Google Scholar]

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

RESOURCES