1887

Microbiology Society logo

Volume 143, Issue 4

Squalene-hopene cyclase from cloning, expression, sequence analysis and comparison to other triterpenoid cyclases Free

Abstract

With the help of a PCR-based screening method, the gene encoding squalene-hopene cyclase (SHC) of USDA 110 was isolated from a cosmid library. The SHC catalyses the cyclization of squalene to hopanoids, a class of triterpenoid lipids recently discovered in nitrogen-fixing, root-nodule-forming bacteria. Hybridization experiments showed that the gene is present in bacteria of all strains tested and in photosynthetic bacteria forming stem nodules on tropical legumes of the genus The gene is 1983 bp in length and encodes a protein of 660 amino acid residues with a calculated molecular mass of 73671 Da. Comparison of the deduced amino acid sequence with the sequences of other SHCs revealed highest similarity (70%) to the SHC from the Gram-negative and lower similarity (48%) to the SHCs from the Gram-positive and SHC also showed similarity (38-43%) to eukaryotic oxidosqualene cyclases. The gene was expressed in The recombinant SHC catalysed the cyclization of squalene to the hopanoids hopene and diplopterol However, the formation of the gammacerane derivative tetrahymanol, which is produced in addition to hopanoids in strains could not be detected Therefore, the presence of a second squalene cyclase in can be assumed. Sequence analysis of 0.5 kb upstream from the gene identified a partial ORF with significant similarity to the C-terminus of an ORF located immediately upstream from the gene in Both ORFs also showed similarity to phytoene desaturases from cyanobacteria and plants. The 3'-end of this ORF from overlaps with 13 bp at the 5'-end of The close proximity of this ORF to suggests that and this ORF may be part of an operon.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): Bradyrhizobium japonicum , hopanoids , squalene-hopene cyclase and triterpenoids
© Society for General Microbiology 1997
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-143-4-1235
1997-04-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/143/4/mic-143-4-1235.html?itemId=/content/journal/micro/10.1099/00221287-143-4-1235&mimeType=html&fmt=ahah

References

  1. Abe I., Rohmer M., Prestwich G. D. 1993; Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes.. Chem Rev 93:2189–2206.
     [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410.
     [Google Scholar]
  3. Ashby M. N., Spear D. H., Edwards P.A. 1990; Prenyltransferases. From yeast to man. In Molecular Biology of Atheriosclerosis Edited by A. D. Attie. Amsterdam:. Elsevier.27–34.
     [Google Scholar]
  4. Berry A. M., Moreau R. A., Jones A. D. 1991; Bacteriohopantetrol: abundant lipid in Frankia cells and in nitrogenfixing tissue.. Plant Physiol 95:111–115.
     [Google Scholar]
  5. Berry A. M., Harriott O. T., Moreau R. A., Osman S. F., Benson, D. R., Jones A. D. 1993; Hopanoid lipids compose the Frankia vesicle envelope, presumptive barrier of oxygen diffusion to nitrogenase.. Proc Natl Acad Sci USA 90:6091–6094.
     [Google Scholar]
  6. Birnboim C., Harriott O. T., Doly J. 1979; Rapid alkaline extraction procedure for screening recombinant plasmid DNA.. Nucleic Acids Res 7:1513–1523.
     [Google Scholar]
  7. Buntel C. J., Griffin J. H. 1992; Nucleotide and deduced amino acid sequences of the oxidosqualene cyclase from Candida albicans.. J Am Chem Soc 114:9711–1913.
     [Google Scholar]
  8. Caspi E., Zander J. M., Greig J. B., Mallory F. B., Conner R. L., Landrey J. R. 1968; Evidence for a nonoxidative cyclization of squalene in the biosynthesis of tetrahymanol.. J Am Chem Soc 90:3563–3564.
     [Google Scholar]
  9. Corey E. J., Matsuda S. P. T., Bartel B. 1993; Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen.. Proc Natl Acad Sci USA 90:11628–11632
     [Google Scholar]
  10. Corey E. J., Matsuda S. P. T., Bartel B. 1994; Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.. Proc Natl Acad Sci USA 91:2211–2215.
     [Google Scholar]
  11. Feil C., Süssmuth R., Jung G., Poralla K. 1996; Site-directed mutagenesis of putative active-site residues in squalene-hopene cyclase.. Eur J Biochem 242:51–55
     [Google Scholar]
  12. Griffin H. G., I’Anson K. J., Gasson M. J. 1993; Rapid isolation of genes from bacterial lambda libraries by direct polymerase chain reaction screening.. FEMS Microbiol Lett 112:49–54.
     [Google Scholar]
  13. Jarvis B. D. W., Gillis M., De Ley J. 1986; Intra- and intergeneric similarities between the ribosomal ribonucleic acid cistrons of Rhizobium and Bradyrhizobium species and some related bacteria.. Int J Syst Bacteriol 36:129–138.
     [Google Scholar]
  14. Jordan D. C. 1982; Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants.. Int J Syst Bacteriol 32:136–139.
     [Google Scholar]
  15. Kannenberg E., Poralla K., Blume A. 1980; A hopanoid from the thermoacidophilic Bacillus acidocaldarius condenses membranes.. Naturwissenschaften 67:458–459.
     [Google Scholar]
  16. Kannenberg E. L., Perzl M., Härtner T. 1995; The occurrence of hopanoid lipids in Bradyrhizobium bacteria.. FEMS Microbiol Lett 127:255–262.
     [Google Scholar]
  17. Kannenberg E. L., Perzl M., Mülller P., Härtner T., Poralla K. 1996; Hopanoid lipids in Bradyrhizobium and other plant associated bacteria and cloning of the Bradyrhizobium squalenehopene cyclase gene.. Plant Soil 186:107–112
     [Google Scholar]
  18. Kleemann G., Poralla K., Englert G., KjØsen H., Liaaen-Jensen S., Neunlist S., Rohmer M. 1990; Tetrahymanol from the phototrophic bacterium Rhodopseudomonas palustris: first report of a gammacerane triterpene from a prokaryote.. J Gen Microbiol 136:2551–2553.
     [Google Scholar]
  19. Kleemann G., Kellner R., Poralla K. 1994; Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol.. Biochim Biophys Acta 1210:317–320.
     [Google Scholar]
  20. Knauf V. C., Nester E. W. 1982; Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid.. Plasmid 8:45–54.
     [Google Scholar]
  21. Martinez-Romero E., Caballero-Mellado J. 1996; Rhizobium phylogenies and bacterial genetic diversity.. Crit Rev Plant Sci 15:113–140.
     [Google Scholar]
  22. Ochs D., Kaletta C., Entian K.-D., Beck-Sickinge A., Poralla K. 1992; Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism.. J Bacteriol 174:298–302.
     [Google Scholar]
  23. Ourisson G., Rohmer M., Poralla K. 1987; Prokaryotic hopanoids and other polyterpenoid sterol surrogates.. Annu Rev Microbiol 41:301–333.
     [Google Scholar]
  24. Parniske M., Kosch K., Werner D., Müller P. 1993; ExoB mutants of Bradyrhizobium japonicum with reduced competitiveness for nodulation of Glycine max.. Mol Plant-Microbe Interact 6:99–106.
     [Google Scholar]
  25. Poralla K. 1994; The possible role of a repetitive amino acid motif in evolution of triterpenoid cyclases.. Bioorg Med Chem Lett 4:285–290.
     [Google Scholar]
  26. Poralla K., Hewelt A., Prestwich G. D., Abe I., Reipen I., Sprenger G. 1994; A specific amino acid repeat in squalene and oxidosqualene cyclases.. Trends Biochem Sci 19:157–158.
     [Google Scholar]
  27. Regensburger B., Hennecke H. 1983; RNA polymerase from Rhizobium japonicum.. Arch Microbiol 135:103–109.
     [Google Scholar]
  28. Reipen I. G., Poralla K., Sahm H., Sprenger G. A. 1995; Zymomonas mobilis squalene-hopene cyclase gene (shc): cloning, DNA sequence analysis, and expression in Escherichia coli.. Microbiology 141:155–161.
     [Google Scholar]
  29. Robinson A. B., Robinson L. R. 1991; Distribution of glutamine and asparagine residues and their near neighbours in peptides and proteins.. Proc Natl Acad Sci USA 88:8880–8884.
     [Google Scholar]
  30. Roessne C. A., Min C., Hardin S. H., Harris-Haller L. W., McCollum J. C., Scott A. I. 1993; Sequence of the Candida albicans erg7 gene.. Gene 127:149–150.
     [Google Scholar]
  31. Saar J., Kader J.-C., Poralla K., Ourisson G. 1991; Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila.. Biochim Biophys Acta 1075:93–101.
     [Google Scholar]
  32. Sahm H., Rohmer M., Bringer-Meyer S., Sprenger G. A., Welle R. 1993; Biochemistry and physiology of hopanoids in bacteria.. Adv Microb Physiol 35:247–273.
     [Google Scholar]
  33. 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]
  34. Sandmann G. 1994; Carotenoid biosynthesis in microorganisms and plants.. Eur J Biochem 223:7–24.
     [Google Scholar]
  35. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering : transposon mutagenesis in Gram negative bacteria.. Biotechnology 1:784–791.
     [Google Scholar]
  36. Somasegaran P., Hoben H. J. 1994; Handbook for Rhizobia: Methods in Legume-Rhizobium Technology.. New York: Springer.
     [Google Scholar]
  37. Tappe C. H. 1993; Squalen-Hopen-Cyclasen: Reinigung, Charakterisierung und Inhibitor-Experimente PhD thesis, University of Tuebingen, Germany...
    [Google Scholar]
  38. Tully R. E., Keister D. L. 1993; Cloning and mutagenesis of a cytochrome P-450 locus from Bradyrhizobium japonicum that is expressed anaerobically and symbiotically.. Appl Environ Microbiol 59:4136–4142
     [Google Scholar]
  39. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.. Gene 19:259–268
     [Google Scholar]
  40. Vilcheze C., Llopiz P., Neunlist S., Poralla K., Rohmer M. 1994; Prokaryotic triterpenoids: new hopanoids from the nitrogen-fixing bacteria Azotobacter vinelandii, Beijerinckia indica and Beijerinckia mobilis.. Microbiology 140:2749–2753
     [Google Scholar]
  41. Vincent J. M. 1970; A Manual for the Practical Study of Root-Nodule Bacteria. IBP Handbook no. 15.. Oxford: Blackwell Scientific.
     [Google Scholar]
  42. Wilcockson J., Werner D. 1976; Nitrogenase activity by Klebsiella and Rhizobium on solid substrata exposed to air.. Ber Dtsch Bot Ges 89:587–607
     [Google Scholar]
  43. Wong F. Y. K., Stackebrandt E., Ladha J. K., Fleischman D. E., Date R. A., Fuerst J. A. 1994; Phylogenetic analysis of Bradyrhizobium japonicum and photosynthetic stem-nodulating bacteria from Aeschynomene species grown in separated geographical regions.. Appl Environ Microbiol 60:940–946
     [Google Scholar]
  44. Young J. P. W., Downer H. L., Eardly B. D. 1991; Phylogeny of the phototrophic Rhizobium strain BTAil by polymerase chain reaction-based sequencing of a 16s rRNA gene segment.. J Bacteriol 173:2271–2277.
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-143-4-1235
Loading
Squalene-hopene cyclase from Bradyrhizobium japonicum: cloning, expression, sequence analysis and comparison to other triterpenoid cyclases
Microbiology 143, 1235 (1997); https://doi.org/10.1099/00221287-143-4-1235
/content/journal/micro/10.1099/00221287-143-4-1235
/content/journal/micro/10.1099/00221287-143-4-1235
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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