Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Oct;179(20):6335–6340. doi: 10.1128/jb.179.20.6335-6340.1997

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4.

M A Stein 1, A Schäfer 1, F Giffhorn 1
PMCID: PMC179547  PMID: 9335280

Abstract

The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.

Full Text

The Full Text of this article is available as a PDF (866.3 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Ames G. F. Bacterial periplasmic transport systems: structure, mechanism, and evolution. Annu Rev Biochem. 1986;55:397–425. doi: 10.1146/annurev.bi.55.070186.002145. [DOI] [PubMed] [Google Scholar]
  3. Ames G. F., Mimura C. S., Shyamala V. Bacterial periplasmic permeases belong to a family of transport proteins operating from Escherichia coli to human: Traffic ATPases. FEMS Microbiol Rev. 1990 Aug;6(4):429–446. doi: 10.1111/j.1574-6968.1990.tb04110.x. [DOI] [PubMed] [Google Scholar]
  4. Armengaud J., Jouanneau Y. Overexpression in Escherichia coli of the fdxA gene encoding Rhodobacter capsulatus ferredoxin II. Protein Expr Purif. 1995 Apr;6(2):176–184. doi: 10.1006/prep.1995.1022. [DOI] [PubMed] [Google Scholar]
  5. Armengaud J., Meyer C., Jouanneau Y. Recombinant expression of the fdxD gene of Rhodobacter capsulatus and characterization of its product, a [2Fe-2S] ferredoxin. Biochem J. 1994 Jun 1;300(Pt 2):413–418. doi: 10.1042/bj3000413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aukhil I., Joshi P., Yan Y., Erickson H. P. Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins. J Biol Chem. 1993 Feb 5;268(4):2542–2553. [PubMed] [Google Scholar]
  7. Bollivar D. W., Jiang Z. Y., Bauer C. E., Beale S. I. Heterologous expression of the bchM gene product from Rhodobacter capsulatus and demonstration that it encodes S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase. J Bacteriol. 1994 Sep;176(17):5290–5296. doi: 10.1128/jb.176.17.5290-5296.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen Z., Jiang J. C., Lin Z. G., Lee W. R., Baker M. E., Chang S. H. Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis. Biochemistry. 1993 Apr 6;32(13):3342–3346. doi: 10.1021/bi00064a017. [DOI] [PubMed] [Google Scholar]
  9. Dahl M. K., Francoz E., Saurin W., Boos W., Manson M. D., Hofnung M. Comparison of sequences from the malB regions of Salmonella typhimurium and Enterobacter aerogenes with Escherichia coli K12: a potential new regulatory site in the interoperonic region. Mol Gen Genet. 1989 Aug;218(2):199–207. doi: 10.1007/BF00331269. [DOI] [PubMed] [Google Scholar]
  10. Das A. Overproduction of proteins in Escherichia coli: vectors, hosts, and strategies. Methods Enzymol. 1990;182:93–112. doi: 10.1016/0076-6879(90)82011-p. [DOI] [PubMed] [Google Scholar]
  11. Dean D. A., Reizer J., Nikaido H., Saier M. H., Jr Regulation of the maltose transport system of Escherichia coli by the glucose-specific enzyme III of the phosphoenolpyruvate-sugar phosphotransferase system. Characterization of inducer exclusion-resistant mutants and reconstitution of inducer exclusion in proteoliposomes. J Biol Chem. 1990 Dec 5;265(34):21005–21010. [PubMed] [Google Scholar]
  12. 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]
  13. Ensor C. M., Tai H. H. Site-directed mutagenesis of the conserved tyrosine 151 of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase yields a catalytically inactive enzyme. Biochem Biophys Res Commun. 1991 Apr 30;176(2):840–845. doi: 10.1016/s0006-291x(05)80262-1. [DOI] [PubMed] [Google Scholar]
  14. Ghosh D., Pletnev V. Z., Zhu D. W., Wawrzak Z., Duax W. L., Pangborn W., Labrie F., Lin S. X. Structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution. Structure. 1995 May 15;3(5):503–513. doi: 10.1016/s0969-2126(01)00183-6. [DOI] [PubMed] [Google Scholar]
  15. Ghosh D., Wawrzak Z., Weeks C. M., Duax W. L., Erman M. The refined three-dimensional structure of 3 alpha,20 beta-hydroxysteroid dehydrogenase and possible roles of the residues conserved in short-chain dehydrogenases. Structure. 1994 Jul 15;2(7):629–640. doi: 10.1016/s0969-2126(00)00064-2. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  18. Hyde S. C., Emsley P., Hartshorn M. J., Mimmack M. M., Gileadi U., Pearce S. R., Gallagher M. P., Gill D. R., Hubbard R. E., Higgins C. F. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature. 1990 Jul 26;346(6282):362–365. doi: 10.1038/346362a0. [DOI] [PubMed] [Google Scholar]
  19. Jacobs M. H., Driessen A. J., Konings W. N. Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides. J Bacteriol. 1995 Apr;177(7):1812–1816. doi: 10.1128/jb.177.7.1812-1816.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jörnvall H., Persson B., Krook M., Atrian S., Gonzàlez-Duarte R., Jeffery J., Ghosh D. Short-chain dehydrogenases/reductases (SDR). Biochemistry. 1995 May 9;34(18):6003–6013. doi: 10.1021/bi00018a001. [DOI] [PubMed] [Google Scholar]
  21. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  22. Klasen R., Bringer-Meyer S., Sahm H. Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans. J Bacteriol. 1995 May;177(10):2637–2643. doi: 10.1128/jb.177.10.2637-2643.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Kühnau S., Reyes M., Sievertsen A., Shuman H. A., Boos W. The activities of the Escherichia coli MalK protein in maltose transport, regulation, and inducer exclusion can be separated by mutations. J Bacteriol. 1991 Apr;173(7):2180–2186. doi: 10.1128/jb.173.7.2180-2186.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Marekov L., Krook M., Jörnvall H. Prokaryotic 20 beta-hydroxysteroid dehydrogenase is an enzyme of the 'short-chain, non-metalloenzyme' alcohol dehydrogenase type. FEBS Lett. 1990 Jun 18;266(1-2):51–54. doi: 10.1016/0014-5793(90)81504-h. [DOI] [PubMed] [Google Scholar]
  29. Obeid J., White P. C. Tyr-179 and Lys-183 are essential for enzymatic activity of 11 beta-hydroxysteroid dehydrogenase. Biochem Biophys Res Commun. 1992 Oct 15;188(1):222–227. doi: 10.1016/0006-291x(92)92373-6. [DOI] [PubMed] [Google Scholar]
  30. Rode H., Giffhorn F. D-(--)-tartrate dehydratase of Rhodopseudomonas sphaeroides: purification, characterization, and application to enzymatic determination of D-(--)-tartrate. J Bacteriol. 1982 Jun;150(3):1061–1068. doi: 10.1128/jb.150.3.1061-1068.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Schauder S., Schneider K. H., Giffhorn F. Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase. Microbiology. 1995 Aug;141(Pt 8):1857–1863. doi: 10.1099/13500872-141-8-1857. [DOI] [PubMed] [Google Scholar]
  33. Schneider K. H., Giffhorn F., Kaplan S. Cloning, nucleotide sequence and characterization of the mannitol dehydrogenase gene from Rhodobacter sphaeroides. J Gen Microbiol. 1993 Oct;139(10):2475–2484. doi: 10.1099/00221287-139-10-2475. [DOI] [PubMed] [Google Scholar]
  34. Schneider K. H., Giffhorn F. Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides. Eur J Biochem. 1989 Sep 1;184(1):15–19. doi: 10.1111/j.1432-1033.1989.tb14984.x. [DOI] [PubMed] [Google Scholar]
  35. Schuler G. D., Altschul S. F., Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. doi: 10.1002/prot.340090304. [DOI] [PubMed] [Google Scholar]
  36. Schäfer A., Stein M. A., Schneider K. H., Giffhorn F. Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme. Appl Microbiol Biotechnol. 1997 Jul;48(1):47–52. doi: 10.1007/s002530051013. [DOI] [PubMed] [Google Scholar]
  37. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  38. Varughese K. I., Skinner M. M., Whiteley J. M., Matthews D. A., Xuong N. H. Crystal structure of rat liver dihydropteridine reductase. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6080–6084. doi: 10.1073/pnas.89.13.6080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Williams S. G., Greenwood J. A., Jones C. W. Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter. Mol Microbiol. 1992 Jul;6(13):1755–1768. doi: 10.1111/j.1365-2958.1992.tb01348.x. [DOI] [PubMed] [Google Scholar]
  41. Yamamoto-Otake H., Koyama Y., Horiuchi T., Nakano E. Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli. Appl Environ Microbiol. 1991 May;57(5):1418–1422. doi: 10.1128/aem.57.5.1418-1422.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zheng S., Haselkorn R. A glutamate/glutamine/aspartate/asparagine transport operon in Rhodobacter capsulatus. Mol Microbiol. 1996 Jun;20(5):1001–1011. doi: 10.1111/j.1365-2958.1996.tb02541.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES