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Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14


The genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter favus H4-14 were identified and characterized. The RuBisC/O genes are separated by 11 by and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS. Primer extension and R-loop experiments with RNA isolated from autotrophically grown X. favus H4-14 showed that transcription of cfxL and cfxS initiated 22 by upstream from cfxL and resulted in a mRNA of at least 2.3 kb. DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators. Downstream from cfxS an additional open reading frame was identified with unknown function. Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS. The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.

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  1. Amachi T, Bowien B (1979) Characterization of two fructosebisphosphatase isoenzymes from the hydrogen bacterium Nocardia opaca 1b. J Gen Microbiol 113:347–356

  2. Andersen K, Caton J (1987) Sequence analysis of the Alcaligenes eutrophus chromosomally encoded ribulose bisphosphate carboxylase large and small subunit and their gene products. J Bacteriol 169:4547–4558

  3. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening of recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

  4. Boczar BA, Delaney TP, Cattolico RA (1989) Gene for the ribulose-1,5-bisphosphate carboxylase small subunit protein of the marine chromophyte Olisthodiscus luteus is similar to that of a chemoautotrophic bacterium. Proc Natl Acad Sci USA 86:4996–4999

  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 73:248–254

  6. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299

  7. Chomczynski P, Qasba PK (1984) Alkaline transfer of DNA to plastic membrane. Biochem Biophys Res Commun 122:340–344

  8. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995

  9. Dijken JP van, Quayle JR (1977) Fructose metabolism in four Pseudomonas species. Arch Microbiol 114:281–286

  10. Dijkhuizen L, Knight M, Harder W (1978) Metabolic regulation in Pseudomonas oxalaticus OX1. Autotrophic and heterotrophic growth on mixed substrates. Arch Microbiol 116:77–83

  11. Douglas SE, Durnford DG (1989) The small subunit of ribulose-1,5-bisphosphate carboxylase is plastid-encoded in the chlorophyll c-containing alga Cryptomonas Φ. Plant Mol Biol 13:13–20

  12. Dron M, Rahire M, Rochaix J-D (1982) Sequence of the chloroplast region of Chlamydomonas reinhardtii containing the large subunit of ribulose bisphosphate carboxylase and parts of its flanking genes. J Mol Biol 162:775–793

  13. Eggink G, van Lelyveld PH, Arnberg A, Arfman N, Witteveen C, Witholt B (1987) Structure of the Pseudomonas putida alk-BAC operon. Identification of transcription and translation products. J Biol Chem 262:6400–6406

  14. Fraenkel DG, Pontremoli S, Horecker BL (1966) The specific fructosediphosphatase of Escherichia coli: properties and partial purification. Arch Biochem Biophys 114:4–12

  15. Gibson JL, Tabita FR (1987) Organization of phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides. J Bacteriol 169:3685–3690

  16. Gibson JL, Tabita FR (1988) Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides. J Bacteriol 170:2153–2158

  17. Glisin F, Crkvenkajov R, Buys C (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633–2636

  18. Goad WB, Kanchisa MI (1982) Pattern recognition in nucleic acids sequences. I. A general method for finding local homologies and symmetries. Nucleic Acids Res 10:247–263

  19. Goldschmidt-Clermont M, Rahire M (1986) Sequence, evolution, and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. J Mol Biol 191:421–432

  20. Gutteridge S, Sigal I, Thomas B, Arendzen R, Cordova A, Lorimer G (1984) A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum. EMBO J 3:2737–2743

  21. Hallenbeck PL, Kaplan S (1987) Cloning of the gene for phosphoribulokinase activity from Rhodobacter sphaeroides and its expression in Escherichia coli: J Bacteriol 169:3669–3678

  22. Hartman FC, Stringer CD, Milanez S, Lee EH (1986) The active site of RuBisC/O. Philos Trans R Soc Lond [Biol] 313:379–395

  23. Henikoff S, Haughn GW, Calvo JM, Wallace JC (1988) A large family of bacterial activator proteins. Proc Natl Acad Sci USA 85:6602–6606

  24. Heusterspreute M, Thi VH, Emery S, Tournis-Gamble S, Kennedy N, Davison J (1985) Vectors with restriction site banks IV. pJRD184, a 3793-bp plasmid vector having 43 unique restriction sites. Gene 39:299–304

  25. Higgens CF, Hiles ID, Salmond GPC, Gill DR, Downie JA, Evans IJ, Holland IB, Gray L, Buckel SD, Bell AW, Hermodson MA (1986) A family of related ATP-binding subunits coupled to many distinct processes in bacteria. Nature 323:448–450

  26. Husemann M, Klintworth R, Büttcher V, Salnikow J, Weissenborn C, Bowien B (1988) Chromosomally and plasmid encoded gene clusters for CO2 fixation (cfx) genes in Alcaligenes eutrophus. Mol Gen Genet 214:112–120

  27. Kaback DB, Angerer LM, Davidson ND (1979) Improved methods for the formation and stabilization of R-loops. Nucleic Acids Res 6:2499–2515

  28. Klintworth R, Husemann M, Weissenborn C, Bowien B (1988) Expression of the plasmid-encoded phosphoribulokinase gene from Alcaligenes eutrophus. FEMS Microbiol Lett 49:1–6

  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  30. Lehmicke LG, Lidstrom ME (1985) Organization of the genes necessary for growth of the hydrogen-methanol autotroph Xanthobacter sp. strain H4-14 on hydrogen and carbon dioxide. J Bacteriol 162:1244–1249

  31. Lidstrom-O'Connor ME, Fulton GL, Wopat AE (1983) Methylobacterium ethanolicum: a syntrophic association of two methylotrophic bacteria. J Gen Microbiol 129:3139–3148

  32. Lipman DJ, Pearson WR (1985) Rapid and sensitive protein similarity searches. Science 227:1435–1441

  33. Lundqvist T, Schneider G (1988) Crystal structure of the binary complex of ribulose-1,5-bisphosphate carboxylase and its product 3-phospho-d-glycerate. J Biol Chem 263:3643–3646

  34. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York

  35. Mazur BJ, Chui C-F (1985) Sequence of a genomic DNA clone for the small subunit of ribulose bisphosphate carboxylase-oxygenase from tobacco. Nucleic Acids Res 13:2373–2386

  36. McKnight SL, Kingsbury R (1982) Transcriptional control signals of a eukaryotic protein-coding gene. Science 217:316–324

  37. Meijer WG, Croes LM, Jenni B, Lehmicke LG, Lidstrom ME, Dijkhuizen L (1990) Characterization of Xanthobacter strains H4-14 and 25a and enzyme profiles after growth under autotrophic and heterotrophic conditions. Arch Microbiol 153:360–367

  38. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York

  39. Nargang F, McIntosh L, Sommerville C (1984) Nucleotide sequence of the ribulose bisphosphate carboxylase oxygenase from Rhodospirillum rubrum. Mol Gen Genet 193:220–224

  40. Reith ME, Cattolico RA (1985) In vivo chloroplast protein synthesis by the chromophytic alga Olisthodiscus luteus. Biochemistry 24:2556–2561

  41. Rodriguez RL, Tait RC (1983) Recombinant DNA techniques. Addison-Wesley, Reading, Mass.

  42. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Nail Acad Sci USA 74:5463–5467

  43. Shinozaki K, Sugiura M (1982) The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-1,5-carboxylase/oxygenase. Gene 20:91–102

  44. Shinozaki K, Sugiura M (1983) The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is located close to the gene for the large subunit in cyanobacterium Anacystis nidulans. Nucleic Acids Res 11:6957–6967

  45. Shinozaki K, Yamada C, Takahata N, Sigiura M (1983) Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 80:4050–4054

  46. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo engineering: transposon mutagenesis in gram-negative bacteria. Biotechnology 1:784–790

  47. Spaink HR, Okker RJH, Wijffelman CA, Pees E, Lugtenberg BJJ (1987) Promoters in the nodulation of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant Mot Biol 9:27–39

  48. Tabita FR (1988) Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms. Microbiol Rev 52:155–189

  49. Woolford JL, Rosbash M (1979) The use of R-looping for structural gene identification and mRNA purification. Nucleic Acids Res 6:2483–2489

  50. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains. Nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–119

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Author information

Correspondence to Lubbert Dijkhuizen.

Additional information

The sequence data will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under the accession number X17252

Communicated by J.W. Lengeler

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Meijer, W.G., Arnberg, A.C., Enequist, H.G. et al. Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14. Molec. Gen. Genet. 225, 320–330 (1991).

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Key words

  • Ribulose-1,5-bisphosphate carboxylase
  • Fructosebisphosphatase
  • Phosphoribulokinase
  • Sequence analysis
  • Xanthobacter favus H4-14