Molecular and General Genetics MGG

, Volume 252, Issue 3, pp 237–248 | Cite as

The hydrogenase gene cluster ofRhizobium leguminosarum bv.viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity

  • L. Rey
  • D. Fernández
  • B. Brito
  • Y. Hernando
  • J. -M. Palacios
  • T. Ruiz-Argüeso
  • J. Imperial
Original Paper

Abstract

Plasmid pAL618 contains the genetic determinants for H2 uptake (hup) fromRhizobium leguminosarum bv.viciae, including a cluster of 17 genes namedhupSLCDEFGHIJK-hypABFCDE. A 1.7-kb segment of insert DNA located downstream ofhypE has now been sequenced, thus completing the sequence of the 20 441-bp insert DNA in plasmid pAL618. An open reading frame (designatedhypX) encoding a protein with a calculated Mr of 62 300 that exhibits extensive sequence similarity with HoxX fromAlcaligenes eutrophus (52% identity) andBradyrhizobium japonicum (57% identity) was identified 10 bp downstream ofhypE. Nodule bacteroids produced byhypX mutants in pea (Pisum sativum L.) plants grown at optimal nickel concentrations (100 µM) for hydrogenase expression, exhibited less than 5% of the wild-type levels of hydrogenase activity. These bacteroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of HupL protein. The Hup-deficient mutants were complemented for normal hydrogenase activity and nickel-dependent maturation of HupL by ahypX gene provided in trans. From expression analysis ofhypX-lacZ fusion genes, it appears thathypX gene is transcribed from the FnrN-dependenthyp promoter, thus placinghypX in thehyp operon (hypBFCDEX). Comparisons of the HypX/HoxX sequences with those in databases provided unexpected insights into their function in hydrogenase synthesis. Similarities were restricted to two distinct regions in the HypX/HoxX sequences. Region I, corresponding to a sequence conserved in N10-formyltetrahydrofolate-dependent enzymes involved in transferring one-carbon units (C1), was located in the N-terminal half of the protein, whereas region II, corresponding to a sequence conserved in enzymes of the enoyl-CoA hydratase/isomerase-family, was located in the C-terminal half. These similarities strongly suggest that HypX/HoxX have dual functions: binding of the C1 donor N10-formyl-tetrahydrofolate and transfer of the C1 to an unknown substrate, and catalysis of a reaction involving polarization of the C=O bond of an X-CO-SCoA substrate. These results also suggest the involvement of a small organic molecule, possibly synthesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hydrogenase.

Key words

Hydrogenase Rhizobium leguminosarum bvviciae N10-formyl tetrahydrofolate hypX Nickel 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Almassy RJ, Janson CA, Kan C-C, Hostomska Z (1992) Structures of apo and complexedEscherichia coli glycinamide ribonucleotide transformylase. Proc Natl Acad Sci USA 89:6114–6118PubMedGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  3. Babbitt PC, Kenyon GL, Martin BM, Charest H, Sylvestre M (1992) Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl: adenyl ligases, enoyl-CoA hydratases/isomerases and acyl-CoA thioesterases. Biochemistry 31:5594–5604CrossRefPubMedGoogle Scholar
  4. Bagley KA, Duin EC, Roseboom W, Albracht SPJ, Woodruff WH (1995) Infrared detectable groups sense changes in charge density on the nickel center in hydrogenase fromChromatium vinosum. Biochemistry 34:5527–5535CrossRefPubMedGoogle Scholar
  5. Bairoch A, Bucher P (1994) Prosite: recent developments. Nucleic Acids Res 22:3583–3589PubMedGoogle Scholar
  6. Brito B, Palacios JM, Hidalgo H, Imperial J, Ruiz-Argüeso T (1994) Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity ofRhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits. J Bacteriol 176:5297–5303PubMedGoogle Scholar
  7. Brito B, Palacios J, Imperial J, Ruiz-Argüeso T, Yang W, Bisseling T, Schmitt H, Kerl V, Bauer T, Kokotek W, Lotz W (1995) Temporal and spatial co-expression of hydrogenase and nitrogenase genes fromRhizobium leguminosarum bv. viciae in pea (Pisum sativum L.) root nodules. Mol Plant-Microbe Interact 8:235–240Google Scholar
  8. Colbeau A, Richaud P, Toussaint B, Caballero JF, Elster C, Delphin C, Smith R, Chabert J, Vignais PM (1993). Organization of the genes necessary for hydrogenase expression inRhodobacter capsulatus. Sequence analysis and identification of twohyp regulatory mutants. Mol Microbiol 8:15–29PubMedGoogle Scholar
  9. Cook RJ, Lloyd RS, Wagner C (1991) Isolation and characterization of complementary DNA clones for rat liver N10-formyltetrahydrofolate dehydrogenase. J Biol Chem 266:4965–4973PubMedGoogle Scholar
  10. Chen P, Schulze-Gahmen U, Stura EA, Inglese J, Johnson DL, Marolewski A, Benkovic SJ, Wilson IA (1992) Crystal structure of glycinamide ribonucleotide transformylase fromEscherichia coli at 3.0 Å resolution: a target enzyme for chemotherapy. J Mol Biol 227:283–292CrossRefPubMedGoogle Scholar
  11. Dernedde J, Eitinger M, Friedrich B (1993) Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases inAlcaligenes eutrophus H16. Arch Microbiol 159:545–553CrossRefPubMedGoogle Scholar
  12. Dunaway-Mariano D, Babbitt PC (1994) On the origins and functions of the enzymes of the 4-chlorobenzoate to 4-hydroxybenzoate converting pathway. Biodegradation 5:259–276CrossRefPubMedGoogle Scholar
  13. Eberz G, Friedrich B (1991) Three trans-acting functions control hydrogenase synthesis inAlcaligenes eutrophus. J Bacteriol 173:1845–1854PubMedGoogle Scholar
  14. Friedrich B, Schwartz E (1993) Molecular biology of hydrogen utilization in aerobic chemolithotrophs. Annu Rev Microbiol 47:351–383CrossRefPubMedGoogle Scholar
  15. Hernando Y, Palacios JM, Imperial J, Ruiz-Argüeso T (1995) ThehypBFCDE operon fromRhizobium leguminosarum bv.viciae is expressed from an Fnr-type promoter that escapes mutagenesis of thefnrN gene. J Bacteriol 177:5661–5669PubMedGoogle Scholar
  16. Hidalgo E, Leyva A, Ruiz-Argüeso T (1990) Nucleotide sequence of the hydrogenase structural genes fromRhizobium leguminosarum. Plant Mol Biol 15:367–370CrossRefPubMedGoogle Scholar
  17. Hidalgo E, Palacios JM, Murillo J, Ruiz-Argüeso T (1992) Nucleotide sequence and characterization of four additional genes of the hydrogenase structural operon fromRhizobium leguminosarum bv.viciae. J Bacteriol 174:4130–4139PubMedGoogle Scholar
  18. Inglese J, Johnson DL, Shiau A, Smith JM, Benkovic SJ (1990a) Subcloning, characterization, and affinity labeling ofEscherichia coli glycinamide ribonucleotide transformylase. Biochemistry 29:1436–1443CrossRefPubMedGoogle Scholar
  19. Inglese J, Smith JM, Benkovic SJ (1990b) Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase fromEscherichia coli. Biochemistry 29:6678–6687CrossRefPubMedGoogle Scholar
  20. Jacobi A, Rossmann R, Böck A (1992) Thehyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes inEscherichia coli. Arch Microbiol 158:444–451CrossRefPubMedGoogle Scholar
  21. Kan C-C, Gehring MR, Nodes BR, Janson CA, Almassy RJ, Hostomska Z (1992) Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain. J Prot Chem 11:467–473CrossRefGoogle Scholar
  22. Keen NT, Tamaki S, DK, Trollinger D (1988) Improved broad-hostrange plasmids for DNA cloning in Gram-negative bacteria. Gene 70:191–197CrossRefPubMedGoogle Scholar
  23. Kim H, Maier RJ (1990) Transcriptional regulation of hydrogenase synthesis by nickel inBradyrhizobium japonicum. J Biol Chem 265:18729–18732PubMedGoogle Scholar
  24. Klein C, Chen P, Arevalo JH, Stura EA, Marolewski A, Warren MS, Benkovic SJ, Wilson IA (1995) Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 Å resolution. J Mol Biol 249:153–175CrossRefPubMedGoogle Scholar
  25. Kortlüke C, Friedrich B (1992) Maturation of membrane-bound hydrogenase ofAlcaligenes eutrophus H16. J Bacteriol 174:6290–6293PubMedGoogle Scholar
  26. Lenz G, Schwartz E, Dernedde J, Eitinger M, Friedrich B (1994) TheAlcaligenes eutrophus H16hoxX gene participates in hydrogenase regulation. J Bacteriol 176:4385–4393PubMedGoogle Scholar
  27. Leyva A, Palacios JM, Mozo T, Ruiz-Argüeso T (1987) Cloning and characterization of hydrogen uptake genes fromRhizobium leguminosarum. J Bacteriol 169:4929–4934PubMedGoogle Scholar
  28. Leyva A, Palacios JM, Murillo J, Ruiz-Argüeso T (1990) Genetic organization of the hydrogen uptake (hup) cluster fromRhizobium leguminosarum. J Bacteriol 172:1647–1655PubMedGoogle Scholar
  29. Lie T, Soe-Agnie J, Muller G, Gsökdan D (1979) Environmental control of symbiotic nitrogen fixation: limitation to flexibility of the legume-Rhizobium system. In: Brougton W (ed). Proc Symp Soil Microbiol Plants Nutrition. University of Malaysia, Kuala Lumpur, pp 194–212Google Scholar
  30. Lutz S, Jacobi A, Schlensog V, Böhm R, Sawers G, Böck A (1991) Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes inEscherichia coli. Mol Microbiol 5:123–135.PubMedGoogle Scholar
  31. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  32. Nakahigashi K, Inokuchi H (1990) Nucleotide sequence of thefadA andfadB genes fromEscherichia coli. Nucleic Acids Res 18:4937PubMedGoogle Scholar
  33. Palacios JM, Murillo J, Leyva A, Ruiz-Argüeso T (1990) Differential expression of hydrogen uptake (hup genes) in vegetative and symbiotic cells ofRhizobium leguminosarum. Mol Gen Genet 221:363–370CrossRefPubMedGoogle Scholar
  34. Palosaari PM, Hiltunen JK (1990) Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl coenzyme A hydratase, 3-hydroxyacyl coenzyme A dehydrogenase, and Δ3, Δ2-enoyl coenzyme A isomerase activities. J Biol Chem 265:2446–2449PubMedGoogle Scholar
  35. Rey L, Hidalgo E, Palacios JM, Ruiz-Argüeso T (1992) Nucleotide sequence and organization of an H2-uptake gene cluster fromRhizobium leguminosarum bv.viciae containing a rubredoxin-like gene and four additional open reading frames. J Mol Biol 228:998–1002CrossRefPubMedGoogle Scholar
  36. Rey L, Murillo J, Hernando Y, Hidalgo E, Cabrera E, Imperial J, Ruiz-Argüeso T (1993) Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis inRhizobium leguminosarum bv.viciae. Mol Microbiol 8:471–481PubMedGoogle Scholar
  37. Rey L, Imperial J, Palacios JM, Ruiz-Argüeso T (1994) Purification ofRhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis. J Bacteriol 176:6066–6073PubMedGoogle Scholar
  38. Richaud P, Culbeau A, Toussaint B, Vignais PM (1991) Identification and sequence analysis of thehupR1 gene which encodes a response regulator of the NtrC family, required for hydrogenase expression inRhodobacter capsulatus. J Bacteriol 173:5928–5932PubMedGoogle Scholar
  39. Ruiz-Argüeso T, Hanus FJ, Evans HJ (1978) Hydrogen production and uptake by pea nodules as affected by strains ofRhizobium leguminosarum. Arch Microbiol 116:113–118CrossRefGoogle Scholar
  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  41. Short JM, Fernandez JM, Sorge JA, Huse WD (1988) λ ZAP: a bacteriophage λ expression vector with in vivo excision properties. Nucleic Acids Res 16:7583–7600PubMedGoogle Scholar
  42. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olso BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefPubMedGoogle Scholar
  43. Spaink HP, Okker RJH, Wijffelman CA, Peers E, Lugtenberg BJJ (1987) Promoters in the regulation region ofRhizobium leguminosarum Sym plasmid pRL1JI. Plant Mol Biol 9:27–39CrossRefGoogle Scholar
  44. Stoker KW, Reijnders NM, Oltmann LF, Stouthamer AH (1989) Initial cloning and squencing ofhydHG, an operon homologous tontrBC and regulating the labile hydrogenase activity inEscherichia coli K-12. J Bacteriol 171:4448–4456PubMedGoogle Scholar
  45. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130CrossRefPubMedGoogle Scholar
  46. Stults LW, Moshiri F, Maier RJ (1986) Aerobic purification of hydrogenase fromRhizobium japonicum by affinity chromatography. J Bacteriol 166:795–800PubMedGoogle Scholar
  47. Van Soom C, Verreth C, Sampaio MJ, Vanderleyden J (1993) Identification of a potential transcriptional regulator of hydrogenase activity in free-livingBradyrhizobium japonicum strains. Mol Gen Genet 239:235–240PubMedGoogle Scholar
  48. Vignais PM, Toussaint B (1994) Molecular biology of membrane-bound H2-uptake hydrogenases. Arch Microbiol 161:1–10PubMedGoogle Scholar
  49. Volbeda A, Charon M, Piras C, Hatchikian E, Frey M, Fontecilla-Camps J (1995) Crystal structure of the nickel-iron hydrogenase fromDesulfovibrio gigas. Nature 373:580–587CrossRefPubMedGoogle Scholar
  50. Wada K, Wada Y, Doi H, Ishibashi F, Gojorobi T, Ikemura T (1990) Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res 19:1981–1986Google Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • L. Rey
    • 1
  • D. Fernández
    • 1
  • B. Brito
    • 1
  • Y. Hernando
    • 1
  • J. -M. Palacios
    • 1
  • T. Ruiz-Argüeso
    • 1
  • J. Imperial
    • 2
  1. 1.Laboratorio de Microbiología, Escuela Técnica Superior de Ingenieros AgrónomosUniversidad Politécnica de MadridMadridSpain
  2. 2.Consejo Superior de Investigaciones CientíficasCiudad Universitaria s/nMadridSpain

Personalised recommendations