1887

Abstract

-Ornithine production in the alfalfa microsymbiont occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in has been little studied. Unlike most bacteria, 1021 is annotated as encoding two enzymes producing ornithine: -acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate -acetyltransferase (ArgJ) transacetylates -glutamate with the acetyl group from NAO, forming ornithine and -acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. and single mutants, and an double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate genes ( (), () and ) all possessed NAO deacetylase activity. and , but not , expressed functionally complemented an Δ : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the mutant. Transcriptional assays of , and , fused to a promoterless gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.

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2015-08-01
2022-01-27
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References

  1. Arteaga A., Dunn M. 2015; Physiological roles of polyamines in rhizobia. Insights Biotechnol. (in press)
    [Google Scholar]
  2. Baetens M., Legrain C., Boyen A., Glansdorff N. 1998; Genes and enzymes of the acetyl cycle of arginine biosynthesis in the extreme thermophilic bacterium Thermus thermophilus HB27. Microbiology 144:479–492 [View Article][PubMed]
    [Google Scholar]
  3. Barnett M.J., Fisher R.F., Jones T., Komp C., Abola A.P., Barloy-Hubler F., Bowser L., Capela D., Galibert F., other authors. 2001; Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc Natl Acad Sci U S A 98:9883–9888 [View Article][PubMed]
    [Google Scholar]
  4. Barnett M.J., Toman C.J., Fisher R.F., Long S.R. 2004; A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote-host interaction. Proc Natl Acad Sci U S A 101:16636–16641 [View Article][PubMed]
    [Google Scholar]
  5. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
    [Google Scholar]
  6. Capela D., Barloy-Hubler F., Gouzy J., Bothe G., Ampe F., Batut J., Boistard P., Becker A., Boutry M., other authors. 2001; Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc Natl Acad Sci U S A 98:9877–9882 [View Article][PubMed]
    [Google Scholar]
  7. Casadaban M.J. 1976; Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 104:541–555 [View Article][PubMed]
    [Google Scholar]
  8. Cho H.-Y., Tanizawa K., Tanaka H., Soda K. 1987; Thermostable aminoacylase from Bacillus thermoglucosidius: purification and characterization. Agric Biol Chem 51:2793–2800 [View Article]
    [Google Scholar]
  9. Cunin R., Glansdorff N., Piérard A., Stalon V. 1986; Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 50:314–352[PubMed]
    [Google Scholar]
  10. Datsenko K.A., Wanner B.L. 2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645 [View Article][PubMed]
    [Google Scholar]
  11. de la Fuente A., Martín J.F., Rodríguez-García A., Liras P. 2004; Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine. J Bacteriol 186:6501–6507 [View Article][PubMed]
    [Google Scholar]
  12. Díaz R., Vargas-Lagunas C., Villalobos M.A., Peralta H., Mora Y., Encarnación S., Girard L., Mora J. 2011; argC Orthologs from Rhizobiales show diverse profiles of transcriptional efficiency and functionality in Sinorhizobium meliloti . J Bacteriol 193:460–472 [View Article][PubMed]
    [Google Scholar]
  13. diCenzo G.C., Finan T.M. 2015; Genetic redundancy is prevalent within the 6.7 Mb Sinorhizobium meliloti genome. Mol Genet Genomics (in press)
    [Google Scholar]
  14. Dunn M.F. 2015; Key roles of microsymbiont amino acid metabolism in rhizobia-legume interactions. Crit Rev Microbiol (in press)
    [Google Scholar]
  15. Dunn M.F., Araíza G., Finan T.M. 2001; Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021. Arch Microbiol 176:355–363 [View Article][PubMed]
    [Google Scholar]
  16. Dunn M.F., Díaz R., Hernández-Lucas I. 2008; Características regulatorias de la N-acetilglutamato cinasa (ArgB) de Sinorhizobium meliloti Rm1021. In VIII Congreso Nacional de la Fijación Biológica del Nitrógeno pp. 69–70
    [Google Scholar]
  17. Fellay R., Frey J., Krisch H. 1987; Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene 52:147–154 [View Article][PubMed]
    [Google Scholar]
  18. Fernández-Murga M.L., Gil-Ortiz F., Llácer J.L., Rubio V. 2004; Arginine biosynthesis in Thermotoga maritima: characterization of the arginine-sensitive N-acetyl-L-glutamate kinase. J Bacteriol 186:6142–6149 [View Article][PubMed]
    [Google Scholar]
  19. Figurski D.H., Helinski D.R. 1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [View Article][PubMed]
    [Google Scholar]
  20. Girard L., Brom S., Dávalos A., López O., Soberón M., Romero D. 2000; Differential regulation of fixN-reiterated genes in Rhizobium etli by a novel fixL-fixK cascade. Mol Plant Microbe Interact 13:1283–1292 [View Article][PubMed]
    [Google Scholar]
  21. Haas D., Leisinger T. 1975a; N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Purification and ligand-directed association-dissociation. Eur J Biochem 52:365–375 [View Article][PubMed]
    [Google Scholar]
  22. Haas D., Leisinger T. 1975b; N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties. Eur J Biochem 52:377–393 [View Article][PubMed]
    [Google Scholar]
  23. Hani E.K., Ng D., Chan V.-L. 1999; Arginine biosynthesis in Campylobacter jejuni TGH9011: determination of the argCOBD cluster. Can J Microbiol 45:959–969 [View Article][PubMed]
    [Google Scholar]
  24. Jacobs M.A., Alwood A., Thaipisuttikul I., Spencer D., Haugen E., Ernst S., Will O., Kaul R., Raymond C., other authors. 2003; Comprehensive transposon mutant library of Pseudomonas aeruginosa . Proc Natl Acad Sci U S A 100:14339–14344 [View Article][PubMed]
    [Google Scholar]
  25. Javid-Majd F., Blanchard J.S. 2000; Mechanistic analysis of the argE-encoded N-acetylornithine deacetylase. Biochemistry 39:1285–1293 [View Article][PubMed]
    [Google Scholar]
  26. Kershaw N.J., McNaughton H.J., Hewitson K.S., Hernández H., Griffin J., Hughes C., Greaves P., Barton B., Robinson C.V., Schofield C.J. 2002; ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activity. Eur J Biochem 269:2052–2059 [View Article][PubMed]
    [Google Scholar]
  27. Kovach M.E., Elzer P.H., Hill D.S., Robertson G.T., Farris M.A., Roop R.M. II, Peterson K.M. 1995; Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [View Article][PubMed]
    [Google Scholar]
  28. Land M., Hauser L., Jun S.-R., Nookaew I., Leuze M.R., Ahn T.-H., Karpinets T., Lund O., Kora G., other authors. 2015; Insights from 20 years of bacterial genome sequencing. Funct Integr Genomics 15:141–161 [View Article][PubMed]
    [Google Scholar]
  29. Lohmeier-Vogel E.M., Loukanina N., Ferrar T.S., Moorhead G.B.G., Thorpe T.A. 2005; N-acetyl glutamate kinase from Daucus carota suspension cultures: embryogenic expression profile, purification and characterization. Plant Physiol Biochem 43:854–861 [View Article][PubMed]
    [Google Scholar]
  30. López-Lara I.M., Gao J.-L., Soto M.J., Solares-Pérez A., Weissenmayer B., Sohlenkamp C., Verroios G.P., Thomas-Oates J., Geiger O. 2005; Phosphorus-free membrane lipids of Sinorhizobium meliloti are not required for the symbiosis with alfalfa but contribute to increased cell yields under phosphorus-limiting conditions of growth. Mol Plant Microbe Interact 18:973–982 [View Article][PubMed]
    [Google Scholar]
  31. Madrid E. 2013 Búsqueda del gen que codifica para la enzima N-acetilglutamato sintasa por mutagénesis PhD thesis, Universidad Autónoma del Estado de Morelos
    [Google Scholar]
  32. Marc F., Weigel P., Legrain C., Almeras Y., Santrot M., Glansdorff N., Sakanyan V. 2000; Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms. Eur J Biochem 267:5217–5226 [View Article][PubMed]
    [Google Scholar]
  33. Martínez-Salazar J.M., Romero D. 2000; Role of the ruvB gene in homologous and homeologous recombination in Rhizobium etli . Gene 243:125–131 [View Article][PubMed]
    [Google Scholar]
  34. Meade H.M., Long S.R., Ruvkun G.B., Brown S.E., Ausubel F.M. 1982; Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122[PubMed]
    [Google Scholar]
  35. Omelchenko M.V., Galperin M.Y., Wolf Y.I., Koonin E.V. 2010; Non-homologous isofunctional enzymes: a systematic analysis of alternative solutions in enzyme evolution. Biol Direct 5:31 [View Article][PubMed]
    [Google Scholar]
  36. Prentki P., Krisch H.M. 1984; In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313 [View Article][PubMed]
    [Google Scholar]
  37. Quandt J., Hynes M.F. 1993; Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene 127:15–21 [View Article][PubMed]
    [Google Scholar]
  38. Sakanyan V., Kochikyan A., Mett I., Legrain C., Charlier D., Piérard A., Glansdorff N. 1992; A re-examination of the pathway for ornithine biosynthesis in a thermophilic and two mesophilic Bacillus species. J Gen Microbiol 138:125–130 [View Article]
    [Google Scholar]
  39. Sakanyan V., Charlier D., Legrain C., Kochikyan A., Mett I., Piérard A., Glansdorff N. 1993a; Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase. J Gen Microbiol 139:393–402 [View Article][PubMed]
    [Google Scholar]
  40. Sakanyan V., Desmarez L., Legrain C., Charlier D., Mett I., Kochikyan A., Savchenko A., Boyen A., Falmagne P., other authors. 1993b; Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus . Appl Environ Microbiol 59:3878–3888[PubMed]
    [Google Scholar]
  41. Sakanyan V., Petrosyan P., Lecocq M., Boyen A., Legrain C., Demarez M., Hallet J.N., Glansdorff N. 1996; Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway. Microbiology 142:99–108 [View Article][PubMed]
    [Google Scholar]
  42. 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]
  43. Schäfer A., Tauch A., Jäger W., Kalinowski J., Thierbach G., Pühler A. 1994; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum . Gene 145:69–73 [View Article][PubMed]
    [Google Scholar]
  44. Shaw F.L. 2011 From prediction to function: Polyamine biosynthesis and formate metabolism in the α- and ϵ- proteobacteria PhD thesis, University of East Anglia, UK
    [Google Scholar]
  45. Shaw F.L., Elliott K.A., Kinch L.N., Fuell C., Phillips M.A., Michael A.J. 2010; Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine. J Biol Chem 285:14711–14723 [View Article][PubMed]
    [Google Scholar]
  46. Soto M.J., Zorzano A., García-Rodriguez F.M., Mercado-Blanco J., López-Lara I.M., Olivares J., Toro N. 1994a; Identification of a novel Rhizobium meliloti nodulation efficiency nfe gene homolog of Agrobacterium ornithine cyclodeaminase. Mol Plant Microbe Interact 7:703–707 [View Article][PubMed]
    [Google Scholar]
  47. Soto M.J., van Dillewijn P., Olivares J., Toro N. 1994b; Ornithine cyclodeaminase activity in Rhizobium meliloti . FEMS Microbiol Lett 119:209–214 [View Article]
    [Google Scholar]
  48. Steele M., Marcone M., Gyles C., Chan V.L., Odumeru J. 2006; Enzymatic activity of Campylobacter jejuni hippurate hydrolase. Protein Eng Des Sel 19:17–25 [View Article][PubMed]
    [Google Scholar]
  49. Stover C.K., Pham X.Q., Erwin A.L., Mizoguchi S.D., Warrener P., Hickey M.J., Brinkman F.S., Hufnagle W.O., Kowalik D.J., other authors. 2000; Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964 [View Article][PubMed]
    [Google Scholar]
  50. Tanimoto K., Higashi N., Nishioka M., Ishikawa K., Taya M. 2008; Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii . FEBS J 275:1140–1149 [View Article][PubMed]
    [Google Scholar]
  51. Thompson J.D., Higgins D.G., Gibson T.J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [View Article][PubMed]
    [Google Scholar]
  52. Vogel H.J., Bonner D.M. 1956; Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106[PubMed]
    [Google Scholar]
  53. Xu M., Rao Z., Dou W., Yang J., Jin J., Xu Z. 2012; Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production. Amino Acids 43:255–266 [View Article][PubMed]
    [Google Scholar]
  54. Xu Y., Labedan B., Glansdorff N. 2007; Surprising arginine biosynthesis: a reappraisal of the enzymology and evolution of the pathway in microorganisms. Microbiol Mol Biol Rev 71:36–47 [View Article][PubMed]
    [Google Scholar]
  55. Yakobson E.A., Guiney D.G. Jr 1984; Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5 . J Bacteriol 160:451–453[PubMed]
    [Google Scholar]
  56. Yuan Z.C., Zaheer R., Morton R., Finan T.M. 2006; Genome prediction of PhoB regulated promoters in Sinorhizobium meliloti and twelve proteobacteria. Nucleic Acids Res 34:2686–2697 [View Article][PubMed]
    [Google Scholar]
  57. Zamorano-Sánchez D., Reyes-González A., Gómez-Hernández N., Rivera P., Georgellis D., Girard L. 2012; FxkR provides the missing link in the fixL-fixK signal transduction cascade in Rhizobium etli CFN42. Mol Plant Microbe Interact 25:1506–1517 [View Article][PubMed]
    [Google Scholar]
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