Skip to main content
SpringerLink
Log in

Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

To elucidate the biosynthetic pathways for all proteinogenic amino acids in Xanthomonas campestris pv. campestris, this study combines results obtained by in silico genome analysis and by 13C-NMR-based isotopologue profiling to provide a panoramic view on a substantial section of bacterial metabolism. Initially, biosynthesis pathways were reconstructed from an improved annotation of the complete genome of X. campestris pv. campestris B100. This metabolic reconstruction resulted in the unequivocal identification of biosynthesis routes for 17 amino acids in total: arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Ambiguous pathways were reconstructed from the genome data for alanine, glycine, and isoleucine biosynthesis. 13C-NMR analyses supported the identification of the metabolically active pathways. The biosynthetic routes for these amino acids were derived from the precursor molecules pyruvate, serine, and pyruvate, respectively. By combining genome analysis and isotopologue profiling, a comprehensive set of biosynthetic pathways covering all proteinogenic amino acids was unraveled for this plant pathogenic bacterium, which plays an important role in biotechnology as a producer of the exopolysaccharide xanthan. The data obtained lay ground for subsequent functional analyses in post-genomics and biotechnology, while the innovative combination of in silico and wet lab technology described here is promising as a general approach to elucidate metabolic pathways.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Alifano P, Fani R, Liò P, Lazcano A, Bazzicalupo M, Carlomagno MS, Bruni CB (1996) Histidine biosynthetic pathway and genes: structure, regulation and evolution. Microbiol Rev 60(1):44–69

    PubMed  CAS  Google Scholar 

  • Berg CM, Whalen WA, Archambault LB (1983) Role of alanine-valine transaminase in Salmonella typhimurium and analysis of an avtA:Tn5 mutant. J Bacteriol 155(3):1009–1014

    PubMed  CAS  Google Scholar 

  • Blom J, Albaum SP, Doppmeier D, Pühler A, Vorhölter FJ, Zakrzewski M, Goesmann A (2009) EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics 10:154

    Article  PubMed  Google Scholar 

  • Bono H, Ogata H, Goto S, Kanehisa M (1998) Reconstruction of amino acid biosynthesis pathways from the complete genome sequence. Genome Res 8(3):203–210

    PubMed  CAS  Google Scholar 

  • Bradbury JF (1984) Xanthomonas. In: Krieg NR, Holt JG (eds) Bergeys manual of systematic bacteriology. Williams and Wikins, Baltimore, pp 199–210

    Google Scholar 

  • Calhoun DH, Pierson DL, Jensen RA (1973) The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa. Mol Gen Genet 121(2):117–132

    Article  PubMed  CAS  Google Scholar 

  • Caspi R, Foerster H, Fulcher CA, Kaipa P, Krummenacker M, Latendresse M, Paley S, Rhee SY, Shearer AG, Tissier C, Walk TC, Zhang P, Karp PD (2008) The MetaCyc Database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases. Nucleic Acids Res 36:D623–D631 (Database issue)

    Article  PubMed  CAS  Google Scholar 

  • Claudel-Renard C, Chevalet C, Faraut T, Kahn D (2003) Enzyme-specific profiles for genome annotation: PRIAM. Nucleic Acids Res 31(22):6633–6639

    Article  PubMed  CAS  Google Scholar 

  • Consortium UniProt (2010) The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res 38:D142–D148 (Database issue)

    Article  Google Scholar 

  • da Silva ACR, Ferro JA, Reinach FC et al (2002) Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417(6887):459–463

    Article  PubMed  Google Scholar 

  • Drevland RM, Waheed A, Graham DE (2007) Enzymology and evolution of the pyruvate pathway to 2-oxobutyrate in Methanocaldococcus jannaschii. J Bacteriol 189(12):4391–4400

    Article  PubMed  CAS  Google Scholar 

  • Eisenreich W, Bacher A (2007) Advances of high-resolution NMR techniques in the structural and metabolic analysis of plant biochemistry. Phytochemistry 68(22–24):2799–2815

    Article  PubMed  CAS  Google Scholar 

  • Eisenreich W, Dandekar T, Heesemann J, Goebel W (2010) Carbon metabolism of intracellular bacterial pathogens and possible links to virulence. Nat Rev Microbiol 8(6):401–412

    Article  PubMed  CAS  Google Scholar 

  • Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2(4):953–971

    Article  PubMed  CAS  Google Scholar 

  • Eylert E, Herrmann V, Jules M, Gillmaier N, Lautner M, Buchrieser C, Eisenreich W, Heuner K (2010) Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates. J Biol Chem 285(29):22232–22243

    Article  PubMed  CAS  Google Scholar 

  • Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer ELL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucleic Acids Res 38:D211–D222 (Database issue)

    Article  PubMed  CAS  Google Scholar 

  • Fontecave M, Ollagnier-de-Choudens S (2008) Iron-sulfur cluster biosynthesis in bacteria: mechanisms of cluster assembly and transfer. Arch Biochem Biophys 474(2):226–237

    Article  PubMed  CAS  Google Scholar 

  • García-Ochoa F, Santos VE, Casas JA, Gómez E (2000) Xanthan gum: production, recovery, and properties. Biotechnol Adv 18(7):549–579

    Article  PubMed  Google Scholar 

  • Gowrishankar J, Pittard J (1982) Regulation of phenylalanine biosynthesis in Escherichia coli K-12: control of transcription of the pheA operon. J Bacteriol 150(3):1130–1137

    PubMed  CAS  Google Scholar 

  • Harder W, Quayle JR (1971) The biosynthesis of serine and glycine in Pseudomonas AM1 with special reference to growth on carbon sources other than C1 compounds. Biochem J 121(5):753–762

    PubMed  CAS  Google Scholar 

  • Humbert R, Simoni RD (1980) Genetic and biomedical studies demonstrating a second gene coding for asparagine synthetase in Escherichia coli. J Bacteriol 142(1):212–220

    PubMed  CAS  Google Scholar 

  • Hunter S, Apweiler R, Attwood TK et al (2009) InterPro: the integrative protein signature database. Nucleic Acids Res 37:D211–D215 (Database issue)

    Article  PubMed  CAS  Google Scholar 

  • Jin JH, Choi KK, Jung US, In YH, Lee SY, Lee J (2004) Regulatory analysis of amino acid synthesis pathway in Escherichia coli: aspartate family. Enzyme Microb Technol 35(6–7):694–706

    Article  CAS  Google Scholar 

  • Karp PD, Paley SM, Krummenacker M et al (2010) Pathway Tools version 13.0: integrated software for pathway/genome informatics and systems biology. Brief Bioinform 11(1):40–79

    Article  PubMed  CAS  Google Scholar 

  • Krishna RV, Beilstein P, Leisinger T (1979) Biosynthesis of proline in Pseudomonas aeruginosa. Properties of γ-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase. Biochem J 181(1):223–230

    PubMed  CAS  Google Scholar 

  • Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305(3):567–580

    Article  PubMed  CAS  Google Scholar 

  • Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y (1993) Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 90(7):3009–3013

    Article  PubMed  CAS  Google Scholar 

  • Lee KH, Park JH, Kim TY, Kim HU, Lee SY (2007) Systems metabolic engineering of Escherichia coli for l-threonine production. Mol Syst Biol 3:149

    Article  PubMed  CAS  Google Scholar 

  • Liu JQ, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H (1998) Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity l-threonine aldolase from Escherichia coli. Eur J Biochem 255(1):220–226

    Article  PubMed  CAS  Google Scholar 

  • Marchler-Bauer A, Lu S, Anderson JB et al (2011) CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res 39:D225–D229 (Database issue)

    Article  PubMed  Google Scholar 

  • Mattern IE, Pittard J (1971) Regulation of tyrosine biosynthesis in Escherichia coli K-12: Isolation and characterization of operator mutants. J Bacteriol 107(1):8–15

    PubMed  CAS  Google Scholar 

  • Meyer F, Goesmann A, McHardy AC, Bartels D, Bekel T, Clausen J, Kalinowski J, Linke B, Rupp O, Giegerich R, Pühler A (2003) GenDB- an open source genome annotation system for prokaryote genomes. Nucleic Acids Res 31(8):2187–2195

    Article  PubMed  CAS  Google Scholar 

  • Miller RE, Stadtman ER (1972) Glutamate synthase from Escherichia coli. J Biol Chem 247(22):7407–7419

    PubMed  CAS  Google Scholar 

  • Morizono H, Cabrera-Luque J, Shi D, Gallegos R, Yamaguchi S, Yu X, Allewell NM, Malamy MH, Tuchman M (2006) Acetylornithine transcarbamylase: a novel enzyme in arginine biosynthesis. J Bacteriol 188(8):2974–2982

    Article  PubMed  CAS  Google Scholar 

  • Orth JD, Thiele I, Palsson BØ (2010) What is flux balance analysis? Nat Biotechnol 28(3):245–248

    Article  PubMed  CAS  Google Scholar 

  • Park JH, Lee KH, Kim TY, Lee SY (2007) Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation. Proc Natl Acad Sci USA 104(19):7797–7802

    Article  PubMed  CAS  Google Scholar 

  • Qian W, Jia Y, Ren S et al (2005) Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris. Genome Res 15(6):757–767

    Article  PubMed  CAS  Google Scholar 

  • Rigano LA, Siciliano F, Enrique R, Sendín L, Filippone P, Torres PS, Qüesta J, Dow JM, Castagnaro AP, Vojnov AA, Marano MR (2007) Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri. Mol Plant Microbe Interact 20(10):1222–1230

    Article  PubMed  CAS  Google Scholar 

  • Risso C, Van Dien SJ, Orloff A, Lovley DR, Coppi MV (2008) Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens. J Bacteriol 190(7):2266–2274

    Article  PubMed  CAS  Google Scholar 

  • Rückert C, Pühler A, Kalinowski J (2003) Genome-wide analysis of the l-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation. J Biotechnol 104(1–3):213–228

    Article  PubMed  Google Scholar 

  • Sambrook J, Russel D (2001) Molecular cloning: a laboratory manual. CSH Press, New York

    Google Scholar 

  • Sayers EW, Barrett T, Benson DA et al (2011) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 39:D38–D51 (Database issue)

    Article  PubMed  Google Scholar 

  • Schlösser T, Gätgens C, Weber U, Stahmann K (2004) Alanine:glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significance. Yeast 21(1):63–73

    Article  PubMed  Google Scholar 

  • Swings JG, Civerolo EL (1993) Xanthomonas. Chapman & Hall, London

    Google Scholar 

  • Umbarger HE, Umbarger MA, Siu PML (1963) Biosynthesis of serine in Escherichia coli and Salmonella typhimurium J. Bacteriol 85(6):1431–1439

    CAS  Google Scholar 

  • Velasco A, Leguina J, Lazcano A (2002) Molecular evolution of the lysine biosynthetic pathways. J Mol Evol 55(4):445–449

    Article  PubMed  CAS  Google Scholar 

  • Vorhölter FJ, Niehaus K, Pühler A (2001) Lipopolysaccharide biosynthesis in Xanthomonas campestris pv. campestris: a cluster of 15 genes is involved in the biosynthesis of the LPS O-antigen and the LPS core. Mol Genet Genomics 266(1):79–95

    Article  PubMed  Google Scholar 

  • Vorhölter F, Thias T, Meyer F, Bekel T, Kaiser O, Pühler A, Niehaus K (2003) Comparison of two Xanthomonas campestris pv. campestris genomes revealed differences in their gene composition. J Biotechnol 106(2–3):193–202

    Article  PubMed  Google Scholar 

  • Vorhölter F, Schneiker S, Goesmann A et al (2008) The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis. J Biotechnol 134(1-2):33–45

    Article  PubMed  Google Scholar 

  • Watt TF, Vucur M, Baumgarth B, Watt SA, Niehaus K (2009) Low molecular weight plant extract induces metabolic changes and the secretion of extracellular enzymes, but has a negative effect on the expression of the type-III secretion system in Xanthomonas campestris pv. campestris. J Biotechnol 140(1–2):59–67

    Article  PubMed  CAS  Google Scholar 

  • Westrop GD, Goodall G, Mottram JC, Coombs GH (2006) Cysteine biosynthesis in Trichomonas vaginalis involves cysteine synthase utilizing O-phosphoserine. J Biol Chem 281(35):25062–25075

    Article  PubMed  CAS  Google Scholar 

  • Wittmann C (2002) Metabolic flux analysis using mass spectrometry. Adv Biochem Eng Biotechnol 74:39–64

    PubMed  CAS  Google Scholar 

  • Xu H, Zhang Y, Guo X, Ren S, Staempfli AA, Chiao J, Jiang W, Zhao G (2004) Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway. J Bacteriol 186(16):5400–5409

    Article  PubMed  CAS  Google Scholar 

  • Yang C, Shapiro BE, Hung S, Mjolsness ED, Hatfield GW (2005) A mathematical model for the branched chain amino acid biosynthetic pathways of Escherichia coli K12. J Biol Chem 280(12):11224–11232

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the DFG SPP1316. We thank Marcus Persicke for helpful discussions and Thomas Patschkowski for carefully reading the manuscript. S. Schatschneider was supported by Hans-Böckler-Stiftung and the Graduate Cluster Industrial Biotechnology (CLIB2021) at Bielefeld University. C. Rückert acknowledges funding by a research grant awarded by the MIWFT within the BIO.NRW initiative (grant 280371902).

Author information

Authors and Affiliations

  1. Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld, Bielefeld, Germany

    Sarah Schatschneider, Frank-Jörg Vorhölter & Karsten Niehaus

  2. Institut für Genomforschung und Systembiologie, Centrum für Biotechnology (CeBiTec), Universität Bielefeld, Bielefeld, Germany

    Christian Rückert & Alfred Pühler

  3. Faculty of Biology and Center for Systems Biology (ZBSA), Albert-Ludwigs-University Freiburg, Freiburg, Germany

    Anke Becker

  4. Lehrstuhl für Biochemie, Technische Universität München, Munich, Germany

    Wolfgang Eisenreich

Authors
  1. Sarah Schatschneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank-Jörg Vorhölter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Rückert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anke Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolfgang Eisenreich
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alfred Pühler
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Karsten Niehaus
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karsten Niehaus.

Additional information

Communicated by D. Andersson.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schatschneider, S., Vorhölter, FJ., Rückert, C. et al. Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling. Mol Genet Genomics 286, 247 (2011). https://doi.org/10.1007/s00438-011-0639-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00438-011-0639-7

Keywords

Search

Navigation