Antonie van Leeuwenhoek

, Volume 93, Issue 1–2, pp 123–132 | Cite as

The distribution of acetohydroxyacid synthase in soil bacteria

Original paper


Most bacteria possess the enzyme acetohydroxyacid synthase, which is used to produce branched-chain amino acids. Enteric bacteria contain several isozymes suited to different conditions, but the distribution of acetohydroxyacid synthase in soil bacteria is largely unknown. Growth experiments confirmed that Escherichia coli, Salmonella enterica serotype Typhimurium, and Enterobacter aerogenes contain isozymes of acetohydroxyacid synthase, allowing the bacteria to grow in the presence of valine (which causes feedback inhibition of AHAS I) or the sulfonylurea herbicide triasulfuron (which inhibits AHAS II) although a slight lag phase was observed in growth in the latter case. Several common soil isolates were inhibited by triasulfuron, but Pseudomonas fluorescens and Rhodococcus erythropolis were not inhibited by any combination of triasulfuron and valine. The extent of sulfonylurea-sensitive acetohydroxyacid synthase in soil was revealed when 21 out of 27 isolated bacteria in pure culture were inhibited by triasulfuron, the addition of isoleucine and/or valine reversing the effect in 19 cases. Primers were designed to target the genes encoding the large subunits (ilvB, ilvG and ilvI) of acetohydroxyacid synthase from available sequence data and a ∼355 bp fragment in Bacillus subtilis, Arthrobacter globiformis, E. coli and S. enterica was subsequently amplified. The primers were used to create a small clone library of sequences from an agricultural soil. Phylogenetic analysis revealed significant sequence variation, but all 19 amino acid sequences were most closely related to published large subunit acetohydroxyacid synthase amino acid sequences within several phyla including the Proteobacteria and Actinobacteria. The results suggested the majority of soil microorganisms contain only one functional acetohydroxyacid synthase enzyme sensitive to sulfonylurea herbicides.


Acetohydroxyacid synthase Diversity ilvB Isozymes Soil Sulfonylureas 



We thank Nick Coleman from the University of Sydney, Australia, and Stefan Radajewski from the University of Warwick, UK, for their help in preparation of this manuscript.


  1. Barak Z, Chipman D, Gollop N (1987) Physiological implications of the specificity of acetohydroxyacid synthase isozymes of enteric bacteria. J Bact 169:3750–3756PubMedGoogle Scholar
  2. Bauerle RH, Freundlich M, Stormer FC, Umbarger HE (1964) Control of isoleucine, valine and leucine biosynthesis II: endproduct inhibition by valine of acetohydroxy acid synthase in Salmonella typhimurium. Biochim Biophys Acta 92:142–149PubMedGoogle Scholar
  3. Boldt T, Jacobsen CS (1998) Different toxic effects of the sulfonylurea herbicides metsulfuron-methyl, chlorsulfuron and thifensulfuron-methyl on fluorescent Pseudomonads isolated from an agricultural soil. FEMS Micro Lett 161:29–35CrossRefGoogle Scholar
  4. Bowen TL, Union J, Tumbula DL, Whitman WB (1997) Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus. Gene 188:77–84PubMedCrossRefGoogle Scholar
  5. Brown HM (1990) Mode of action, crop selectivity and soil relations of the sulfonylurea herbicides. Pest Sci 29:263–281CrossRefGoogle Scholar
  6. Brown HM, Cotterman JC (1994) Recent advances in sulfonylurea herbicides. In: Stetter J (ed) Chemistry of plant protection volume 10: herbicides inhibiting branched chain amino acid biosynthesis. Springer Verlag, Berlin, pp 47–81Google Scholar
  7. Bugg T, Foght JM, Pickard MA, Gray MR (2000) Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a. Appl Env Micro 66:5387–5392CrossRefGoogle Scholar
  8. Burnet M, Hodgson B (1991) Differential effects of the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl on microorganisms. Arch Micro 155:521–525CrossRefGoogle Scholar
  9. Chipman D, Barak Z, Schloss JV (1998) Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases. Biochim Biophys Acta 1385:401–419PubMedGoogle Scholar
  10. De Felice M, Griffo G, Carmine T, Limauro D, Ricca E (1988) Detection of the acetolactate synthase isozymes I and III from Escherichia coli K-12. Meth Enz 166:241–244CrossRefGoogle Scholar
  11. De Felice M, Levinthal M, Iaccarino M, Guardiola J (1979) Growth inhibition as a consequence of antagonism between related amino acids: effect of valine in Escherichia coli K12. Micro Rev 43:42–58Google Scholar
  12. Duggleby RG, Pang SS (2000) Acetohydroxyacid synthase. J Biochem Mol Biol 33:1–36Google Scholar
  13. Epelbaum S, Chipman DM, Barak Z (1996) Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium. J Bact 178:1187–1196PubMedGoogle Scholar
  14. Epelbaum S, LaRossa RA, VanDyk TK, Elkayam T, Chipman DM, Barak Z (1998) Branched-chain amino acid biosynthesis in Salmonella typhimurium: a quantitative analysis. J Bact 180:4056–4067PubMedGoogle Scholar
  15. Forlani G, Mantelli M, Branzoni M, Nielsen E, Favilli F (1995) Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides. Plant Soil 176:243–253CrossRefGoogle Scholar
  16. Forlani G, Riccardi G, De Rossi E, De Felice M (1991) Biochemical evidence for multiple forms of acetohydroxy acid synthase in Spirulina platensis. Arch Micro 155:298–302CrossRefGoogle Scholar
  17. Gollop N, Damri B, Chipman DM, Barak Z (1990) Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms. J Bact 172:3444–3449PubMedGoogle Scholar
  18. Irvin RT, McAlister TJ, Costerton JW (1981) Tris(hydroxymethyl)aminomethane buffer modification of E. coli outer membrane permeability. J Bact 145:1397–1403PubMedGoogle Scholar
  19. Kim YR, Lee SE, Kim CM, Kim SY, Shin EK, Shin DH, Chung SS, Choy HE, Progulske-Fox A, Hillman JD, Handfield M, Rhee JH (2003) Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun 71:5461–5471PubMedCrossRefGoogle Scholar
  20. Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucl Acids Res 18:999–1005PubMedCrossRefGoogle Scholar
  21. LaRossa RA, Schloss JV (1984) The sulfonylurea herbicide sulfometuron-methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium. J Biol Chem 259:8753–8757PubMedGoogle Scholar
  22. LaRossa RA, Smulski DR (1984) ilvB-Encoded acetolactate synthase is resistant to the herbicide sulfometuron methyl. J Bact 160:391–394PubMedGoogle Scholar
  23. LaRossa RA, Van Dyk TK, Smulski DR (1987) Toxic accumulation of α-ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme acetolactate synthase in Salmonella typhimurium. J Bact 169:1372–1378PubMedGoogle Scholar
  24. Lawther R, Calhoun D, Gray J, Adams C, Hauser C, Hatfield G (1982) DNA sequence fine-structure analysis of ilvG (ilvG+) mutations of Escherichia coli K-12. J Bact 149:294–298PubMedGoogle Scholar
  25. Levitt G (1983) Sulfonylureas: new high potency herbicides. In: Miyamoto J, Kearney PC (eds) Pesticide chemistry: human welfare and the environment, vol. 1. Pergamon Press, Oxford, pp 243Google Scholar
  26. Macek J, Berden B (1994) The influence of primi- and triasulfuron herbicides on mycelial growth and sporulation of some Fusarium fungi. Acta Phyto Entomol Hung 29:371–375Google Scholar
  27. Methe BA, Nelson KE, Deming JW, Momen B, Melamud E, Zhang X, Moult J, Madupu R, Nelson WC, Dodson RJ, Brinkac LM, Daugherty SC, Durkin AS, DeBoy RT, Kolonay JF, Sullivan SA, Zhou L, Davidsen TM, Wu M, Huston AL, Lewis M, Weaver B, Weidman JF, Khouri H, Utterback TR, Feldblyum TV, Fraser CM (2005) The psychrophilic lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses. Proc Natl Acad Sci 102:10913–10918PubMedCrossRefGoogle Scholar
  28. Naqvi SM, Hawkins RH (1989) Responses and LC50 values for selected microcrustaceans exposed to Spartan, Malathion, Sonar, Weedtrine-D and Oust pesticides. Bull Env Cont Tox 43:386–393CrossRefGoogle Scholar
  29. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci 86:2766–2770PubMedCrossRefGoogle Scholar
  30. Page RDM (2001) Treeview., University of Glasgow, Glasgow
  31. Pang SS, Duggleby RG (1999) Expression, purification, characterization and reconstitution of the large and small subunits and yeast acetohydroxyacid synthase. Biochem 38:5222–5231CrossRefGoogle Scholar
  32. Porat I, Vinogradov M, Vyazmensky M, Lu CD, Chipman DM, Abdelal AT, Barak Z (2004) Cloning and characterization of acetohydroxyacid synthase from Bacillus stearothermophilus. J Bact 186:570–574PubMedCrossRefGoogle Scholar
  33. Powley CR, de Bernard PA (1998) Screening method for nine sulfonylurea herbicides in soil and water by liquid chromatography with ultraviolet detection. J Agric Food Chem 46:514–519PubMedCrossRefGoogle Scholar
  34. Sabater C, Carrasco JM (1997) Effects of chlorsulfuron on growth of three freshwater species of phytoplankton. Bull Env Cont Tox 58:807–813CrossRefGoogle Scholar
  35. Saitou N, Nei M (1987) The neigbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp E3–E4Google Scholar
  37. Schloss J, Van Dyk D, Vasta J, Kutny R (1985) Purification and properties of Salmonella typhimurium acetolactate synthase isozyme II from Escherichia coli HB101/pDU9. Biochem 24:4952–4959CrossRefGoogle Scholar
  38. Schloss JV (1990) Acetolactate synthase, mechanism of action and its herbicide binding site. Pest Sci 29:283–292CrossRefGoogle Scholar
  39. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  40. Van-Moppes D, Barak Z, Chipman DM, Gollop N (1989) An herbicide (sulfometuron-methyl) resistant mutant in Porphyridium (Rhodophyta). J Phycol 25:108–112CrossRefGoogle Scholar
  41. Yeates C, Gillings M (1998) Rapid purification of DNA from soil for molecular biodiversity analysis. Lett Appl Micro 27:49–53CrossRefGoogle Scholar
  42. Zohar Y, Einav M, Chipman DM, Barak Z (2003) Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones. Biochim Biophys Acta 1649:97–105PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.School of Molecular and Microbial BiosciencesUniversity of SydneySydneyAustralia
  2. 2.School of Civil Engineering and GeosciencesUniversity of Newcastle upon TyneNewcastle upon TyneUK

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