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Cloning, Expression and Characteristics of the Acetohydroxyacid Synthase (AHAS) Gene from Pseudomonas sp. L19(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2012 01
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Cloning, Expression and Characteristics of the Acetohydroxyacid Synthase (AHAS) Gene from Pseudomonas sp. L19
LANG Zhifei YU Xinyan HUANG Xing ZHANG Juan HE Jian
(1College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China)
(2Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China)
acetohydroxyacid synthase inhibitor herbicide-resistant acetohydroxyacid synthase (AHAS) Pseudomonas enzymatic characteristics
Q936 : Q78 : S482.4

Acetohydroxyacid synthase (AHAS) is the target of AHAS-inhibitor herbicides, such as sulfonylurea, imidazolinone, triazolopyrimidine sulfonamide and pyrimidinyl oxybenzoate. It was recognized that AHAS herbicide-resistant mutant has potential application in the construction of transgenetic herbicide-resistant crop and the development of new potent herbicidal compounds. In this study, an AHAS inhibitor-resistant bacterium L19 was isolated from farm soil treated with metsulfuron-methyl for many years. The strain was preliminarily identified as Pseudomonas sp. according to its morphological, physiological and biochemical properties and the homology analysis of its 16S rDNA sequence. The gene coding for acetohydroxyacid synthase was cloned from Pseudomonas sp. L19 by PCR technique. The AHAS regulatory subunit of Pseudomonas sp. L19 was identical to that of P. putida KT2440, whereas four different sites were found in the catalytic subunit: [Val 29→Ala (L19→KT2440), Pro93→Ser, Val 345→Ala, Pro 484→Arg]. The AHAS of Pseudomonas sp. L19 and P. putida KT2440 were over-expressed, purified and characterized. The AHAS of Pseudomonas sp. L19 showed multiple resistance to four types of AHAS-inhibitor herbicides. The resistance factors of rAHAS were 53.6-fold, 9.3-fold, 8.2-fold and 9.5-fold to the AHAS of P. putida KT2440 when metsulfuron-methyl, imazethapyr, flumetsulam and pyriminobac-methyl used as inhibitor, respectively. The specific activity, Kc for ThDP and Mg2+ of Pseudomonas sp. L19 AHAS were lower than that of P. putida KT2440 AHAS, while the Km value of Pseudomonas sp. L19 AHAS for pyruvate was approximately 1-fold higher than the corresponding value of P. putida KT2440. Fig 1, Ref 4, Ref 30


1 Bernasconi P, Woodworth AR, Rosen BA, Subramanian MV, Siehl DL. A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase. J Biol Chem, 1995, 270: 17381~17385
2 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248~254
3 Chang AK, Duggleby RG. Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: Characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants. Biochem J, 1998, 333: 765~777
4 Duggleby RG, Pang SS. Acetohydroxyacid synthase. J Biochem Mol Biol, 2000, 33: 1~36
5 Duggleby RG, Pang SS, Yu HQ, Guddat LW. Systematic characterization of mutations in yeast acetohydroxyacid synthase. Eur J Biochem, 2003, 270: 2895~2904
6 Falco SC, McDevitt RE, Chui CF, Hartnett ME, Knowlton S, Mauvais CJ, Smith JK, Mazur BJ. Engineering herbicide-resistant acetolactate synthase. Dev Ind Microbiol, 1989, 30: 187~194
7 Harms CT, Armour SL, DiMaio JJ, Middlesteadt LA, Murray D, Negrotto DV, Thompson-Taylor H, Weymann K, Montoya AL, Shillito RD, Jen GC. Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene. Mol Gen Genet, 1992, 233: 427~435
8 Hattori J, Brown D, Mourad G, Labbé H, Ouellet T, Sunohara G, Rutledge R, King J, Miki B. An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance. Mol Gen Genet, 1995, 246: 419~425
9 Hattori J, Rutledge R, Labbé H, Brown D, Sunohara G, Miki B. Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance. Mol Gen Genet, 1992, 232: 167~173
10 Haughn GW, Smith J, Mazur B, Somerville C. Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides. Mol Gen Genet, 1988, 211: 266~271
11 Hill CM, Duggleby RG. Mutagenesis of E. coli acetohydroxyacid synthase isozyme II and characterization of three herbicide-resistant forms. Biochem J, 1998, 335: 653~661
12 Huang X, He J, Sun XF, Sun JQ, Li YF, Shen JJ, Li SP. Characterization and molecular mechanism of a naturally occurring metsulfuron-methyl resistant strain of Pseudomonas aeruginosa. World J Microbiol Biotechnol, 2010, 26: 515~521
13 Ibdah M, Bar-Ilan A, Livnah O, Schloss JV, Barak Z, Chipman DM. Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry, 1996, 35: 16282~16291
14 Lee YT, Chang AK, Duggleby RG. Effect of mutagenesis at serine 653 of Arabidopsis thaliana acetohydroxyacid synthase on the sensitivity to imidazolinone and sulfonylurea herbicides. FEBS Lett, 1999, 452: 341~345
15 Lee KY, Townsend J, Tepperman J, Black M, Chui CF, Mazur B, Dunsmuir P, Bedbrook J. The molecular basis of sulfonylurea herbicide resistance in tobacco. EMBO J, 1988, 7: 1241~1248
16 Mourad G, King J. Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana. Planta, 1992, 188: 491~497
17 Pang SS, Duggleby RG, Guddat LW. Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors. J Mol Biol, 2002, 317: 249~262
18 Pang SS, Guddat LW, Duggleby RG. Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase. J Biol Chem, 2003, 278: 7639~7644
19 Rajasekaran K, Grula JW, Anderson DM. Selection and characterization of mutant cotton (Gossypium hirsutum L.) cell lines resistant to sulfonylurea and imidazolinone herbicides. Plant Sci, 1996, 119: 115~124
20 Sambrook J, Russell DW. Molecular cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory, 2001
21 Sathasvian K, Haughn GW, Murai N. Molecular basis of imidazolinone herbicide resistance in Arabidopsis thaliana var. columbia. Plant Physiol, 1991, 97: 1044~1050
22 Wang BZ, Guo P, Hang BJ, Li L, He J, Li SP. Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from sphingobium sp. strain JZ-1 and characterization of the gene product. Appl Environ Microbiol, 2009, 75: 5496~5500
23 Yoon TY, Chung SM, Chang SI, Yoon MY, Hahn TR, Choia JD. Roles of lysine 219 and 255 residues in tobacco acetolactate synthase. Biochem Biophys Res Commun, 2002, 293: 433~439
24 Yadav N, McDevitt RE, Benard S, Falco SC. Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl. Proc Natl Acad Sci, 1986, 83: 4418~4422
25 Xie Q, Jiménez A. Molecular cloning of a novel allele of SMR1 which determines sulfometuron methyl resistance in Saccharomyces cerevisiae. FEMS Microbiol Lett, 1996, 137: 165~168
26 Dong XZ (东秀珠), Cai MY (蔡妙英). 常见细菌系统鉴定手册. Beijing, China: Science Press (北京: 科学出版社), 2001. 370~410
27 Fang GH (房桂华), Li LT (李联泰), Li R (李荣), Zhu JC (朱建春), Hong Q (洪青), Li SP (李顺鹏). Isolation and characterization of a malachite green-degrader Arthrobacter sp. M6. Chin J Appl Environ Biol (应用与环境生物学报), 2010, 16 (4): 581~584
28 He LJ (何丽娟), Li ZH (李正华), Hong Q (洪青), Li SP (李顺鹏). Characterization of a phenanthrene-degrading strain and cloning of degradation-related gene. Chin J Appl Environ Biol (应用与环境生物学报), 2009, 15 (5): 682~685
29 Shen JJ (沈晶晶), Huang X (黄星), Yu XY (俞欣妍), Li SP (李顺鹏), He J (何健). Isolation of two bacterial strains resistant to acetohydroxyacid synthase (AHAS) inhibitor herbicides and cloning of AHAS genes ilvIH. Chin J Appl Environ Biol (应用与环境生物学报), 2008, 14 (5): 663~667
30 Sun XF (孙笑非), Huang X (黄星), 陈博, Li SP (李顺鹏), He J (何健). Isolation of a metsulfuron-methyl-resistant bacteria and cloning and expression of the acetohydroxyacid synthase genes ilvIH. Acta Microbiol Sin (微生物学报), 2008, 48 (11): 1493~1498


Last Update: 2012-02-29