Nelson Darryl R, Duxbury Trevor
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW, 2006, Australia.
Antonie Van Leeuwenhoek. 2008 Jan-Feb;93(1-2):123-32. doi: 10.1007/s10482-007-9186-y. Epub 2007 Jul 11.
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 approximately 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.
大多数细菌都拥有乙酰羟酸合酶，该酶用于生产支链氨基酸。肠道细菌含有几种适合不同条件的同工酶，但土壤细菌中乙酰羟酸合酶的分布情况在很大程度上尚不清楚。生长实验证实，大肠杆菌、鼠伤寒沙门氏菌血清型鼠伤寒菌和产气肠杆菌含有乙酰羟酸合酶的同工酶，这使得这些细菌能够在缬氨酸（会对乙酰羟酸合酶I产生反馈抑制）或磺酰脲类除草剂嗪草酸甲酯（会抑制乙酰羟酸合酶II）存在的情况下生长，不过在后一种情况下生长会出现轻微的滞后期。几种常见的土壤分离菌受到嗪草酸甲酯的抑制，但荧光假单胞菌和红平红球菌不受嗪草酸甲酯和缬氨酸任何组合的抑制。当27株纯培养的分离细菌中有21株受到嗪草酸甲酯抑制时，揭示了土壤中对磺酰脲类敏感的乙酰羟酸合酶的程度，添加异亮氨酸和/或缬氨酸可在19例中逆转这种效应。根据现有序列数据设计引物，靶向编码乙酰羟酸合酶大亚基（ilvB、ilvG和ilvI）的基因，随后在枯草芽孢杆菌、球形节杆菌、大肠杆菌和肠炎沙门氏菌中扩增出一个约355 bp的片段。这些引物被用于构建一个来自农业土壤的小序列克隆文库。系统发育分析显示出显著的序列变异，但所有19个氨基酸序列与包括变形菌门和放线菌门在内的几个门中已发表的大亚基乙酰羟酸合酶氨基酸序列关系最为密切。结果表明，大多数土壤微生物仅含有一种对磺酰脲类除草剂敏感的功能性乙酰羟酸合酶。
