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首页> 外文期刊>Journal of bacteriology >Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds in Agrobacterium tumefaciens: Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene
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Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds in Agrobacterium tumefaciens: Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene

机译：影响根癌土壤杆菌中芳香族化合物生物转化的突变的正选择：原儿茶酸3,4-双加氧酶基因的自发突变分析

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摘要

A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain ofAgrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and β-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described forAcinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciensfor spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (?1) frameshifts, one (+1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG from Pseudomonas putida andAcinetobacter sp. as well as of residues in other parts of the enzyme.
机译：将影响芳香族化合物生物转化的突变的正选择方法应用于根癌农杆菌A348的突变菌株。 A348 pcaHGB 基因的核苷酸序列，编码原儿茶酸3,4-双加氧酶（PcaHG）和β-羧基-顺式，顺式-粘液酸环异构酶（PcaB）。确定了二酚原儿茶酸酯分解代谢的前两个步骤。在A348的 pcaB 基因中引入了一个ω元素，从而产生了ADO2077菌株。在可以用作碳源的酚类化合物的存在下，由于三羧酸盐中间体的积累，ADO2077的生长受到抑制。如前所述，不动杆菌属的毒性作用为上游分解代谢步骤所需的基因中的抑制子突变提供了有力的选择。通过监测 pcaB 中标记的丢失，可以确定与不动杆菌 sp获得的结果相比，缺失的形成是最小的。因此，三羧酸trick俩本身似乎并未选择大的缺失突变。通过针对 A的 pcaHG 基因证明了选择的力量。 tumefaciens 用于自发突变。对16个在 pcaH 或- G 中带有假定的第二位点突变的菌株进行序列分析。所有的单点事件及其突变均未显示出对多碱基缺失或异常模式的特殊偏向：五（？1）移码，一（+1）移码，一串重复的88 bp，一串删除的92 bp，一个无意义的突变，和七个错义突变。 PcaHG被认为是原型铁内二醇双加氧酶。错义突变可证实由恶臭假单胞菌和不动杆菌属的PcaHG晶体结构推导的活性位点氨基酸残基的重要性。以及酶其他部分的残基。

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