Parke D
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
J Bacteriol. 2000 Nov;182(21):6145-53. doi: 10.1128/JB.182.21.6145-6153.2000.
A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain of Agrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and beta-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 for Acinetobacter 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. tumefaciens for 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 and Acinetobacter sp. as well as of residues in other parts of the enzyme.
一种用于筛选影响芳香族化合物生物转化突变的正向选择方法方法方法应用于根癌农杆菌A348的突变菌株。测定了A348 pcaHGB基因的核苷酸序列，该基因编码二酚原儿茶酸分解代谢前两步中的原儿茶酸3,4-双加氧酶（PcaHG）和β-羧基-顺,顺-粘康酸环异构酶（PcaB）。将一个ω元件引入A348的pcaB基因，构建了ADO2077菌株。在存在可作为碳源的酚类化合物时，由于三羧酸中间体的积累，ADO2077的生长受到抑制。先前针对不动杆菌属描述的这种毒性效应，为上游分解代谢步骤所需基因中的抑制突变提供了有力的选择。通过监测pcaB中标记的丢失，与不动杆菌属的结果相比，可以确定缺失的形成极少。因此，三羧酸技巧本身似乎不会选择大的缺失突变。通过针对根癌农杆菌的pcaHG基因进行自发突变，证明了这种选择的有效性。对16株在pcaH或-G中携带假定的第二位点突变的菌株进行了序列分析。所有单点事件，其突变对多碱基缺失或异常模式没有特别的偏向：5个（-1）移码突变、1个（+1）移码突变、1个88 bp的串联重复、1个92 bp的缺失、1个无义突变和7个错义突变。PcaHG被认为是典型的铁内二醇双加氧酶。错义突变证实了从恶臭假单胞菌和不动杆菌属的PcaHG晶体结构推断出的活性位点氨基酸残基以及该酶其他部分残基的重要性。
