Thöny-Meyer L, Künzler P
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
J Bacteriol. 1996 Nov;178(21):6166-72. doi: 10.1128/jb.178.21.6166-6172.1996.
The Bradyrhizobium japonicum acnA gene encoding the tricarboxylic acid cycle enzyme aconitase was cloned and characterized. The gene was mapped immediately upstream of the cytochrome c biogenesis gene cycV and found to be transcribed in the opposite direction. The nucleotide sequence of acnA was determined; the derived amino acid sequence shared a significant similarity with bacterial aconitases and with the human iron-responsive-element-binding protein. The level of expression of the acnA gene under aerobic growth conditions was 10-fold higher than that under anaerobic conditions. The start of transcription was mapped by primer extension experiments, and the putative promoter was found to contain a typical -10 but no -35 consensus sequence for a sigma70-type RNA polymerase. A 5' deletion removing all but 19 nucleotides upstream of the start of transcription completely abolished gene expression. An acnA mutant was constructed by gene disruption, and the mutant phenotype was characterized. Growth of the mutant was severely affected and could not be corrected by the addition of glutamate as a supplement. Although aconitase activity in free-living cells was decreased by more than 70%, the ability of the mutant to establish an effective root nodule symbiosis with soybean plants was not affected. This suggested either the existence of a second aconitase or the compensation for the mutant defect by symbiosis-specific metabolites synthesized in the root nodules.
编码三羧酸循环酶乌头酸酶的日本慢生根瘤菌acnA基因被克隆并进行了特性分析。该基因定位于细胞色素c生物合成基因cycV的紧邻上游，且发现其转录方向相反。测定了acnA的核苷酸序列；推导的氨基酸序列与细菌乌头酸酶以及人类铁反应元件结合蛋白具有显著相似性。在有氧生长条件下acnA基因的表达水平比无氧条件下高10倍。通过引物延伸实验确定了转录起始位点，发现推定的启动子含有典型的-10序列，但没有sigma70型RNA聚合酶的-35共有序列。一个5'缺失突变体去除了转录起始位点上游除19个核苷酸以外的所有序列，完全消除了基因表达。通过基因破坏构建了一个acnA突变体，并对其突变表型进行了表征。突变体的生长受到严重影响，添加谷氨酸作为补充剂也无法纠正。尽管自由生活细胞中的乌头酸酶活性降低了70%以上，但该突变体与大豆植株建立有效根瘤共生的能力并未受到影响。这表明要么存在第二种乌头酸酶，要么根瘤中合成的共生特异性代谢产物对突变体缺陷起到了补偿作用。
