Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, Miyagi 981-8555, Japan.
Appl Environ Microbiol. 2011 Jun;77(12):4027-34. doi: 10.1128/AEM.00003-11. Epub 2011 Apr 29.
We previously isolated a mutant hypersensitive to L-alanyl-L-alanine from a non-L-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular L-alanine and a reduction in the L-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the L-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of L-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular L-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-L-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular L-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for L-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export.
我们之前从一个非代谢 L-丙氨酸的大肠杆菌菌株中分离出一种对 L-丙氨酰-L-丙氨酸敏感的突变体，并发现它缺乏可诱导的 L-丙氨酸输出系统。因此，与亲株相比，该突变体表现出显著的细胞内 L-丙氨酸积累和 L-丙氨酸输出率降低。当该突变体被用作克隆互补二肽敏感表型的基因（s）的宿主时，鉴定出两个未鉴定的基因 ygaW 和 ytfF 以及两个已鉴定的基因 yddG 和 yeaS。在突变体中过表达每个基因都会导致细胞内 L-丙氨酸水平降低，并在存在二肽时增强 L-丙氨酸的输出率，表明它们的产物作为 L-丙氨酸的输出物发挥作用。由于 ygaW 在鉴定的四个基因中对细胞内和细胞外 L-丙氨酸水平都有最显著的影响，我们在非代谢 L-丙氨酸的菌株中敲除了 ygaW 基因。由此产生的同源突变体显示出与通过化学诱变获得的二肽敏感突变体相同的细胞内和细胞外 L-丙氨酸水平。当每个基因在本身不分泌丙氨酸的野生型菌株中过表达时，只有 ygaW 基因赋予细胞分泌丙氨酸的能力。此外，在存在二肽的情况下，ygaW 基因的表达被诱导。基于这些结果，我们得出结论，YgaW 可能是大肠杆菌中 L-丙氨酸最相关的生理输出物，并提议将该基因重新命名为 alaE 用于 L-丙氨酸的输出。
