Andrade A C, Van Nistelrooy J G, Peery R B, Skatrud P L, De Waard M A
Laboratory of Phytopathology, Wageningen University, The Netherlands.
Mol Gen Genet. 2000 Jul;263(6):966-77. doi: 10.1007/pl00008697.
This paper describes the characterization of atrC and atrD (ABC transporters C and D), two novel ABC transporter-encoding genes from the filamentous fungus Aspergillus nidulans, and provides evidence for the involvement of atrD in multidrug transport and antibiotic production. BLAST analysis of the deduced amino acid sequences of AtrCp and AtrDp reveals high homology to ABC transporter proteins of the P-glycoprotein cluster. AtrDp shows a particularly high degree of identity to the amino acid sequence of Afu Mdr1p, a previously characterized ABC transporter from the human pathogen A. fumigatus. Northern analysis demonstrates an increase in transcript levels of atrC and atrD in fungal germlings upon treatment with natural toxic compounds and xenobiotics. The atrC gene has a high constitutive level of expression relative to attrD, which suggests its involvement in a metabolic function. Single knock-out mutants for atrC and atrD were generated by gene replacement using pyrG from A. oryzae as a selectable marker. DeltatrD mutants display a hypersensitive phenotype to compounds such as cycloheximide, the cyclosporin derivative PSC 833, nigericin and valinomycin, indicating that AtrDp is involved in protection against cytotoxic compounds. Energy-dependent efflux of the azole-related fungicide fenarimol is inhibited by substrates of AtrDp (e.g. PSC 833, nigericin and valinomycin), suggesting that AtrDp plays a role in efflux of this fungicide. Most interestingly, (delta)atrD mutants display a decrease in penicillin production, measured indirectly as antimicrobial activity against Micrococcus luteus. These results suggest that ABC transporters may be involved in secretion of penicillin from fungal cells.
本文描述了来自丝状真菌构巢曲霉的两个新的ABC转运蛋白编码基因atrC和atrD（ABC转运蛋白C和D）的特征，并提供了atrD参与多药转运和抗生素生产的证据。对AtrCp和AtrDp推导的氨基酸序列进行的BLAST分析显示，它们与P-糖蛋白簇的ABC转运蛋白具有高度同源性。AtrDp与来自人类病原体烟曲霉的先前鉴定的ABC转运蛋白Afu Mdr1p的氨基酸序列具有特别高的同一性。Northern分析表明，在用天然有毒化合物和外源性物质处理后，真菌幼芽中atrC和atrD的转录水平增加。相对于atrD，atrC基因具有较高的组成型表达水平，这表明它参与了代谢功能。使用来自米曲霉的pyrG作为选择标记，通过基因替换产生了atrC和atrD的单敲除突变体。DeltatrD突变体对诸如环己酰亚胺、环孢菌素衍生物PSC 833、尼日利亚菌素和缬氨霉素等化合物表现出超敏表型，表明AtrDp参与了对细胞毒性化合物的保护。AtrDp的底物（如PSC 833、尼日利亚菌素和缬氨霉素）可抑制与唑类相关的杀菌剂苯菌灵的能量依赖性外排，这表明AtrDp在这种杀菌剂的外排中起作用。最有趣的是，（delta）atrD突变体的青霉素产量下降，通过对藤黄微球菌的抗菌活性间接测量。这些结果表明，ABC转运蛋白可能参与了青霉素从真菌细胞中的分泌。
