Abstract:
The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.
摘要:
五价有机砷化镓丝菌素(AST)是由根际细菌伯克霍尔德氏菌gladioliGSRB05合成的天然产物。AST是一种广谱抗生素,可有效对抗耐碳青霉烯类阴沟肠杆菌等人类病原体。它是抑制细菌谷氨酰胺合成酶的非蛋白氨基酸和谷氨酸模拟物。AST生物合成途径由三个基因簇组成,arsQML。ArsL催化合成还原的三价羟基虫草素(R-AST-OH),其被ArsM甲基化为AST的还原三价形式(R-AST)。在剑兰芽孢杆菌的培养基中,两种三价物种都表现为相应的五价砷化物,可能是由于空气中的氧化。ArsQ是一种外排通透酶,用于将AST或相关物种转运出细胞,但实际运输底物的化学性质尚不清楚。在这项研究中,剑兰芽孢杆菌arsQ在大肠杆菌中表达,并显示出对AST及其衍生物的抗性。大肠杆菌的细胞积累R-AST,表达arsQ的指数生长细胞吸收较少的R-AST。细胞表现出很少的五价形式的运输。运输与细胞能量无关,似乎是平衡的。ArsQ的同源性模型表明Ser320在底物结合位点中。S320A突变体表现出减少的R-AST-OH转运,表明它在ArsQ功能中起作用。ArsQ通透酶被认为是一种与能量无关的单向转运蛋白,负责将三价形式的AST从细胞中下坡运输。它在细胞外被氧化成抗生素的活性形式。