Physicochemical Studies on multi-drug Efflux Transporters of Bacteria

(1)分离到耐多柔比星突变株Haloferax Volcanii，证实其抗性是由ATP-水解酶驱动的外排泵引起的。(2)火山嗜盐杆菌野生型细胞表达该外排泵，当细胞在过量的葡萄糖和氨基酸存在下培养时，其活性增强。(3)测定了编码该外排泵的部分DNA序列，与ABC转运蛋白有很高的相似性。(4)分离到一株对溴化乙啶具有高抗性的Hirae绿球藻突变体。利用大肠杆菌作为HAST细胞进行枪击克隆，获得了一个与抗性有关的因子为165rRNA，这是一个新的发现。(5)枯草芽孢杆菌ISW1214在葡萄糖的存在下外排活性降低，当用非金属糖代替葡萄糖时，没有发现这种现象。详细分析表明，胞内6-磷酸果糖浓度与外排活性呈良好的负相关。(6)克隆了枯草芽孢杆菌ISW1214外排转运蛋白的调控蛋白基因。有趣的是，该基因的一部分与己糖激酶的一部分以及葡萄糖代谢酶的一部分具有很高的相似性。

(1) A mutant of Haloferax volcanii resistant for doxorubicin was isolated and its resistance was proved to be caused by an efflux pump driven by ATP-hydrolysis.(2) Wild-type cells of H.volcanii expressed this efflux pump, and its activity increased when cells were cultured in the presence of excess qlucose and amino acids.(3) Partial DNA sequence was determined which may code this efflux pump, and had high similarity with that of ABC transporters.(4) A mutant of Enoterococcus hirae was isolated which showed high resistance against ethidium bromide. By shot-gun cloning using E.coli as a hast cell, a factor conferring the resistance was 165 rRNA.The fact that 165 rRNA is related to the resistance is a new finding.(5) An efflux activity of Bacillus subtilis ISW1214 was decreased when cultured in the presence of glucose.When non-metacolic sugars were used instead of glucose, this phenomena were not found. Detailed analysis showed a good negative correlation between intracellular concentration of fructose 6-phosphate and the efflux activity.(6) We cloned a gene coding a requlator protein that controls an expression of the efflux transporter in Bacillus subtilis ISW1214. It is quite interesting that a part of this gene has high similarity with a part of hexokinase, and enzyme of qlucose metabolism.