Physicochemical Studies on multi-drug Efflux Transporters of Bacteria

(1)分离到一株沃氏盐藻（Haloferax volcano ii）的阿霉素抗性突变株，证明其抗性是由ATP水解驱动的外排泵引起的。(2)火山血吸虫的野生型细胞表达这种外排泵，当细胞在过量的葡萄糖和氨基酸存在下培养时，其活性增加。(3)确定了可能编码该外排泵的部分DNA序列，并且与ABC转运蛋白的序列具有高度相似性。(4)从嗜热链球菌中分离到一株对溴化乙锭具有高抗性的突变株。通过以大肠杆菌为接种细胞的鸟枪克隆，发现了165 rRNA基因是产生抗性的一个因子，这是一个新的发现。(5)枯草芽孢杆菌ISW 1214在葡萄糖存在下外排活性降低，用非代谢糖代替葡萄糖时，这种现象没有出现，详细的分析表明，胞内6-磷酸果糖浓度与外排活性呈良好的负相关。(6)我们克隆了编码调节蛋白的基因，该蛋白控制枯草杆菌ISW 1214中外排转运蛋白的表达。有趣的是，该基因的一部分与己糖激酶和葡萄糖代谢酶的一部分有很高的相似性。

(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.