Physicochemical Studies on multi-drug Efflux Transporters of Bacteria

(1)分离到一株对阿霉素耐药的火山盐铁霉突变体，证实其耐药是由atp水解驱动的外排泵引起的。(2)火山芽胞杆菌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，并与ABC转运蛋白序列具有较高的相似性。(4)分离到一株对溴化乙啶具有高抗性的腺球菌（Enoterococcus hirae）突变体。通过以大肠杆菌为宿主细胞的“霰弹枪”克隆，获得了具有耐药性的165 rRNA因子。165 rRNA与耐药性有关的事实是一个新发现。(5)枯草芽孢杆菌ISW1214在葡萄糖存在下培养时，外排活性降低。当使用非代谢糖代替葡萄糖时，没有发现这种现象。详细分析表明，胞内果糖6-磷酸浓度与外排活性呈良好的负相关关系。(6)我们克隆了枯草芽孢杆菌ISW1214中调控外排转运蛋白表达的基因。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.