GENETIC REGULATION OF HEAT LABILE ENTEROTOXIN SYNTHESIS IN E COLI

产肠毒素大肠杆菌（BTEC）是引起人和农业动物腹泻的主要病原之一。对LT的遗传调控的研究将有望使人们更清楚地了解ETEC菌株如何与宿主相互作用并导致疾病，以及改进疫苗的开发。LT获得的信息可能为其他大肠杆菌引起的其他肠道疾病提供有用的信息。大肠杆菌和志贺氏菌的毒力因子也受相同的调节蛋白H-NS的温度调节。电泳迁移率变动分析（EMSA）和体外转录实验将用于确定H-NS是否通过直接结合LT操纵子DNA（特别是下游调节元件（DRE））来介导其对LT表达的温度控制。LT操纵子DNA的进一步表征将通过分离具有改变的LT表达的突变体来实现，所述突变体已经通过进行化学诱变然后进行阿糖胞苷定向诱变而获得。H-NS与突变DNA的相互作用将通过EMSA和体外转录（如果合适）进行评价。几个H-NS敏感的毒力因子已被证明有它们的表达改变的DNA拓扑结构的变化。通过评价促旋酶抑制剂的作用，通过使用在编码顶端异构酶1和促旋酶的两个亚基的基因中具有突变的菌株，以及通过评价已知改变超螺旋的温度以外的环境条件，将研究超螺旋对LT表达的影响。由于许多H-NS结合位点已显示出固有曲率，因此还将研究LT操纵子DNA的这种特性。将对LT DRE进行研究，以观察该DNA片段在低温下是否通过琼脂糖或丙烯酰胺凝胶发生异常移动。如果发现LT DRE是弯曲的，抗生素偏端霉素可用于确定这种弯曲是否是H-NS或其他调节蛋白（尚未确定）结合所必需的。

Enterotoxinogenic E.coli (BTEC) which produce heat-labile enterotoxin (LT are among the leading causes of diarrheal disease in man and agricultural animals. Investigation of the genetic regulation of LT will lead hopefully lead to a clearer understanding of how ETEC strains interacts with the host and causes disease as well as to the development of improved vaccines. Information gained with LT may provide information that is usefiil for other enteric diseases caused by other E. coli strains and Shigella whose virulence factors are also thermoregulated by the same regulatory protein, H-NS. Electrophoretic mobility shift assays (EMSA) and in vitro transcription experiments will be used to determine if H-NS mediates its temperature control on LT expression by directly binding to LT operon DNA, specifically the downnstream regulatory element (DRE). Further characterization of the LToperon DNA will be achieved by isolating mutants with altered LT expression that have been obtained by carrying out chemical mutagenesis and then oligonucleotide-directed mutagenesis. H- NS interactions with the mutated DNA will be evaluated by EMSA and in vitro transcription if appropriate. Several H-NS-sensitive virulence factors have been shown to have their expression alterated by changes in DNA topolgy. Supercoiling effects on LT expression will be investigated by evaluating the effects of gyrase inhibitors , by using strains that have mutations in the genes encoding top oisomerase l and the two subunits of gyrase, and by evaluating environmental conditions beside temperature which are known to alter supercoiling Since many H-NS binding sites have been shown to display intrinsic curvature, this characteristic of LT operon DNA will also be investigated. The LT DRE will be studied to see if this DNA fragment moves aberrantly through agarose or acrylamide gels at low temperatures. If the LT DRE is found to be curved, the antibiotic distamycin can be used to determine if this curvature is essential for binding of H-NS or other regulatory proteins (yet to be determined).