GENETIC REGULATION OF HEAT LABILE ENTEROTOXIN SYNTHESIS IN E COLI

产肠毒素的大肠杆菌（BTEC）产生不耐热的肠毒素（LT），是人类和农业动物腹泻病的主要原因之一。对LT基因调控的研究有望使我们更清楚地了解ETEC菌株如何与宿主相互作用并导致疾病，以及开发改进的疫苗。通过LT获得的信息可能为其他大肠杆菌菌株和志贺氏菌引起的其他肠道疾病提供有用的信息，这些大肠杆菌菌株和志贺氏菌的毒力因子也受相同的调节蛋白H-NS的热调节。电泳迁移迁移试验（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，特别是下游调控元件（DRE）介导其温度控制LT表达。LToperon DNA的进一步表征将通过分离通过化学诱变和寡核苷酸定向诱变获得的LT表达改变的突变体来实现。H- NS与突变DNA的相互作用将通过EMSA和体外转录（如果合适）进行评估。一些对h - ns敏感的毒力因子已被证明其表达随DNA拓扑结构的变化而改变。超卷曲对LT表达的影响将通过评估回转酶抑制剂的作用，通过使用编码顶部异构酶1和回转酶两个亚基的基因突变的菌株，以及通过评估已知改变超卷曲的温度以外的环境条件来研究。由于许多H-NS结合位点已显示出固有曲率，因此也将研究LT操纵子DNA的这一特性。研究人员将研究LT DRE，以确定该DNA片段在低温下是否会在琼脂糖或丙烯酰胺凝胶中异常移动。如果发现LT DRE是弯曲的，抗生素去霉素可以用来确定这种弯曲是否是H-NS或其他调节蛋白结合所必需的（尚待确定）。

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).