Degradative Plasmids: Comparative Regulatory Sequences

几十年来，化学工业一直在制造高氯化化合物，导致它们释放到环境中。虽然简单的氯化化合物是可生物降解的，但由于天然微生物群的分解有限，高氯化化合物具有相当的持久性，这反映了这些微生物中缺乏针对此类化合物的降解基因。因此，我们对了解编码简单氯化化合物降解的新基因如何在自然界中进化很感兴趣，希望这种理解可以帮助我们加速在实验室环境中对抗高氯化化合物的进化过程。编码3氯儿茶酚降解酶的三个结构基因和一个正向调节这三个结构基因表达的调控基因，已经被定位在一个主要的腐臭假单胞菌的质粒pAC27上，作为一个操纵子（clc操纵子），类似于另一组允许腐臭假单胞菌降解天然亲本化合物的染色体基因。儿茶酚（猫的操纵子）。该资助旨在确定调节基因catR和clcR的产物如何与两个操纵子catBC和clcABD的操作子/启动子区域结合，从而允许这两个操纵子在诱导剂cis， cis-muconate和2 - chloro-cis， cis-muconate存在的情况下转录。由于这两个操纵子的遗传组织非常相似，并且这两个操纵子的单个基因表现出广泛的同源性，因此了解调控途径的基因（包括氯化化合物作为诱导剂）如何从编码类似非氯化化合物异化的祖先基因进化而来是很重要的。catBC和clcABD操作子/启动子区域中参与与CatR和ClcR结合的特定核苷酸的分子细节，以及参与操作子/启动子区域中特定碱基接触的调节蛋白CatR和ClcR的氨基酸。将提供相当大的见解关于进化机制的调控系统，以响应氯化合物的释放。

Highly chlorinated compounds have been manufactured by the chemical industry for several decades now, resulting in their release into the environment. While simple chlorinated compounds are biodegradable, highly chlorinated compounds are quite persistent due to limited breakdown by natural microflora which reflects the absence of degradative genes against such compounds in these microorganisms. We have thus been interested in understanding how new genes encoding degradation of simple chlorinated compounds evolve in nature with the hope that such understanding may help us to accelerate the process of evolution in a laboratory setting against highly chlorinated compounds. Three structural genes encoding the degradative enzymes of 3 chlorocatechol and a regulatory gene that regulates positively the expression of the three structural genes, have been mapped on a plasmid pAC27 in a main strain of Pseudomonas putida as an operon ( the clc operon), similar to another set of chromosomal genes that allow degradation by P. putida of the natural parent compound, catechol ( the cat operon). This grant seeks to determine how the products of the regulatory genes catR and clcR bind with the operator/promoter regions of the two operons catBC and clcABD to allow transcription of these two operons in presence of the inducers cis, cis-muconate and 2 chloro-cis, cis-muconate. Since the genetic organization of the two operons is quite similar, and the individual genes of these two operons demonstrate extensive homology, it is important to understand how genes for a regulated pathway, which includes a chlorinated compound as an inducer, evolve from ancestral genes that encode dissimilation of an analogous non-chlorinated compound. The molecular details of specific nucleotides in the catBC and clcABD operator/promoter regions that are involved in the binding with CatR and ClcR in presence of the non-chlorinated and chlorinated muconates as well as the amino acids of the regulatory proteins CatR and ClcR that are involved in specific base contacts in the operator/promoter region, will provide considerable insight regarding the mechanism of evolution of regulatory systems in response to the release of chlorinated compounds.