Degradative Plasmids: Comparative Regulatory Sequences

几十年来，化学工业一直在生产高度氯化的化合物，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，但高度氯化的化合物是相当持久的，这是由于自然微生物区系的有限分解，这反映了这些微生物中没有针对此类化合物的降解基因。因此，我们一直有兴趣了解编码简单氯化化合物降解的新基因在自然界中是如何进化的，希望这种了解可能有助于我们在实验室环境中加速针对高氯化化合物的进化过程。恶臭假单胞菌主要菌株pAC27上编码3-氯代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被定位为操纵子(CLC操纵子)，类似于恶臭假单胞菌降解天然母体化合物儿茶酚(猫操纵子)的另一组染色体基因。这项资助旨在确定调控基因CatR和CLcr的产物如何与两个操纵子catBC和clcABD的操纵子/启动子区域结合，以允许这两个操纵子在诱导剂顺式、顺式和2氯-顺式、顺式-弱酸存在的情况下转录。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显示出广泛的同源性，因此了解包括氯化化合物作为诱导剂的调控途径的基因是如何从编码类似的非氯化化合物异化的祖先基因进化而来的是很重要的。在非氯代和氯代变异体存在的情况下，涉及与CatR和CLcR结合的catBC和clcABD操纵子/启动子区域中的特定核苷酸的分子细节，以及参与操纵子/启动子区域中特定碱基接触的调节蛋白CatR和CLcR的氨基酸，将提供相当大的洞察力，以了解监管系统在应对氯化化合物排放方面的演变机制。

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.