Regulation of Acinetobacter calcoaceticus benzoate degradation

9507393土壤细菌Acinetobacter calcoaceticus对Neidle Benzoate的降解为研究芳香族化合物的降解规律提供了理想的体系。了解控制环境中大量芳香族化合物降解的因素对于生物修复的成功至关重要，因为许多污染物是容易被细菌降解的芳香族化合物的结构类似物。与苯甲酸盐结构相似的有毒污染物包括氯代苯甲酸盐、苯、甲苯、乙苯和二甲苯。在自然界中，细菌通过有限数量的分解代谢途径汇集许多底物，因此，可以从对几种代表性途径的深入研究中得出概括。这些代表性途径中最具特征的是B-酮己二酸途径，苯甲酸酯以及许多不同的芳香族化合物可以通过该途径被A降解。醋酸钙 这些研究表征了一种新的调控电路，该电路控制至少14个A的表达。将苯甲酸盐转化为三羧酸循环中间体所需的醋酸钙菌Ben和Cat基因。BenM和CatM都是LysR型转录激活因子，其响应于诱导剂顺式，顺式-粘康酸，苯甲酸降解的中间体。此外，BenM，而不是CatM，可以响应苯甲酸作为诱导剂，即使这种化合物在结构上不同于粘康酸。该系统的独特之处在于每个激活剂控制两个非连锁基因（catA和catB）表达的能力，每个基因都响应于粘康酸。BenM/CatM系统是独特的：没有其他两个同源的LysR型蛋白质调节相同的基因以响应相同的诱导物的例子。 通过确定CatM和BenM的独特和相互作用，该系统的新功能可以用于深入了解LysR型转录激活的更一般的方面，LysR型转录激活是细菌基因调控的最常见和最重要的手段之一。这项工作的目的是确定如何BenM和CatM都控制catA和catB的表达，并测试的假设，BenM，而不是CatM，调节苯甲酸盐的ben基因。为了满足这些目标，CatM和BenM蛋白将被纯化并用于蛋白质-DNA结合相互作用和蛋白质-诱导剂相互作用的研究。ben和cat转录本的分析以及转录因子基因融合将用于表征转录控制并确定诱导物特异性。第三个目标是表征多种芳香族化合物的同时代谢。与乔治盖恩斯博士（Isogenetics inc.，芝加哥，IL）的研究发现，当存在多种芳族碳源时，粘康酸酯参与特定化合物的优先降解。这些NMR技术将用于表征这种现象，并将阐明粘康酸盐，CatM和BenM在优先碳源利用中的具体作用。 在环境中，总是存在多种碳源，控制降解偏好的因素对于确定哪些化合物将自然降解以及哪些化合物将作为污染物保留至关重要。对A.醋酸钙应该是一般土壤细菌所使用的那些的代表。株供试菌可以降解多种化合物的醋酸钙菌已经从大多数天然和污染的环境中分离出来。它们与假单胞菌和变形菌门伽马亚门的其他革兰氏阴性成员密切相关，这些成员主要负责芳香族化合物的有氧降解。用于这些研究的实验室菌株自然能够通过DNA转化，从而促进遗传研究。遗传“工具”已经为A.这些系统已经为β-酮己二酸途径的生物化学、生理学和遗传组织提供了大量信息。以前的研究提供了必要的背景，这些调查的ben和猫基因，这将有助于更好地了解细菌的调控机制和一个合理的方法对生物修复。 ***

9507393 Neidle Benzoate degradation by the soil bacterium Acinetobacter calcoaceticus provides an ideal system for investigating the regulation of aromatic compound degradation. An understanding of the factors controlling the degradation of the vast number of aromatic compounds in the environment is central to the success of bioremediation, since many pollutants are structural analogs of aromatic compounds that are readily degraded by bacteria. Toxic pollutants similar in structure to benzoate include chlorinated benzoates, benzene, toluene, ethyl benzene and xylenes. In nature, bacteria funnel many substrates through a limited number of catabolic pathways, and, therefore, generalizations can be drawn from in-depth studies of several representative pathways. The best-characterized of these representative pathways is the B-ketoadipate pathway, the route by which benzoate as well as many diverse aromatic compounds can be degraded by A. calcoaceticus. These studies are characterizing a novel regulatory circuit governing the expression of at least 14 A. calcoaceticus ben and cat genes needed for the conversion of benzoate to tricarboxylic acid cycle intermediates. BenM and CatM are both LysR-type transcriptional activators that respond to the inducer cis,cis-muconate, an intermediate in the degradation of benzoate. In addition, BenM, but not CatM, can respond to benzoate as an inducer even though this compound is structurally unlike muconate. The distinctive aspect of this system is the ability of each activator to control the expression of two unlinked genes, catA and catB, each in response to muconate. The BenM/CatM system is unique: there are no other examples of two homologous LysR-type proteins regulating the same genes in response to the same inducer. By determining the distinct and interactive roles of CatM and BenM, the novel features of this system can be used to gain insights into more general aspects of LysR-type transcriptional activation, one of the most common and important means o f bacterial gene regulation. The objectives of this work are to determine how BenM and CatM both control catA and catB expression, and to test the hypothesis that BenM, but not CatM, regulates the ben genes in response to benzoate. In order to meet these objectives, the CatM and BenM proteins will be purified and used in studies of protein-DNA binding interactions and protein-inducer interactions. Analyses of the ben and cat transcripts, as well as reporter-gene fusions will be used to characterize transcriptional controls and to determine inducer specificities. A third objective is to characterize the simultaneous metabolism of multiple aromatic compounds. The use of novel Nuclear Magnetic Resonance (NMR) techniques, in collaboration with Dr. George Gaines (Isogenetics inc., Chicago, IL) led to the discovery that muconate is involved in the preferential degradation of specific compounds when multiple aromatic carbon sources are present. These NMR techniques will be used to characterize this phenomenon, and the specific roles of muconate, CatM and BenM in preferential carbon source utilization will be clarified. %%% In the environment, where multiple carbon sources are always present, factors controlling degradative preferences are critical in determining which compounds will be naturally degraded and which will remain as pollutants. The regulatory controls of A. calcoaceticus should be representative of those used by soil bacteria in general. Strains of A. calcoaceticus which can degrade a wide variety of compounds have been isolated from most natural and polluted environments. They are closely related to pseudomonads and other Gram-negative members of the gamma subdivision of the Proteobacteria which are largely responsible for the aerobic degradation of aromatic compounds. The laboratory strains to be used in these studies are naturally competent for transformation by DNA, thereby facilitating genetic investigations. Genetic "tools" have been well-developed for A. calcoaceticus studies, and these systems have contributed to a significant amount of information on the biochemistry, physiology and genetic organization of the B-ketoadipate pathway. Previous studies provide the necessary background for these investigations of the ben and cat genes which will contribute both to a better understanding of bacterial regulatory mechanisms and to a rational approach towards bioremediation. ***