Regulation of Acinetobacter calcoaceticus benzoate degradation

9507393土壤细菌醋酸钙不动杆菌对苯甲酸酯的降解为研究芳香族化合物的降解规律提供了一个理想的体系。了解控制环境中大量芳香族化合物降解的因素是生物修复成功的关键，因为许多污染物是芳香族化合物的结构类似物，很容易被细菌降解。在结构上与苯甲酸盐相似的有毒污染物包括氯代苯甲酸酯、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输送许多底物，因此，通过对几种典型途径的深入研究可以得出结论。这些具有代表性的途径中最具代表性的是B-酮基己二酸途径，通过该途径，苯甲酸酯和许多不同的芳香族化合物可以被醋酸钙降解。这些研究描述了一种新的调控电路，控制着至少14个醋酸钙和CAT基因的表达，这些基因是将苯甲酸转化为三羧酸循环中间体所需的。BenM和CATM都是LysR型转录激活剂，它们对苯甲酸降解的中间产物顺式-松果酸做出反应。此外，苯丙胺，但不是CATM，可以作为诱导剂对苯甲酸作出反应，尽管这种化合物在结构上不同于变形酸。这个系统的独特之处在于，每个激活子都有能力控制两个不相连的基因的表达，每个基因都对互锥酸做出反应。BenM/CATM系统是独一无二的：没有其他例子表明，两个同源的LysR型蛋白调节相同的基因，以响应相同的诱导剂。通过确定CATM和BenM的不同和相互作用的角色，该系统的新特征可以用来深入了解LysR型转录激活的更一般方面，LysR型转录激活是细菌基因调控最常见和最重要的手段之一。本工作的目的是确定BenM和CATM如何控制CATA和CatB的表达，并检验假设，即BenM，而不是CATM，调节BEN基因对苯甲酸的反应。为了达到这些目标，CATM和BenM蛋白将被提纯，并用于蛋白质-DNA结合作用和蛋白质-诱导剂相互作用的研究。对Ben和CAT转录本以及报告基因融合的分析将用于表征转录控制和确定诱导剂的特异性。第三个目标是表征多种芳香族化合物的同时代谢。与伊利诺伊州芝加哥的George Gaines博士合作，使用新的核磁共振(核磁共振)技术发现，当存在多种芳香族碳源时，Muconate参与特定化合物的优先降解。这些核磁共振技术将被用来表征这一现象，并将阐明Muconate、CATM和BenM在优先利用碳源中的具体作用。在总是存在多种碳源的环境中，控制降解偏好的因素在决定哪些化合物将被自然降解以及哪些将作为污染物保留下来方面至关重要。醋酸钙的调控措施应代表土壤细菌一般使用的调控措施。从大多数自然和污染的环境中分离出能降解多种化合物的醋酸钙菌株。它们与假单胞菌和变形杆菌伽玛细分的其他革兰氏阴性成员密切相关，它们主要负责芳香族化合物的好氧降解。在这些研究中使用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”已经为醋酸钙的研究开发得很好，这些系统为B-酮己二酸途径的生化、生理学和遗传组织提供了大量的信息。以前的研究为这些ben和cat基因的研究提供了必要的背景，这将有助于更好地理解细菌的调节机制，并为生物修复提供合理的方法。***

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. ***