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. ***
