These studies of the soil bacterium Acinetobacter sp. strain ADP1 focus on transcriptional regulation of the beta-ketoadipate pathway for aromatic compound degradation. The importance of this pathway is emphasized not only by the abundance of naturally occurring aromatic compounds but also by a wide variety of structurally similar aromatic pollutants that persist in the environment. Rational strategies for engineering bacteria to degrade toxic compounds rely on a thorough understanding of carbon source utilization, and studies of ADP1 will strengthen that foundation. Furthermore, these investigations will provide insights into transcriptional and metabolic regulation in a naturally transformable bacterium ideally suited for genetic engineering. The first objective of this project is to determine how two homologous LysR-type transcriptional activators, BenM and CatM, control the expression of several operons involved in benzoate degradation. BenM and CatM are 57% identical at the amino acid level, and both respond to the co-inducer cis,cis-muconate, a metabolite of benzoate degradation. Despite this similarity, BenM and CatM carry out distinct functions in activating ben and cat gene transcription. Since the endogenous level of cis cis-muconate is controlled by differential ben and cat gene expression, the inducer regulates its own synthesis and degradation. It also regulates the degradation of an alternative carbon source, 4-hydroxybenzoate (4HB). Specific hypotheses concerning BenM- and CatM-mediated regulation of the benABCDE, catA and catBCIJFD genes will be tested. The small differences that distinguish CatM from BenM will be determined, including those that differentiate DNA-protein binding and protein-inducer binding. Unusual aspects of this system include: the ability of BenM to substitute for CatM (albeit poorly) but not CatM for BenM; the ability of BenM, but not CatM, to respond to multiple co-inducers; the ability of benzoate and cis,cis-muconate to increase BenM-mediated ben gene expression to a much greater extent than either alone; and, the ability of both BenM and CatM to control catA expression. The second objective of the project is to determine how 4HB degradation is inhibited during growth with preferred carbon sources. A better understanding is needed of bacterial catabolism in multi-substrate environments, a topic of critical importance for improving bioremediation techniques to treat pollution. Previous studies showed that ADP1 consumes different aromatic compounds in a preferred order. During benzoate consumption, 4HB degradation is inhibited at the transcriptional level, and the pobA gene, needed for the initial step in 4HB oxidation, is not expressed. Although the endogenous generation of cis, cis-muconate is required for this inhibition, the mechanisms by which it occurs are not currently known. The possible involvement of BenM. Pcak and PobR in regulating carbon-source dependent pobA expression will be tested. PobR is the transcriptional activator of pobA, and PcaK is a permease involved in 4HB uptake. Novel mechanisms for controlling carbon source utilization in ADP1 will be investigated by selecting and characterizing mutants that express pobA during growth with normally repressive carbon sources such as benzoate, protocatechuate, shikimate and anthranilate. To meet these objectives, molecular biological approaches will be used, including transcript analyses, DNA footprinting and in vitro transcription assays. Genetic and mutational analyses will exploit the natural transformability of the laboratory strains. Metabolites will be monitored to determine the significance of transcriptional controls. These studies will determine important transcriptional mechanisms involved in multiple carbon source utilization that coordinate the expression of distinct operons involved in common physiological functions. Moreover, they will facilitate the development of Acinetobacter sp. strain ADP1 for biotechnology applications such as biotransformations and bioremediation.
