Aromatic Compound Degradation by Acinetobacter sp. Strain ADP1

土壤细菌不动杆菌属菌株ADP 1的这些研究集中在芳香族化合物降解的β-酮己二酸途径的转录调控。这一途径的重要性不仅通过天然存在的芳香族化合物的丰富来强调，而且还通过环境中存在的各种结构相似的芳香族污染物来强调。工程菌降解有毒化合物的合理策略依赖于对碳源利用的透彻理解，而对ADP 1的研究将加强这一基础。此外，这些调查将提供见解的转录和代谢调控的自然可转化的细菌非常适合基因工程。 本项目的第一个目标是确定两个同源LysR型转录激活因子BenM和CatM如何控制参与苯甲酸酯降解的几个操纵子的表达。BenM和CatM在氨基酸水平上有57%的相同性，并且都对共诱导物顺式，顺式-粘康酸盐（苯甲酸降解的代谢物）有反应。尽管有这种相似性，BenM和CatM在激活ben和cat基因转录中发挥不同的功能。由于内源性顺式粘康酸水平受ben和cat基因表达的差异控制，因此诱导剂调节其自身的合成和降解。它还调节替代碳源4-羟基苯甲酸酯（4 HB）的降解。将检验关于BenM和CatM介导的benABCDE、catA和catBCIJFD基因调控的特定假设。将确定区分CatM和BenM的微小差异，包括区分DNA-蛋白结合和蛋白-诱导剂结合的差异。该系统的不寻常的方面包括：BenM取代CatM的能力（尽管很差），但不是CatM取代BenM的能力; BenM而不是CatM响应多种共诱导剂的能力;苯甲酸酯和顺，顺-粘康酸酯增加BenM介导的ben基因表达的能力比单独使用时大得多;以及BenM和CatM两者控制catA表达的能力。 该项目的第二个目标是确定在使用首选碳源的生长过程中如何抑制4 HB降解。需要更好地了解多基质环境中的细菌催化剂，这是改善生物修复技术以处理污染的一个至关重要的主题。以前的研究表明，ADP 1消耗不同的芳香族化合物的优先顺序。在苯甲酸消耗过程中，4 HB降解在转录水平上受到抑制，并且4 HB氧化的初始步骤所需的pobA基因不表达。尽管这种抑制需要内源性产生顺式，顺式-粘康酸，但其发生的机制目前尚不清楚。BenM的可能参与。将测试Pcak和PobR在调节碳源依赖性pobA表达中的作用。PobR是pobA的转录激活因子，PcaK是参与4 HB摄取的通透酶。本研究将利用转录本分析、DNA足迹分析和体外转录分析等分子生物学方法，通过选择和鉴定在苯甲酸、原儿茶酸、莽草酸和邻氨基苯甲酸等抑制性碳源条件下生长的pobA突变体，研究ADP 1中控制碳源利用的新机制。遗传和突变分析将利用实验室菌株的自然转化能力。将监测代谢产物以确定转录控制的意义。这些研究将确定参与多碳源利用的重要转录机制，这些机制协调参与共同生理功能的不同操纵子的表达。 此外，它们将促进不动杆菌属菌株ADP 1的生物技术应用，如生物转化和生物修复的发展。

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.