Aromatic Compound Degradation by Acinetobacter sp. Strain ADP1

本文对土壤细菌不动杆菌进行了研究。菌株ADP1专注于芳香族化合物降解的β-酮基己二酸途径的转录调控。这一途径的重要性不仅被自然产生的芳香族化合物的丰富所强调，而且还被环境中持续存在的各种结构相似的芳香族污染物所强调。工程菌降解有毒化合物的合理策略依赖于对碳源利用的彻底了解，而对ADP1的研究将加强这一基础。此外，这些研究将为一种适合基因工程的自然可转化细菌的转录和代谢调节提供洞察力。这个项目的第一个目标是确定两个同源的LysR型转录激活子BenM和CATM如何控制参与苯甲酸降解的几个操纵子的表达。BenM和CATM在氨基酸水平上有57%的相同之处，并且都对共同诱导剂顺式-松果酸做出反应，顺式-松果酸是苯甲酸降解的代谢物。尽管有这些相似之处，BenM和CATM在激活Ben和CAT基因转录方面执行不同的功能。由于顺式互补酸的内源水平受ben和cat基因差异表达的控制，因此诱导剂调节其自身的合成和降解。它还调节另一种碳源--4-羟基苯甲酸(4HB)的降解。将测试关于BenM和CATM介导的benABCDE、CATA和catBCIJFD基因调控的特定假设。区分CATM和BenM的细微差别将被确定，包括那些区分DNA-蛋白质结合和蛋白质诱导结合的区别。该系统的不同寻常之处包括：BenM能够替代CATM(尽管效果不佳)，但CATM不能替代BenM；BenM能够响应多种共诱导物，但不能响应CATM；苯甲酸和顺式、顺式-松果酸能够比单独使用更大程度地提高BenM介导的BEN基因的表达；以及BenM和CATM都能够控制CATA的表达。该项目的第二个目标是确定在生长期间如何在首选碳源的情况下抑制4HB的降解。需要更好地了解多底物环境中的细菌分解代谢，这是改进生物修复技术以处理污染的一个至关重要的主题。以往的研究表明，ADP1以优先顺序消耗不同的芳香族化合物。在苯甲酸的消耗过程中，4HB的降解在转录水平上受到抑制，4HB氧化的初始步骤所需的POBA基因没有表达。虽然这种抑制需要内源性顺式，顺式-松果酸的产生，但其发生的机制目前尚不清楚。BenM可能参与其中。Pcak和PobR在调节碳源依赖的POBA表达中的作用将得到检验。PobR是PobA的转录激活剂，PcaK是参与4Hb摄取的通透酶。通过筛选和鉴定在生长过程中表达POBA的突变体，如苯甲酸盐、原儿茶酸盐、莽草酸和邻氨基苯甲酸酯，将研究控制ADP1碳源利用的新机制。为了实现这些目标，将使用分子生物学方法，包括转录分析、DNA足迹和体外转录检测。遗传和突变分析将利用实验室菌株的自然转化能力。将对代谢产物进行监测，以确定转录调控的重要性。这些研究将确定涉及多种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，它们还将促进不动杆菌属的发展。用于生物转化和生物修复等生物技术应用的菌株ADP1。

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.