Molecular Analysis of Polycyclic Aromatic Hydrocarbon Degradation by Mycobacteria

9723921 Zylstra多环芳烃（PAH）是一类由两个或多个稠合苯环以线性、角形和簇状排列组成的芳烃。由于在环境中发现的许多芳烃具有天然热解来源，因此它们在整个进化时期都必须与微生物接触。因此，进化出能够降解芳香族化合物的微生物并不令人惊讶。几乎所有关于多环芳烃代谢的知识都是基于快速生长的革兰氏阴性生物和萘（二环）和菲（三环）作为模型底物。生长缓慢的革兰氏阳性微生物，如分枝杆菌，不仅能降解萘和菲，而且能降解由四个或更多芳香环组成的多环芳烃。事实上，这类生物可能是对它们矿化高分子量多环芳烃能力研究得最好的群体。然而，不幸的是，人们对这些生物体代谢多环芳烃的能力知之甚少。已经提出了分解代谢途径，但这些途径是基于仅鉴定了在培养上清液中鉴定的少数中间化合物。本研究利用分子遗传学和生物化学的方法，对分枝杆菌菌株PYO 1和PYO 5C降解高分子量多环芳烃的能力进行了详细的研究。已获得分解代谢途径中初始双加氧酶的克隆。假设PAH降解的基因是成簇的，则可以通过核苷酸测序来定位途径中的剩余基因。序列分析与表达的、克隆的基因和敲除突变体的生物学分析相结合将鉴定每个鉴定的开放阅读框的功能。这些实验将导致确定分解代谢途径中的每一步，并鉴定每一个分解代谢中间体。如果PAH降解基因在分枝杆菌中不连锁，则通过分枝杆菌菌株的转座子诱变鉴定基因。这将通过以下事实得到帮助：已在靶分枝杆菌菌株中鉴定并克隆了插入序列。 分枝杆菌属的细菌及其亲缘种在降解环境中的高挥发性、稳定的多环芳香族化合物的能力方面是独特的。然而，人们对这种活性的分子和生物化学基础知之甚少。本项目旨在从基因和酶水平研究分枝杆菌菌株降解多环化合物的能力。彻底了解分枝杆菌如何降解这些化合物将有助于设计生物修复过程，从环境中去除多环化合物。

9723921 Zylstra Polycyclic aromatic hydrocarbons (PAHs) constitute a class of aromatic hydrocarbons consisting of two or more fused benzene rings in linear, angular, and cluster arrangements. Since many of the aromatic hydrocarbons found in the environment have a natural pyrolytic origin they must have been in contact with microorganisms throughout evolutionary periods of time. It is not surprising therefore that microorganisms have evolved that are capable of degrading aromatic compounds. Almost all of what is known about the metabolism of PAHs is based on fast-growing gram negative organisms and naphthalene (two rings) and phenanthrene (three rings) as model substrates. Slow-growing gram positive organisms such as Mycobacterium have been shown not only to degrade naphthalene and phenanthrene but also to degrade PAHs consisting of four or more aromatic rings. In fact, this class of organisms is perhaps the best studied group for their ability to mineralize high molecular weight PAHs. Unfortunately, however, very little is known about the ability of these organisms to metabolize PAHs. Catabolic pathways have been proposed but these are based on the identification of only a few intermediate compounds that have been identified in culture supernatants. This project involves the use of molecular genetics and biochemistry to investigate the ability of Mycobacterium strains PYO1 and PYO5C to degrade high molecular weight PAHs in more detail. A clone for the initial dioxygenase in the catabolic pathway(s) has been obtained. Assuming that the genes for PAH degradation are clustered, the remaining genes in the pathway can be located by nucleotide sequencing. Sequence analysis coupled with biological analyses with expressed, cloned genes and with knock-out mutants will identify the function of each identified open reading frame. These experiments will result in the determination of every step in the catabolic pathway with the identification of every catabolic intermediate. In the case that the genes fo r PAH degradation are not linked in Mycobacterium then genes will be identified through transposon mutagenesis of the Mycobacterium strain. This will be aided by the fact that an insertion sequence has been identified in and cloned from the target Mycobacterium strain. Mycobacterium species of bacteria and their relatives are unique in their ability to degrade highly-recalcitrant, stable polycyclic aromatic compounds in the environment. However, little is known about the molecular and biochemical basis of this activity. This project is designed to investigate the ability of Mycobacterium strains to degrade polycyclic compounds at the gene and enzyme level. A thorough knowledge of how Mycobacterium degrade these compounds will aid in the design of bioremediative processes for removing polycyclic compounds from the environment.