Molecular Analysis of Polycyclic Aromatic Hydrocarbon Degradation by Mycobacteria

分枝杆菌降解多环芳烃的分子分析

• 批准号: 9723921
• 负责人: Gerben Zylstra
• 金额: $ 30万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723921&HistoricalAwards=false
• 关键词: Molecular; Analysis; Polycyclic; Aromatic; Hydrocarbon

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.

多环芳烃（PAHs）是一类由两个或两个以上呈线性、角状和簇状排列的融合苯环组成的芳烃。由于在环境中发现的许多芳香烃具有自然热解的起源，它们在进化过程中一定与微生物接触过。因此，微生物进化到能够降解芳香族化合物也就不足为奇了。几乎所有关于多环芳烃代谢的已知知识都是基于快速生长的革兰氏阴性生物和萘（双环）和菲（三环）作为模式底物。生长缓慢的革兰氏阳性菌，如分枝杆菌，不仅能降解萘和菲，还能降解由四个或更多芳环组成的多环芳烃。事实上，这类生物可能是研究得最好的群体，因为它们具有矿化高分子量多环芳烃的能力。然而，不幸的是，我们对这些生物代谢多环芳烃的能力知之甚少。已经提出了分解代谢途径，但这些都是基于在培养上清中鉴定的少数中间化合物的鉴定。本项目涉及利用分子遗传学和生物化学方法，更详细地研究分枝杆菌菌株PYO1和PYO5C降解高分子量多环芳烃的能力。已经获得了分解代谢途径中初始双加氧酶的克隆。假设多环芳烃降解的基因聚集在一起，则可以通过核苷酸测序来定位该途径中剩余的基因。序列分析结合表达基因、克隆基因和敲除突变体的生物学分析将确定每个已确定的开放阅读框的功能。这些实验将确定分解代谢途径中的每一步，并确定每一个分解代谢中间体。在分枝杆菌中降解多环芳烃的基因没有关联的情况下，将通过分枝杆菌菌株的转座子诱变来鉴定基因。这将是辅助的事实，插入序列已确定，并从目标分枝杆菌菌株克隆。分枝杆菌种类的细菌和他们的亲戚是独特的，在他们的能力降解环境中高度顽固的，稳定的多环芳香族化合物。然而，人们对这种活性的分子和生化基础知之甚少。本项目旨在研究分枝杆菌菌株在基因和酶水平上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从环境中去除多环化合物的生物修复工艺。