Molecular Assessment of Hydrocarbon Degradation

该项目的主要目标是通过关注关键的初始加氧酶来获得有关群落功能的信息，这些加氧酶是芳香族化合物在有氧和亚氧环境中的生产性降解和最终矿化的限速步骤。本计画将探讨加氧酶基因序列的多样性，并在不考虑宿主生物的情况下，监测其族群的变化。拟议的实验结果将确定环境中加氧酶遗传多样性的广度，并将这种多样性与已知和未知的生物体联系起来。特别是，这些数据将确定基因靶点，以便更好地对环境中的碳氢化合物氧化进行分子评估。 从这个为期两年的探索性研究计划中，我们预计将开发一个大型的加氧酶基因序列数据库。预计这些序列中的大多数将是新的序列，来源于尚未在实验室培养的生物体。我们成功获得这些序列的能力将来自于使用新的加氧酶PCR引物。全长加氧酶基因序列将通过反向PCR和环境DNA粘粒克隆测序获得。 开发和使用这些分子方法的目标是评估其在实验室规模的环境扰动实验或补救处理应用中跟踪加氧酶基因群体变化的稳健性。这将通过集中精力从一个单一的超级基金网站的物理，化学和地质的网站参数的差异，可以评估其对“分子指纹”的微生物加氧酶的模式，从实验处理的影响源材料。来自这种类型的探索性研究的数据应使我们能够确定哪些分子特征（在这种情况下，双加氧酶模式）可预测总体生物降解性 系统的性能。由此，我们计划构建新一代的“加氧酶微阵列”，它可以提供有关微生物群落中那些在生物降解和生物修复过程中变得重要的成员的功能信息。

The primary goal of this project is to obtain information about community function by focusing on the critical initial oxygenases that are the rate-limiting step in productive degradation and eventual mineralization of aromatic compounds in oxic and suboxic environments. This project will explore the diversity of oxygenase gene sequences and monitor shifts in their population without regard to host organism. Results of the proposed experiments will identify the breadth of genetic diversity of oxygenases in the environment and associate that diversity with known and unknown organisms. In particular the data will identify gene targets for better molecular assessment of hydrocarbon oxidation in the environment. From this two-year exploratory research program we anticipate the development of a large database of oxygenase gene sequences. It is anticipated that the majority of these will be novel sequences, derived from organisms that are not yet in laboratory cultivation. Our ability to successfully obtain such sequences will result from the use of novel oxygenase PCR primers. Full length oxygenase gene sequences will be obtained through inverse PCR and sequencing of environmental DNA cosmid clones. The goal of developing and using these molecular approaches is to assess their robustness for tracking changes in oxygenase gene populations in laboratory-scale environmental perturbation experiments or in remediation treatment applications. This will be accomplished by focusing intensively on source materials from a single Superfund site in which differences in physical, chemical and geological site parameters can be assessed for their impact on the "molecular fingerprints" of the microbial oxygenase patterns that result from the experimental treatments. Data from this type of exploratory study should allow us to determine which molecular signatures (in this case, dioxygenase patterns) are predictive of overall biodegradative perforrnance of the system. From this, we plan to construct a new generation of "oxygenase microarray" that can provide functional information about those members of the microbial community that become important in biodegradative and bioremediative processes.