Stable Isotope Probing-metagenomics of river microbial populations degrading the aromatic pollutant para-nitrophenol (PNP)

对硝基苯酚（PNP）是一种广泛分布于环境中的芳香污染物。这是由于它被用作染料和广泛用于有机合成的化合物，例如炸药和杀虫剂。由于其工业用途，PNP在生产现场和附近造成土壤和水道的环境污染，而且在农业土壤中一系列农药的降解过程中也造成污染。虽然PNP在化学上是稳定的，但微生物可以降解它。细菌负责环境中化学物质的生物修复，但导致PNP降解的特定种群和代谢途径的身份尚未确定。为了进一步加强PNP的生物降解，例如在水处理厂或农业土壤等工程系统中，了解PNP降解微生物的身份是主要的兴趣。细菌的PNP降解主要是利用实验室培养的纯化菌株进行的研究。已经在许多细菌中发现了赋予PNP降解能力的基因及其功能，但这些基因和功能是否代表了在自然环境中PNP降解的关键途径和细菌尚不确定。这主要是因为生活在环境中的绝大多数细菌抵抗培养。使用特定的技术，人们可以识别出这种“不可培养”的种群。这是通过给自然种群的微生物喂食一种与正常基质化学成分相同的基质，但实际上更重。在我们的例子中，我们将使用PNP，其中碳原子的原子量为13，而不是更传统的12。当微生物消化如此重的分子时，重碳会被整合到包括DNA在内的分子中。通过从轻形式中纯化这种重DNA并寻找基因中的特征序列，可以识别使用PNP的微生物，并且可以通过测序其基因组DNA推断出它们这样做的机制。我们将用沃里克郡德尼河的河水做这些实验。我们已经研究了这条河中的PNP降解细菌，并且已经能够分离出降解PNP的特定菌株。我们还开发了许多针对PNP降解关键基因的遗传探针，并分析了这些标记在PNP降解微观环境中的多样性。这些分析表明，在这条河中有许多不可培养的细菌有助于PNP降解-很可能使用以前未被证明参与PNP降解的酶。这个项目将使我们能够应用尖端的分子工具来识别这些群体和一些可能有助于PNP降解的候选新酶。这些结果将是有益的，因为它们将增加我们对一个重要的微生物过程的理解，该过程负责从环境中去除有毒的人为芳香族污染物。更好地了解生物修复过程可能会导致改善这一环境过程的功效，例如在废水处理厂或农业土壤中，这是特别重要的。

Para-nitrophenol (PNP) is an aromatic pollutant that is widely distributed in the environment. This is due to its utilisation as a dye and as a compound used in a wide range of organic syntheses, for instance of explosives and pesticides. As a result of its industrial use PNP causes environmental pollution of soils and water courses, at and near the site of manufacture but also during degradation of a range of pesticids in agricultural soil. While PNP is chemically stable, microorganisms can degrade it. Bacteria are responsible for bioremediation of the chemical in the environment, but the identity of the specific populations and metabolic pathways that contribute to PNP degradation are as yet uncharacterised. Knowledge of the identity of PNP degrading microorganisms is of major interest in order to further enhance the biodegradation of PNP, for instance in engineered systems such as water treatment works or in agricultural soils.PNP degradation of bacteria has been mostly studied using purified strains that are grown in the lab. The genes and their functions that confer PNP degradation capability have been identified in a number of bacteria, but whether these represent the most important pathways and bacteria that are the pivotal for PNP degradation in in natural environments is uncertain. This is mainly because the great majority of bacteria that live in the environment resist cultivation. Using specific techniques, one can identify such 'unculturable' populations. This is done by feeding natural populations of microbes with a version of the substrate that is chemically identical to the normal one but which is, literally, heavier. In our case, we will use PNP in which the carbon atoms have an atomic weight of 13, not the more conventional 12. When a microbe digests such a heavy molecule, the heavy carbon is incorporated into its molecules, including DNA. By purifying this heavy DNA from the light form and by looking for signature sequences in the genes, the microorganisms that used the PNP can be identified and the mechanisms by which they do so can be inferred from sequencing their genomic DNA.We will do these experiments using river water from the River Dene, in Warwickshire. We have already studied PNP degrading bacteria in this river and have been able to isolate particular strains that degraded PNP. We have also developed a number of genetic probes that target key genes of PNP degradation in a wide range of bacteria and analysed the diversity of these markers in the PNP degradation microcosms. These analyses have shown that there are a number of unculturable bacteria that contribute to PNP degradation in this river - very likely using enzymes that have not previously been shown to be involved in PNP degradation. This project would allow us to apply cutting-edge molecular tools to identify these populations and some of the candidate novel enzymes that may contribute to PNP degradation in addition to the ones already known. The results will be beneficial in that they will increase our understanding of an important microbial process that is responsible for the removal of toxic anthropogenic aromatic pollutants from the environment. Understanding the process of bioremediation better may lead to improvement of the efficacy of this environmental process, for instance in waste water treatment plants or agricultural soils where it is particularly important.