Understanding aromatic hydrocarbon uptake as the first step in biodegradation

描述（由申请人提供）：芳香烃（AH）是自然、工业和社会活动中广泛存在的环境污染物。许多AH在环境中具有毒性、致癌性和顽固性，因此清除它们对公共卫生极为重要。微生物，特别是细菌的生物降解被认为是从污染土壤中去除AH的最具成本效益和最合适的方法。虽然有大量的文献研究AH和其他外源药物在细胞内的命运，但实际上对这些分子的摄取一无所知。在革兰氏阴性菌中，由于细胞外部存在脂多糖（LPS），外膜（OM）形成了一个非常有效的屏障，阻止疏水分子的自发渗透。因此，需要OM蛋白通道来介导疏水分子进入细胞。该项目的目标是了解AH如何通过革兰氏阴性细菌的外膜（OM）运输，并评估在类似自然环境的条件下这些通道对生物降解的重要性。此外，我们将进行全基因组分析，以确定在AH生物降解过程中重要的其他蛋白质。FadL家族：吸收疏水分子的OM通道。迄今为止，唯一确定在疏水分子运输中起作用的OM蛋白属于FadL家族，该家族成员广泛存在于生物降解革兰氏阴性菌中，包括恶臭假单胞菌F1 （PpF1）。该家族的原型，来自大肠杆菌的FadL (EcFadL)，介导长链脂肪酸（LCFAs）在OM中的摄取。我们最近发现，ecfadl介导的LCFA摄取通过一种独特的横向扩散机制发生。此外，我们已经证明EcFadL作为配体门控通道起作用。除了我们对EcFadL的研究，我们还确定了生物降解细菌中参与单芳香烃（MAH）运输的FadL通道的晶体结构。与EcFadL相比，MAH通道显示出实质性的结构差异，表明与LCFA相比，MAH摄取是通过不同的机制发生的。此外，初步数据表明，FadL通道是衬底特异性的。当前的提案将建立在我们对EcFadL和其他OM通道的研究基础上，通过确定PpF1的三个FadL类似物如何在OM中传输各种AH。我们还将评估哪些结构特征是这些FadL通道的衬底特异性的基础。最后，我们将确定在模拟自然环境的条件下，FadL通道介导的AH的OM摄取的重要性。具体而言，我们将努力实现以下目标：目的：阐明单芳香烃（MAH）在OM中的转运机制。2. 确定FadL通道底物特异性的结构基础。3. 评估FadL通道在生物降解中的重要性。4. 确定哪些蛋白在PpF1对甲苯的生物降解过程中起重要作用。为了回答这些问题，我们将结合广泛的实验方法，包括遗传学，生物化学，结构生物学和微观实验。这些实验将增加我们对疏水分子如何通过OM运输以及在运输疏水底物的通道中如何产生底物特异性的认识。该结果可能用于设计更有效的生物降解剂和利用疏水底物的生物催化剂菌株。

DESCRIPTION (provided by applicant): Aromatic hydrocarbons (AH) are widespread pollutants within the environment owing to natural, industrial and social activities. Many AH are toxic, carcinogenic and recalcitrant within the environment, making their removal extremely important for public health. Biodegradation by microorganisms, especially bacteria, is considered the most cost-effective and appropriate way to remove AH from contaminated soils. While a huge body of literature exists for the intracellular fate of AH and other xenobiotics, virtually nothing is known about the uptake of such molecules. In Gram-negative bacteria, the outer membrane (OM) forms a very effective barrier against the spontaneous permeation of hydrophobic molecules, due to the presence of lipopolysaccharide (LPS) on the outside of the cell. As a consequence, OM protein channels are required to mediate passage of hydrophobic molecules into the cell. The goal of this project is to understand how AH are transported across the outer membrane (OM) of Gram-negative bacteria and to assess the importance of those channels for biodegradation under conditions resembling the natural environment. In addition we will perform genome-wide analyses to identify other proteins that are important during biodegradation of AH. The FadL family: OM channels for uptake of hydrophobic molecules. To date, the only OM proteins with an established role in the transport of hydrophobic molecules belong to the FadL family, members of which are widespread in biodegrading Gram-negative bacteria, including Pseudomonas putida F1 (PpF1). The archetype of the family, FadL from Escherichia coli (EcFadL), mediates uptake of long-chain fatty acids (LCFAs) across the OM. We have recently discovered that EcFadL-mediated LCFA uptake across the OM occurs via a unique, lateral diffusion mechanism. In addition, we have shown that EcFadL functions as a ligand-gated channel. Besides our work on EcFadL, we have determined crystal structures of FadL channels involved in mono-aromatic hydrocarbon (MAH) transport in biodegrading bacteria. The MAH channels show substantial structural differences compared to EcFadL, suggesting that MAH uptake occurs via a different mechanism compared to LCFA uptake. Moreover, preliminary data show that FadL channels are substrate specific. The current proposal will build on our work on EcFadL and other OM channels by determining how various AH are transported across the OM by the three FadL paralogs of PpF1. We will also assess which structural features underlie the substrate specificities of those FadL channels. Finally, we will determine the importance of FadL channel-mediated OM uptake of AH under conditions that mimic closely the natural environment. More specifically, we will pursue the following Aims: 1. To elucidate the transport mechanism of mono-aromatic hydrocarbons (MAH) across the OM. 2. To determine the structural basis for the substrate specificity of FadL channels. 3. To assess the importance of FadL channels for biodegradation. 4. To determine which proteins are important during toluene biodegradation by PpF1. To answer these questions we will combine a wide range of experimental approaches, including genetics, biochemistry, structural biology and microcosm experiments. The experiments will increase our knowledge about how hydrophobic molecules are transported across the OM and how substrate specificity is generated within channels that transport hydrophobic substrates. The results could potentially be used to design more efficient biodegrader and biocatalyst strains that utilize hydrophobic substrates.