Understanding aromatic hydrocarbon uptake as the first step in biodegradation

描述(申请人提供)：芳香烃(AH)是由于自然、工业和社会活动而在环境中广泛存在的污染物。许多苯系物在环境中具有毒性、致癌性和顽固性，因此它们的去除对公众健康极其重要。微生物，特别是细菌的生物降解被认为是从受污染的土壤中去除环己烷的最经济和最合适的方法。虽然有大量文献研究了AH和其他外源生物物质在细胞内的命运，但对这些分子的吸收几乎一无所知。在革兰氏阴性菌中，由于细胞外存在脂多糖(LPS)，外膜(OM)形成了一种非常有效的屏障，阻止疏水分子的自发渗透。因此，OM蛋白通道需要介导疏水分子进入细胞。该项目的目标是了解AH是如何通过革兰氏阴性细菌的外膜(OM)运输的，并评估这些通道在类似自然环境的条件下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：摄取疏水分子的OM通道。到目前为止，唯一在疏水分子运输中已确定作用的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)。该家族的原型是来自大肠杆菌的Fadl(EcFadL)，它介导了长链脂肪酸(LCFAs)通过OM的摄取。我们最近发现，EcFadL介导的LCFA跨OM的摄取是通过一种独特的横向扩散机制发生的。此外，我们还证明了EcFadL作为配基门控通道的功能。除了我们在EcFadL方面的工作外，我们还确定了生物降解菌中参与单芳烃(MAH)运输的FADL通道的晶体结构。与EcFadL相比，MAH通道显示出显著的结构差异，表明与LCFA摄取相比，MAH摄取通过不同的机制发生。此外，初步数据表明，FADL通道是底物特异性的。目前的提案将建立在我们对EcFadL和其他OM通道的工作的基础上，通过确定PpF1的三个Fadl平行对数如何通过OM传输各种AH。我们还将评估哪些结构特征是这些FADL通道底物特异性的基础。最后，我们将确定在模拟自然环境的条件下，Fadl通道介导的OM吸收AH的重要性。更具体地说，我们将追求以下目标：1.阐明单芳烃(MAH)在有机质中的传输机制。2.确定FADL通道底物特异性的结构基础。3.评价Fadl通道在生物降解中的重要性。4.确定PpF1降解甲苯过程中哪些蛋白质起重要作用。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关于疏水分子如何通过OM运输以及底物专一性如何在运输疏水底物的通道中产生的知识。这些结果可能被用于设计更有效的利用疏水底物的生物降解菌和生物催化剂菌株。

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.