Role of tripartite efflux pumps in pyoverdine secretion in Pseudomonas putida KT2440

铁对大多数生物体来说是必不可少的，但细菌往往很难获得铁，因为铁离子在自然栖息地的溶解度较低，或者与宿主中的蛋白质结合。假单胞菌属的细菌通过合成、分泌和重新摄取清除铁的铁载体吡咯烷(PVD)来解决这一问题。PVD的合成和摄取对PVD-铁复合体的研究相对较好，然而，对PVD分泌的机制还只有部分了解。本课题以土壤细菌恶臭假单胞菌KT2440为实验模型，探索PVD的分泌机制。该菌株是研究最深入的腐生型假单胞菌，是工业生物催化的功能底盘，它只产生一种类型的铁载体--PVD。我们的初步结果表明，PVD的分泌不依赖于一个特定的外排系统，而是依赖于几个所谓的三分外排泵，这在一定程度上是已知的导致耐药性的因素。我们计划(1)确定恶臭假单胞菌KT2440中参与PVD分泌的外排泵；(2)研究这些泵在PVD介导的铁获取以及对药物和其他有毒化合物的耐药性中的特定作用；以及(3)首次在生化水平上表征PVD的运输，以测试三方外排泵在PVD运输中的直接参与，并获得关于能量耦合、底物特异性和动力学的信息。这一结果有望有助于更好地理解铁限制条件下假单胞菌的生理学，并为阐明三方外排泵的生理意义提供新的线索。

Iron is essential for most organisms, but often difficult for bacteria to acquire because iron ions have a low solubility in natural habitats or are bound to proteins in hosts. Bacteria of the genus Pseudomonas solve this problem by the synthesis, secretion and re-uptake of the iron scavenging siderophore pyoverdine (PVD). PVD synthesis and uptake of the PVD-iron complex are relatively well investigated, however, the mechanism of PVD secretion is only partially understood. This project explores the mechanism of PVD secretion using the soil bacterium Pseudomonas putida KT2440 as experimental model. The strain is the best studied saprophytic pseudomonad, a functional chassis for industrial biocatalysis, and it produces only one type of siderophore, PVD. Our preliminary results suggest that PVD secretion does not depend on a particular efflux system, but on several so-called tripartite efflux pumps, which are in part known to contribute to drug resistance. We plan (1) to identify the efflux pumps involved in PVD secretion in P. putida KT2440; (2) to investigate the specific role of these pumps in PVD-mediated iron acquisition and in the resistance against drugs and other toxic compounds; and (3) to characterize PVD transport for the first time at the biochemical level to test a direct involvement of tripartite efflux pumps in PVD transport and to obtain information on the mechanism of energy coupling, substrate specificity and kinetics. The results are expected to contribute to a better understanding of the physiology of pseudomonads under iron limitation and to shed new light on the physiological significance of tripartite efflux pumps.