Genome-wide analysis of multidrug efflux in the plant-pathogenic bacterium Pseudomonas syringae

多药外排（MDE）转运体是细菌对抗生素产生耐药性的主要因素。与MDE在临床相关微生物中的作用相比，人们对环境细菌中的MDE转运体知之甚少。本项目旨在鉴定和表征植物病原体丁香假单胞菌中所有MDE泵，并深入了解其调控和自然功能。MDE泵可能在丁香假单胞菌对其宿主植物的适应中发挥重要作用，保护它们免受植物抗菌剂的侵害。丁香假单胞菌被细分为致病变异（病原体），根据其宿主范围而变化。现有的三种不同丁香假单胞菌病原菌的基因组序列将用于开发包含转运蛋白家族所有成员（包括MDE系统）基因的微阵列。微阵列将用于鉴定抗生素处理后表达的转运蛋白，抗菌植物代谢物，以及植物。待测试的丁香假单胞菌菌株感染属于不同植物科的植物，每种菌株产生不同的抗菌代谢物谱。因此，植物代谢物的多样性将被筛选为MDE转运体的底物，这可能会揭示MDE系统对宿主特异性的贡献。将克隆假定的MDE蛋白，以确定底物特异性，寻找天然底物，并研究其转录调控。由于并不是所有的MDE转运体在抗生素作用下都表现出表达增加，我们将进一步克隆和DFG植物病原体中的多药物外排表征与其他生物已知的MDE泵序列相似的基因。诱变将用于揭示这些转运体在发病过程中的作用。

Multidrug efflux (MDE) transporters are major contributors to bacterial resistance towards antibiotics. In contrast to the well-understood role of MDE in clinically relevant microbes, only little is known about MDE transporters in environmental bacteria. This project aims to identify and characterize all MDE pumps in the plant pathogen Pseudomonas syringae and to gain in-depth knowledge about their regulation and natural functions. MDE pumps may play an important role in the adaptation of P. syringae to its respective host plants by protecting them against plant antimicrobials. P. syringae is sub-divided into pathogenic variants (pathovars) which vary according to their host range. Available genome sequences of three different P. syringae pathovars will be used to develop a microarray containing genes of all members of transport protein families that include MDE systems. The microarrays will be used to identify transporters that are expressed after treatment with antibiotics, antimicrobial plant metabolites, and in planta. The to-be-tested P. syringae strains infect plants belonging to different plant families, each producing a different spectrum of antimicrobial metabolites. Thus, the diversity of plantborne metabolites will be screened as substrates of MDE transporters potentially shedding light on the contribution of MDE systems on host specificity. Putative MDE proteins will be cloned to determine substrate specificity, to search for natural substrates, and to study their transcriptional regulation. Because not all MDE transporters might show an increased expression in response to antibiotics, we will furthermore clone and DFG Research proposal - Matthias Ullrich - Multidrug efflux in plant pathogens 3 characterize genes that show sequence similarities to known MDE pumps from other organisms. Mutagenesis will be used to reveal the role of these transporters during pathogenesis.