Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants.IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.

中文翻译：

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丁香假单胞菌mexB突变体的全基因组转座子筛选揭示了外向转运蛋白的底物。

细菌表达大量外排转运蛋白，赋予其环境中存在的多种有毒物质的抗性。由于这些转运蛋白的高水平功能冗余，很难鉴定出在赋予对特定化合物的抗性中最重要的那些转运蛋白。耐药结节区分开（RND）蛋白家族就是在革兰氏阴性细菌中广泛分布的冗余转运蛋白的一个例子。在该家族中，MexAB-OprM蛋白复合物在假单胞菌属物种中高度表达和保守。我们将丁香假单胞菌B728a的等基因野生型和ΔmexB背景中的条形码转座子突变体文库暴露于多种有毒化合物，以鉴定对这些化合物敏感性增加的突变体。在ΔmexB背景中观察到了编码已知和新型冗余转运蛋白的基因中具有突变但具有部分重叠的底物特异性的突变体。Psyr_0228，转运蛋白的主要促进者超家族的一个未表征的成员，优先有助于耐受orange啶橙和a啶黄。位于内膜的另一种转运蛋白Psyr_0541有助于耐受tolerance啶黄和小ber碱。多个冗余的，基因组编码的外排转运蛋白的存在似乎使细菌菌株能够耐受多种环境毒素。在高敏感性突变株中进行的全基因组筛选揭示了许多转运蛋白，否则这些转运蛋白在这些条件下是可有可无的。细菌菌株，例如P。可能在环境中遇到各种毒素（例如与许多不同植物物种相关联）的丁香科植物，可能受益于拥有多个冗余转运蛋白，这些转运蛋白在耐受新型有毒物质方面具有多功能性。重要细菌利用蛋白质泵从细胞中去除有毒化合物内部，可以在各种环境中生存。这些蛋白质泵可能是高度冗余的，因此很难进行目标检测。在这项研究中，我们将丁香假单胞菌的突变种群暴露于多种有毒物质，以鉴定有助于在这些条件下存活的泵。同时，我们通过测试缺乏负责毒素耐受性的主要外排转运蛋白的人群的突变体来检查泵的冗余度。我们确定了多余转运蛋白的部分底物重叠，以及一些看起来对底物更具特异性的泵。对于在不同环境中发现的细菌，拥有多个部分冗余的外排泵可能会允许在栖息地定居中提供灵活性。