Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

革兰氏阴性细菌病原体具有限制分子进入细胞的外膜。外膜中充满水的蛋白质通道，即所谓的孔蛋白，可促进营养物质的吸收，并被认为可以使抗生素进入。在这里，我们确定了孔蛋白在主要病原体铜绿假单胞菌中的作用，方法是构建一个缺乏所有 40 个可识别孔蛋白的菌株和 15 个仅携带一种独特类型的孔蛋白的菌株，并用 NMR 代谢组学和抗微生物药敏试验表征这些菌株。与常见假设相反，所有孔蛋白对于假单胞菌都是可有可无的在丰富的培养基中生长并消耗多种亲水性营养素。然而，在没有孔蛋白的情况下，具有两个或多个羧酸盐基团的优选营养素如琥珀酸盐和柠檬酸盐的渗透性很差。孔蛋白为这些具有广泛且重叠的底物选择性的营养素提供了有效的易位途径，同时有效地排除了除碳青霉烯类以外的所有测试抗生素，部分通过 OprD 进入。羧酸盐基团阻碍了抗生素通过外膜脂质双层的不依赖孔蛋白的渗透，这与我们的营养数据一致。总之，这些结果通过证明外膜脂质双层的孔蛋白非依赖性渗透是营养物和药物进入细菌细胞的主要途径，挑战了关于孔蛋白作用的常见假设。