Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoprotein ^EcLpoB was identified as a critical activator of Escherichia coli PBP1b (^EcPBP1b), one of the major PG synthases of this organism. Activation of ^EcPBP1b is mediated through the association of ^EcLpoB with a regulatory domain on ^EcPBP1b called UB2H. Notably, Pseudomonas aeruginosa also encodes PBP1b (^PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potential ^PaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity of ^PaPBP1b. We named this protein LpoP and found that it interacts directly with ^PaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated that ^PaLpoP-^PaPBP1b as well as an equivalent protein pair from Acinetobacter baylyi can fully substitute for ^EcLpoB-^EcPBP1b in E. coli for PG synthesis. Furthermore, we show that amino acid changes in ^PaPBP1b that bypass the ^PaLpoP requirement map to similar locations in the protein as changes promoting ^EcLpoB bypass in ^EcPBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

青霉素结合蛋白 (PBP) 是构建围绕大多数细菌细胞的必需肽聚糖 (PG) 细胞壁所需的合成酶。鉴于它们作为关键抗生素靶点的地位，调节这些酶的活性以控制 PG 合成的机制仍然令人惊讶地不明确。几年前，外膜脂蛋白^Ec LpoB 被确定为大肠杆菌PBP1b ( ^Ec PBP1b) 的关键激活剂，大肠杆菌是该生物体的主要 PG 合酶之一。^Ec PBP1b 的激活是通过 Ec LpoB 与 Ec PBP1b 上称为 UB2H 的调节域的关联来^介导的。值得注意的是，铜绿假单胞菌也编码 PBP1b（^Pa PBP1b)，它拥有一个 UB2H 结构域，但这种细菌缺乏可识别的 LpoB 同源物。因此，我们寻找潜在的^{Pa PBP1b 激活剂，并确定了一种与 LpoB 无关的脂蛋白，它是Pa} PBP1b体内活性所必需的。我们将这种蛋白质命名为 LpoP，发现它在体外直接与^Pa PBP1b 相互作用，并且在许多革兰氏阴性菌中是保守的。重要的是，我们还证明了^Pa LpoP- ^Pa PBP1b 以及来自Acinetobacter baylyi的等效蛋白质对可以完全替代大肠杆菌中的^Ec LpoB- ^Ec PBP1b用于 PG 合成。^{此外，我们表明， Pa} PBP1b 中的氨基酸变化绕过^Pa LpoP 要求映射到蛋白质中的相似位置，因为变化促进^{Ec PBP1b 中的Ec} LpoB 绕过。总体而言，我们的结果表明，尽管不同的革兰氏阴性细菌用不同的脂蛋白激活其 PBP1b 合酶，但它们使用保守机制刺激这些重要药物靶标的活性。