多重耐药的医院病原体鲍曼不动杆菌的DNA损伤反应具有多种特征,可将其与常用的LexA抑制系统区分开。这些包括该属中不存在LexA,UmuD聚合酶管理器进化为易错聚合酶的UmuDAb阻遏物,使用不动杆菌(DdrR)特有的辅助抑制物,和异常大的UmuDAb结合位点。我们定义了UmuDAbDNA结合和基因抑制所需的顺式和反式作用因子,并测试DdrR是否直接增强其DNA结合。我们使用DNA结合测定法来表征UmuDAb与六个共抑制的umuDC或umuC基因上游的拟议操纵子的结合。UmuDAb与该位点紧密且合作地结合,亲和力比LexA低约10倍。DdrR增强了天然和二聚化缺陷型UmuDAb形式的结合,但仅相对于UmuDAb大于等摩尔比。无法二聚化或影响基因抑制的UmuDAb突变体显示受损的DNA结合,表达G124D二聚化突变体的菌株不能抑制UmuDAb-DdrR调节子的转录。用突变的操纵子探针进行的竞争电泳迁移率变化测定表明,与典型的SOS盒不同,UmuDAb操纵子具有一个五碱基对的中心核心,其序列对结合比侧翼回文更重要。回文的两个侧翼臂中只有一个存在对于UmuDAb结合是必需的。总的来说,数据支持具有两个UmuDAb绑定位点的运算符模型。UmuDAb及其调控启动子的特征与典型的LexA抑制模型不同,展示了一种新的镇压方法。鲍曼不动杆菌是引起医院获得性感染的革兰氏阴性菌。其独特的DNA损伤反应可以激活多个易错的聚合酶基因,允许它获得可以增加其毒力和抗生素抗性的突变。携带多个抗生素抗性基因的感染性菌株的出现,包括耐碳青霉烯,迫切需要发现和开发对抗已知抗生素治疗耐药的感染的方法。解读调节剂UmuDAb和DdrR如何抑制易错聚合酶可能导致开发互补治疗以停止这种产生抗性的机制。
The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb\'s binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.