Division of Infectious Diseases and Medical Services, Massachusetts General Hospital, and Harvard Medical School, Boston, MA, USA.
J Bacteriol. 2012 Apr;194(7):1823-34. doi: 10.1128/JB.06503-11. Epub 2012 Jan 27.
We previously showed that at acid pH, the transcription of norB, encoding the NorB efflux pump, increases due to a reduction in the phosphorylation level of MgrA, which in turn leads to a reduction in bacterial killing by moxifloxacin, a substrate of the NorB efflux pump. In this study, we demonstrated that reduced oxygen levels did not affect the transcript levels of mgrA but modified the dimerization of the MgrA protein, which remained mostly in its monomeric form. Under reduced aeration, we also observed a 21.7-fold increase in the norB transcript levels after 60 min of growth that contributed to a 4-fold increase in the MICs of moxifloxacin and sparfloxacin for Staphylococcus aureus RN6390. The relative proportions of MgrA in monomeric and dimeric forms were altered by treatment with H(2)O(2), but incubation of purified MgrA with extracts of cells grown under reduced but not normal aeration prevented MgrA from being converted to its dimeric DNA-binding form. This modification was associated with cleavage of a fragment of the dimerization domain of MgrA without change in MgrA phosphorylation and an increase in transcript levels of genes encoding serine proteases in cells incubated at reduced aeration. Taken together, these data suggest that modification of MgrA by proteases underlies the reversal of its repression of norB and increased resistance to NorB substrates in response to reduced-aeration conditions, illustrating a third mechanism of posttranslational modification, in addition to oxidation and phosphorylation, that modulates the regulatory activities of MgrA.
我们之前的研究表明，在酸性 pH 值下，由于 MgrA 的磷酸化水平降低，编码 NorB 外排泵的 norB 转录增加，从而导致莫西沙星（NorB 外排泵的底物）对细菌的杀伤作用降低。在这项研究中，我们证明低氧水平不会影响 mgrA 的转录水平，但会改变 MgrA 蛋白的二聚化，使其主要以单体形式存在。在低通气条件下，我们还观察到生长 60 分钟后 norB 转录水平增加了 21.7 倍，这导致金黄色葡萄球菌 RN6390 对莫西沙星和司帕沙星的 MIC 增加了 4 倍。用 H₂O₂处理会改变 MgrA 单体和二聚体的相对比例，但在低通气条件下培养的细胞提取物孵育纯化的 MgrA 可防止 MgrA 转化为其二聚体 DNA 结合形式。这种修饰与 MgrA 二聚化结构域的片段切割有关，而 MgrA 的磷酸化没有变化，并且在低通气条件下孵育的细胞中编码丝氨酸蛋白酶的基因转录水平增加。总的来说，这些数据表明，蛋白酶对 MgrA 的修饰是其对 norB 的抑制作用逆转以及对 NorB 底物的耐药性增加的基础，这表明了除氧化和磷酸化之外的第三种翻译后修饰机制，调节 MgrA 的调节活性。
