Department of Plant Pathology, China Agricultural University, Beijing 100193, China. Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
The Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
Sci Signal. 2016 Jan 26;9(412):ra11. doi: 10.1126/scisignal.aad0446.
The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology.
细菌细胞的形态对于毒力、逃避宿主免疫系统和应对环境压力都很重要。广泛分布的 Fic 蛋白（环腺苷酸诱导的丝化）由于存在 HPFx[D/E]GN[G/K]R 基序，被注释为参与细胞分裂的蛋白质。我们表明，荧光假单胞菌的 Fic-1 的存在显著降低了在大肠杆菌或荧光假单胞菌中表达时质粒 DNA 的产量。Fic-1 与 DNA 拓扑异构酶的一个亚基 GyrB 相互作用，GyrB 对于细菌 DNA 复制是必不可少的。Fic-1 催化 GyrB 在 Tyr(109)上的 AMP 化，该残基对于结合 ATP 至关重要，并表现出自 AMP 化活性。Fic-1 自动 AMP 化位点的突变大大降低了对自身和 GyrB 的 AMP 化活性。Fic-1 依赖性的 GyrB AMP 化引发 SOS 反应，表明存在 DNA 复制应激或 DNA 损伤。当 SOS 反应被阻断时，Fic-1 还促进了长形细胞的形成。我们鉴定了一种α-抑制剂蛋白，我们将其命名为抗 Fic-1（AntF），由 Fic-1 上游的一个基因编码。AntF 与 Fic-1 相互作用，在体外抑制 Fic-1 对 GyrB 的 AMP 化活性，并阻断 Fic-1 介导的细菌中 DNA 复制抑制，表明 Fic-1 和 AntF 构成一个毒素-抗毒素模块。我们的工作确立了 Fic-1 作为一种 AMP 化酶，它靶向 GyrB 以抑制 DNA 复制，并可能靶向其他蛋白质来调节细菌形态。
