Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation
Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation
复制标题
鉴定 Fic-1 是一种通过 AMPylation GyrB 抑制细菌 DNA 复制、促进细丝形成的酶
DOI：
10.1126/scisignal.aad0446
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发表时间：
2016-01-26
影响因子：
7.3
通讯作者：
Luo, Zhao-Qing
中科院分区：
文献类型：
--
作者：
Lu, Canhua;Nakayasu, Ernesto S.;Luo, Zhao-Qing
Fic-1通过AMPylating DNA促旋酶抑制细菌复制。AMP下调DNA复制细菌Fic蛋白催化多种翻译后修饰，如AMP化、磷酸化和UMP化，并参与调节细菌丝状体的形成。Lu等人发现，与来自编码Fic-2或Fic-3质粒的细菌的DNA产量相比，来自表达编码Fic-1的质粒的细菌培养物的DNA产量降低。这表明Fic-1参与抑制DNA复制。Fic-1AMP化细菌DNA复制所需的DNA拓扑异构酶GyrB，其抑制GyrB活性并诱导SOS反应。Fic-1自身腺苷酸化抑制其活性。作者还鉴定了一种Fic-1抗毒素，他们将其命名为AntF。Fic-1表达还通过独立于SOS反应的机制导致细菌中的丝状体形成。因此，本研究确定了Fic-1抑制DNA复制的一个分子靶点，并表明其他靶点介导了Fic-1对细菌丝状体形成的影响。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是重要的。由于存在HPFx[D/E]GN[G/K]R基序，广泛分布的Fic蛋白（由cAMP诱导的抑制）被注释为参与细胞分裂的蛋白质。我们发现，来自荧光假单胞菌的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GyrB相互作用，GyrB是DNA旋转酶的一个亚基，是细菌DNA复制所必需的。Fic-1催化GyrB在Tyr 109（结合ATP的关键残基）处的AMP化，并表现出自身AMP化活性。Fic-1自身AMP化位点的突变大大降低了其自身和GyrB的AMP化活性。GyrB的Fic-1依赖性AMP化触发SOS反应，指示DNA复制应激或DNA损伤。当SOS反应被阻断时，Fic-1也促进细长细胞的形成。我们确定了一种抑制蛋白，我们命名为抗Fic-1（AntF），由Fic-1的上游基因编码。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DNA复制抑制，表明Fic-1和AntF组成了一个毒素-抗毒素模块。我们的工作建立了Fic-1作为一种腺苷酸化酶，靶向GyrB抑制DNA复制，并可能靶向其他蛋白质来调节细菌形态。
Fic-1 inhibits bacterial replication by AMPylating DNA gyrase. AMPing down DNA replication Bacterial Fic proteins catalyze diverse posttranslational modifications, such as AMPylation, phosphorylation, and UMPylation, and are involved in regulating bacterial filament formation. Lu et al. found that DNA yield from bacterial cultures expressing plasmids encoding Fic-1 was reduced compared to DNA yield from bacteria encoding Fic-2 or Fic-3 plasmids. This suggested that Fic-1 is involved in inhibiting DNA replication. Fic-1 AMPylated the DNA topoisomerase, needed for bacterial DNA replication, GyrB, which inhibited GyrB activity and induced SOS response. Self-AMPylation of Fic-1 inhibited its activity. The authors also identified a Fic-1 antitoxin, which they named AntF. Fic-1 expression also resulted in filament formation in bacteria through a mechanism independent of the SOS response. Thus, this study identifies one molecular target for the inhibition of DNA replication by Fic-1 and suggests that additional targets mediate the effect of Fic-1 on bacterial filament formation. 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 Tyr109, 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 &agr;-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.