噬菌体(噬菌体)已经进化出有效的手段来接管细菌宿主的机器。可用的分子工具可用于操纵细菌并随意转移分子途径。这里,我们描述了一种细菌生长抑制剂,基因产物T5.015,由T5噬菌体编码。细菌突变体基因组DNA的高通量测序,对这种抑制剂有抗性,揭示了大肠杆菌ung基因的破坏性突变,表明T5.015介导的生长抑制取决于Ung的尿嘧啶切除活性。我们验证了在不存在ung的情况下生长抑制被消除,并通过T5.015证实了Ung的物理结合。此外,使用T5.015和Ung的生化测定表明,T5.015介导由Ung的碱基切除活性产生的无碱基位点的核酸内切活性。重要的是,由核酸内切活性导致的生长抑制表现为DNA复制和细胞分裂停滞。我们推测噬菌体利用这种蛋白质选择性地导致宿主DNA的切割,比噬菌体拥有更多的尿嘧啶。这种蛋白质还可以增强噬菌体对感染细胞中可用资源的利用,因为停止复制可以节省核苷酸,停止细胞分裂可以维持分裂细胞的两个女儿。
Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli
ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of
Ung. We validated that growth inhibition is abrogated in the absence of
ung and confirmed physical binding of
Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of
Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.