Chen Yi, Young Ry
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA Center for Phage Technology, Texas A&M University, College Station, Texas, USA.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA Center for Phage Technology, Texas A&M University, College Station, Texas, USA
J Bacteriol. 2016 Aug 25;198(18):2448-57. doi: 10.1128/JB.00294-16. Print 2016 Sep 15.
The latent period of phage T4, normally ∼25 min, can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon, designated lysis inhibition (LIN), was first described in the 1940s and is genetically defined by mutations in diverse T4 r genes. RI, the main effector of LIN, has been shown to be secreted to the periplasm, where, upon activation by superinfection with a T-even virion, it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Another r locus, rIII, has been the subject of conflicting reports. In this study, we show that RIII, an 82-amino-acid protein, is also required for LIN in both Escherichia coli B strains and E. coli K-12 strains. In T4ΔrIII infections, LIN was briefly established but was unstable. The overexpression of a cloned rIII gene alone impeded T-mediated lysis temporarily. However, coexpression of rIII and rI resulted in a stable LIN state. Bacterial two-hybrid assays and pulldown assays showed that RIII interacts with the cytoplasmic N terminus of T, which is a critical domain for holin function. We conclude that RIII is a T4 antiholin that blocks membrane hole formation by interacting directly with the holin. Accordingly, we propose an augmented model for T4 LIN that involves the stabilization of a complex of three proteins in two compartments of the cell: RI interacting with the C terminus of T in the periplasm and RIII interacting with the N terminus of T in the cytoplasm.
Lysis inhibition is a unique feature of phage T4 in response to environmental conditions, effected by the antiholin RI, which binds to the periplasmic domain of the T holin and blocks its hole-forming function. Here we report that the T4 gene rIII encodes a cytoplasmic antiholin that, together with the main antiholin, RI, inhibits holin T by forming a complex of three proteins spanning two cell compartments.
噬菌体T4的潜伏期通常约为25分钟，如果在感染后5分钟对感染细胞进行超感染，潜伏期可无限延长。这种现象被称为裂解抑制（LIN），最早在20世纪40年代被描述，并且在基因上由多种T4 r基因的突变所定义。LIN的主要效应因子RI已被证明分泌到周质中，在那里，通过被T偶数病毒粒子超感染而激活后，它与T4溶菌酶T的C末端周质结构域结合，并阻止其对细胞质膜的致命通透作用。另一个r位点rIII一直是相互矛盾报道的主题。在本研究中，我们表明RIII是一种82个氨基酸组成的蛋白质，在大肠杆菌B菌株和大肠杆菌K-12菌株中LIN过程中也是必需的。在T4ΔrIII感染中，LIN可短暂建立但不稳定。单独克隆的rIII基因的过表达会暂时阻碍T介导的裂解。然而，rIII和rI的共表达会导致稳定的LIN状态。细菌双杂交试验和下拉试验表明，RIII与T的细胞质N末端相互作用，这是溶菌酶功能的关键结构域。我们得出结论，RIII是一种T4抗溶菌酶，通过直接与溶菌酶相互作用来阻止膜孔形成。因此，我们提出了一个T4 LIN的增强模型，该模型涉及细胞两个区室中三种蛋白质复合物的稳定：RI在周质中与T的C末端相互作用，RIII在细胞质中与T的N末端相互作用。
裂解抑制是噬菌体T4响应环境条件的一个独特特征，由抗溶菌酶RI介导，RI与T溶菌酶的周质结构域结合并阻止其孔形成功能。在这里我们报道，T4基因rIII编码一种细胞质抗溶菌酶，它与主要抗溶菌酶RI一起，通过形成跨越两个细胞区室的三种蛋白质复合物来抑制溶菌酶T。
