Chamakura Karthik R, Tran Jennifer S, Young Ry
Center for Phage Technology, Texas A&M AgriLife, Texas A&M University, College Station, Texas, USA.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
J Bacteriol. 2017 May 25;199(12). doi: 10.1128/JB.00058-17. Print 2017 Jun 15.
The L protein of the single-stranded RNA phage MS2 causes lysis of without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (nsensitivity to ysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the mutant was recessive and could be suppressed by vercomes ( ) alleles selected for restoration of lysis. All three alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the L truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions. Small single-stranded nucleic acid lytic phages ( and ) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.
单链RNA噬菌体MS2的L蛋白可导致细胞裂解，而不会诱导溶菌活性或抑制肽聚糖（PG）的净合成。为了找到L介导的细胞裂解所需的宿主基因，通过选择L表达的幸存者获得了自发的Ill（对裂解不敏感）突变体，并发现其在DnaJ的C末端结构域中高度保守的脯氨酸发生了错义变化（P330Q）。在突变宿主中，L介导的细胞裂解在30°C时完全受阻，而不影响L的细胞内水平。在较高温度（37°C和42°C）下，细胞裂解和L积累均延迟。突变体在30°C时的裂解阻断是隐性的，并且可以通过为恢复裂解而选择的vercomes（）等位基因来抑制。所有三个等位基因都缺少裂解蛋白高度碱性的N末端一半，并且比全长L提前约20分钟导致细胞裂解。发现DnaJ与全长L形成复合物。该复合物被P330Q突变消除，并且在L截短体中不存在。这些结果表明，在缺乏与DnaJ相互作用的情况下，L的N末端结构域会干扰其与未知靶标的结合能力。L的非必需、高度碱性的N末端结构域赋予的裂解延迟和对DnaJ伴侣的依赖性类似于ϕX174的E裂解蛋白高度碱性的C末端结构域赋予的对SlyD伴侣的依赖性，这表明了一个共同的主题，即单基因裂解可以受到受生理条件影响的宿主因子的调节。小型单链核酸裂解噬菌体（和）通过表达单一的“蛋白质抗生素”来裂解其宿主。三种原型小型裂解病毒中的两种产生的蛋白质抗生素已被证明可抑制保守的PG生物合成途径中的两个不同步骤。然而，第三种原型病毒MS2的裂解蛋白L引起裂解的分子基础尚不清楚。我们研究的意义在于鉴定出DnaJ是MS2 L裂解途径中的一种伴侣，并鉴定出MS2 L的最小裂解结构域。此外，我们的研究突出了DnaJ的C末端结构域中高度保守的P330残基对于特定蛋白质相互作用的重要性。
