The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage φX174
The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage φX174
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DOI：
10.1046/j.1365-2958.2002.02984.x
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发表时间：
2002-07-01
影响因子：
3.6
通讯作者：
Young, R
中科院分区：
文献类型：
--
作者：
Bernhardt, TG;Roof, WD;Young, R
大多数噬菌体通过引起宿主细胞的裂解而突然终止它们的营养循环。单链DNA噬菌体Phi x174使用一个单一的裂解基因E，编码91个氨基酸的膜蛋白，通过抑制mray(毛霉素素生物合成的一种保守酶)导致大肠杆菌的裂解。宿主基因slyD(裂解敏感性)的隐性突变完全阻断了E介导的裂解和phi x174斑块的形成。SlyD基因编码一种FKBP-型肽-脯氨酰顺-反式异构酶(PPIase)。为了研究这种独特的FKBP依赖的分子基础，在slyD草坪上分离了phiX174的自发空斑形成突变体。所有这些Epos(slyD‘上的平板)抑制蛋白编码具有R3H或L19F变化的蛋白质。该双突变体也被分离出来，并在slyD草坪上产生了最大的斑块。C-myc表位标签序列被整合到亲本E和Epos基因中，而不影响裂解功能。蛋白质印迹和脉冲追逐标记实验表明，Epos和E在slyD背景下都是高度不稳定的，但Epos的合成速度更快，允许足够裂解水平的Epos积累。我们的结果表明，SlyD是稳定E蛋白并使其积累到发挥其裂解作用所需的水平所必需的。这些数据是根据SlyD PPIase在E折叠中的特定作用的模型以及使用非常严格的SlyD依赖表型来识别PPIase选择性的元件来讨论的。
Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage phi X174 uses a single lysis gene, E, encoding a 91-amino-acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E-mediated lysis and phi X174 plaque formation. The slyD gene encodes a FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP-dependence, spontaneous plaque-forming mutants of phiX174 were isolated on a slyD lawn. All of these Epos (plates on slyD') suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c-myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse-chase labelling ex-periments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis-sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD- dependence phenotype for identifying elements of PPIase selectivity.