Center of Phage Technology, Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
University of Texas Medical Branch at Galveston, Galveston, Texas, USA.
J Virol. 2018 Oct 29;92(22). doi: 10.1128/JVI.00380-18. Print 2018 Nov 15.
Spanins are bacteriophage lysis proteins responsible for disruption of the outer membrane, the final step of Gram-negative host lysis. The absence of spanins results in a terminal phenotype of fragile spherical cells. The phage T1 employs a unimolecular spanin gp that has an N-terminal lipoylation signal and a C-terminal transmembrane domain. Upon maturation and localization, gp ends up as an outer membrane lipoprotein with a C-terminal transmembrane domain embedded in the inner membrane, thus connecting both membranes as a covalent polypeptide chain. Unlike the two-component spanins encoded by most of the other phages, including lambda, the unimolecular spanins have not been studied extensively. In this work, we show that the gp mutants lacking either membrane localization signal were nonfunctional and conferred a partially dominant phenotype. Translation from internal start sites within the gp coding sequence generated a shorter product which exhibited a negative regulatory effect on gp function. Fluorescence spectroscopy time-lapse videos of gp-GFP expression showed gp accumulated in distinct punctate foci, suggesting localized clusters assembled within the peptidoglycan meshwork. In addition, gp was shown to mediate lysis in the absence of holin and endolysin function when peptidoglycan density was depleted by starvation for murein precursors. This result indicates that the peptidoglycan is a negative regulator of gp function. This supports a model in which gp acts by fusing the inner and outer membranes, a mode of action analogous to but mechanistically distinct from that proposed for the two-component spanin systems. Spanins have been proposed to fuse the cytoplasmic and outer membranes during phage lysis. Recent work with the lambda spanins Rz-Rz1, which are similar to class I viral fusion proteins, has shed light on the functional domains and requirements for two-component spanin function. Here we report, for the first time, a genetic and biochemical approach to characterize unimolecular spanins, which are structurally and mechanistically different from two-component spanins. Considering similar predicted secondary structures within the ectodomains, unimolecular spanins can be regarded as a prokaryotic version of type II viral membrane fusion proteins. This study not only adds to our understanding of regulation of phage lysis at various levels but also provides a prokaryotic genetically tractable platform for interrogating class II-like membrane fusion proteins.
类脂转移蛋白是负责破坏外膜的噬菌体裂解蛋白，这是革兰氏阴性宿主裂解的最后一步。类脂转移蛋白的缺失会导致脆弱的球形细胞的终末表型。噬菌体 T1 使用一种单分子类脂转移蛋白 gp，它具有 N 端脂酰化信号和 C 端跨膜结构域。在成熟和定位后，gp 最终成为一种外膜脂蛋白，其 C 端跨膜结构域嵌入内膜中，从而将两个膜连接为一个共价多肽链。与包括 lambda 在内的大多数其他噬菌体编码的双组分类脂转移蛋白不同，单分子类脂转移蛋白尚未得到广泛研究。在这项工作中，我们表明，缺乏任何一种膜定位信号的 gp 突变体均无功能，并表现出部分显性表型。在 gp 编码序列的内部起始位点进行翻译会产生较短的产物，该产物对 gp 功能具有负调控作用。gp-GFP 表达的荧光光谱延时视频显示，gp 聚集在明显的点状焦点中，这表明在肽聚糖网格内组装了局部聚集物。此外，当通过饥饿耗尽肽聚糖前体来耗尽肽聚糖密度时，gp 在没有 holin 和内溶素功能的情况下也介导裂解。这一结果表明，肽聚糖是 gp 功能的负调节剂。这支持了一种模型，即 gp 通过融合内膜和外膜起作用，这种作用方式类似于但在机制上不同于双组分类脂转移蛋白系统所提出的作用方式。类脂转移蛋白被提议在噬菌体裂解过程中外膜和细胞质膜融合。最近使用类似于 I 类病毒融合蛋白的 lambda 类脂转移蛋白 Rz-Rz1 的工作揭示了双组分类脂转移蛋白功能的功能域和要求。在这里，我们首次报道了一种用于表征单分子类脂转移蛋白的遗传和生化方法，这些蛋白在结构和机制上与双组分类脂转移蛋白不同。考虑到外显子结构域内类似的预测二级结构，单分子类脂转移蛋白可以被视为 II 类病毒膜融合蛋白的原核版本。这项研究不仅增加了我们对不同水平噬菌体裂解调控的理解，而且为研究 II 类类似膜融合蛋白提供了一个原核遗传上可操作的平台。
