留蛋白是参与双链DNA(dsDNA)噬菌体裂解周期的最后阶段的小跨膜蛋白。它们与内溶素合作实现细菌裂解,从而将噬菌体后代释放到细胞外环境中。除了它们作为膜透化剂的作用,允许内溶素转移和/或激活,holins还调节裂解时间。在这项工作中,我们提供了三种针对蜡状芽孢杆菌组的噬菌体编码的holin的功能表征。syphovirusDeep-Purple有一个裂解盒,其中holP30和holP33编码两种具有holin特性的蛋白质,包括跨膜结构域。holin基因在大肠杆菌中表达并诱导细菌裂解,HolP30比HolP33毒性更大.在苏云金芽孢杆菌中,两个holins的同时表达对于观察裂解是必要的,表明它们可能相互作用形成功能性孔。深蓝和Vp4肌病毒都编码一个候选holin(HolB和HolV,分别)具有两个跨膜结构域,其基因不位于细胞内溶素基因附近。它们作为holin蛋白的功能被证实为它们在大肠杆菌中的表达受损的细胞生长和活力。HolV在苏云金芽孢杆菌中的表达也导致细菌裂解,通过将holin与其同源内溶素共表达而增强。尽管类似的组织和预测的拓扑结构,HolB和HolV蛋白的截短突变体表现出不同的毒性水平,这表明氨基酸组成的差异会影响它们的裂解特性。重要性噬菌体生命周期以宿主细胞裂解结束,从而将新的病毒粒子释放到环境中进行下一轮细菌感染。如今,人们对噬菌体作为生物防治剂重新产生了兴趣,主要是由于它们通过裂解引起细菌死亡的能力。而内溶素,介导肽聚糖降解,已经被很好地描述了,成孔蛋白,被称为霍林斯,只有少数模式噬菌体被广泛表征,主要感染革兰氏阴性菌。在这项工作中,我们表征了由一个syphovirus和两个针对革兰氏阳性蜡状芽孢杆菌组成员的肌病毒编码的holins,其中包括密切相关的物种,包括著名的炭疽杆菌,B.严格意义的蜡状细胞,和苏云金芽孢杆菌.总的来说,本文提供了由B.cereus噬菌体编码的holins的第一个实验特征,并揭示了这些噬菌体使用的通用裂解机制。
Holins are small transmembrane proteins involved in the final stage of the lytic cycle of double-stranded DNA (dsDNA) phages. They cooperate with endolysins to achieve bacterial lysis, thereby releasing the phage progeny into the extracellular environment. Besides their role as membrane permeabilizers, allowing endolysin transfer and/or activation, holins also regulate the lysis timing. In this work, we provide functional characterization of the holins encoded by three phages targeting the Bacillus cereus group. The siphovirus Deep-Purple has a lysis cassette in which holP30 and holP33 encode two proteins displaying holin properties, including a transmembrane domain. The holin genes were expressed in Escherichia coli and induced bacterial lysis, with HolP30 being more toxic than HolP33. In Bacillus thuringiensis, the simultaneous expression of both holins was necessary to observe lysis, suggesting that they may interact to form functional pores. The myoviruses Deep-Blue and Vp4 both encode a single candidate holin (HolB and HolV, respectively) with two transmembrane domains, whose genes are not located near the endolysin genes. Their function as holin proteins was confirmed as their expression in E. coli impaired cell growth and viability. The HolV expression in B. thuringiensis also led to bacterial lysis, which was enhanced by coexpressing the holin with its cognate endolysin. Despite similar organizations and predicted topologies, truncated mutants of the HolB and HolV proteins showed different toxicity levels, suggesting that differences in amino acid composition influence their lysis properties. IMPORTANCE The phage life cycle ends with the host cell lysis, thereby releasing new virions into the environment for the next round of bacterial infection. Nowadays, there is renewed interest in phages as biocontrol agents, primarily due to their ability to cause bacterial death through lysis. While endolysins, which mediate peptidoglycan degradation, have been fairly well described, the pore-forming proteins, referred to as holins, have been extensively characterized in only a few model phages, mainly infecting Gram-negative bacteria. In this work, we characterized the holins encoded by a siphovirus and two myoviruses targeting members of the Gram-positive Bacillus cereus group, which comprises closely related species, including the well-known Bacillus anthracis, B. cereus sensu stricto, and Bacillus thuringiensis. Overall, this paper provides the first experimental characterization of holins encoded by B. cereus phages and reveals versatile lysis mechanisms used by these phages.