Mu噬菌体是一种典型的大肠杆菌噬菌体,主要是因为它使用转座进行基因组复制。然而,在广泛的废话突变体屏幕中,只鉴定了一个裂解基因,内溶素gp22.这是令人惊讶的,因为在革兰氏阴性宿主中,Caudovirales噬菌体的裂解已显示需要破坏细胞包膜的所有三层的蛋白质。通常这涉及到一个holin,一种内溶素,和一个靶向细胞质膜的span蛋白,肽聚糖(PG),和外膜(OM),分别,用holin确定裂解开始的时间。这里,我们证明gp22是信号锚释放(SAR)内溶素,并将gp23和gp23.1鉴定为两组分spanin亚基。然而,我们发现Mu缺乏holin,而是编码膜束缚的细胞质蛋白,gp25,这是释放SAR内溶素所必需的。突变分析表明,这种对gp25的依赖性是由gp22的短胞质结构域的6和7位的赖氨酸残基赋予的。gp25,我们指定为释放,也有助于从其他噬菌体释放SAR内溶素。此外,gp25的全长,包括其N端跨膜结构域,属于蛋白质家族,DUF2730,在许多类似Mu的噬菌体中发现,包括细胞质内溶素。根据释放功能的演变和机制模型以及无holin控制的Mu裂解的基本原理来讨论这些结果。重要性宿主细胞裂解是噬菌体感染周期的终末事件。在革兰氏阴性宿主中,裂解需要破坏三种细胞包膜成分中每一种的蛋白质,在Mu中只发现了一种:内溶素gp22。我们证明gp22可以被表征为SAR内溶素,一种从细胞膜释放后激活以降解细胞壁的胞壁酶。此外,我们将基因23和23.1鉴定为用于破坏外膜的spanin亚基。重要的是,我们证明了Mu是第一个已知的Caudovirales噬菌体,一种破坏内膜的蛋白质,传统上已知会释放内溶素。取而代之的是,我们报道了一种裂解蛋白的发现,被称为释放,Mu用于SAR内溶素释放。这是一个系统的例子,其中一种蛋白质的动态膜定位由第二种蛋白质控制。
Bacteriophage Mu is a paradigm coliphage studied mainly because of its use of transposition for genome replication. However, in extensive nonsense mutant screens, only one lysis gene has been identified, the endolysin gp22. This is surprising because in Gram-negative hosts, lysis by Caudovirales phages has been shown to require proteins which disrupt all three layers of the cell envelope. Usually this involves a holin, an endolysin, and a spanin targeting the cytoplasmic membrane, peptidoglycan (PG), and outer membrane (OM), respectively, with the holin determining the timing of lysis initiation. Here, we demonstrate that gp22 is a signal-anchor-release (SAR) endolysin and identify gp23 and gp23.1 as two-component spanin subunits. However, we find that Mu lacks a holin and instead encodes a membrane-tethered cytoplasmic protein, gp25, which is required for the release of the SAR endolysin. Mutational analysis showed that this dependence on gp25 is conferred by lysine residues at positions 6 and 7 of the short cytoplasmic domain of gp22. gp25, which we designate as a releasin, also facilitates the release of SAR endolysins from other phages. Moreover, the entire length of gp25, including its N-terminal transmembrane domain, belongs to a protein family, DUF2730, found in many Mu-like phages, including those with cytoplasmic endolysins. These results are discussed in terms of models for the evolution and mechanism of releasin function and a rationale for Mu lysis without holin control. IMPORTANCE Host cell lysis is the terminal event of the bacteriophage infection cycle. In Gram-negative hosts, lysis requires proteins that disrupt each of the three cell envelope components, only one of which has been identified in Mu: the endolysin gp22. We show that gp22 can be characterized as a SAR endolysin, a muralytic enzyme that activates upon release from the membrane to degrade the cell wall. Furthermore, we identify genes 23 and 23.1 as spanin subunits used for outer membrane disruption. Significantly, we demonstrate that Mu is the first known Caudovirales phage to lack a holin, a protein that disrupts the inner membrane and is traditionally known to release endolysins. In its stead, we report the discovery of a lysis protein, termed the releasin, which Mu uses for SAR endolysin release. This is an example of a system where the dynamic membrane localization of one protein is controlled by a secondary protein.