目的:在本研究中,我们使用荧光视频显微镜在噬菌体裂解前的几分钟内跟踪绿色荧光蛋白(GFP)标记的holin。我们的工作将先前的遗传和生化数据背景化,显示孔形成的开始时间以及与溶解破裂部位相关的holin低聚物形成的位置。此外,先前的工作表明,λ感染细胞的形态特征是在细胞极点开始的爆炸性事件;然而,其依据尚不明确。这项研究表明,holin最常在细胞极处寡聚化,并且寡聚化的位置与裂解爆裂的位置在空间上相关。因此,holin是噬菌体λ极性裂解形态的关键贡献者。
The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of
polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to
polar lysis morphology for phage lambda.