噬菌体诱导的革兰氏阴性细菌宿主的裂解通常需要一组噬菌体裂解蛋白,一个holin,内肽酶,和一个spanin系统,破坏三个细胞包膜层中的每一个。基因组注释和先前的研究确定了ShewanellaoneidenisprophageLambdaSo中的一个基因区域,其中包含潜在的holin和内溶素编码基因,但缺乏明显的spanin系统。通过候选方法的组合,突变筛选,表征,和显微镜,我们发现LambdaSo使用pinholin/信号锚释放(SAR)内溶素系统来诱导质子泄漏和细胞壁降解。在相应的基因之间,我们发现两个广泛嵌套的开放阅读框编码一个双组分Spanin模块Rz/Rz1。出乎意料的是,我们确定了LambdaSo诱导的细胞裂解严格需要的另一个因素,噬菌体蛋白Lcc6.Lcc6是一种具有65个氨基酸残基的跨膜蛋白,具有迄今为止未知的功能,它在细胞质膜中的holin水平起作用以允许内溶素释放。因此,LambdaSo介导的细胞裂解至少需要四种蛋白质因子(Pinholin,SAR内溶素,Spanin,和Lcc6)。该发现进一步扩展了参与宿主裂解和噬菌体出口的噬菌体蛋白的已知库。
目的:细菌的裂解可以产生多种后果,例如释放宿主DNA以培养强大的生物膜。噬菌体诱导的革兰氏阴性细胞裂解需要三层的破坏,外膜和内膜以及细胞壁。在大多数情况下,感染革兰氏阴性细胞的噬菌体的裂解系统包含使细胞膜破坏或去极化的holins,从而释放或激活内溶素,然后降解细胞壁。这个,反过来,允许spanins变得活跃并融合内外膜,完成细胞包膜破坏并允许噬菌体排出。这里,我们表明,这三种成分的存在可能不足以使细胞裂解,在已知的噬菌体中也暗示了这一点,可能需要进一步的因素。
Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase, and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization, and microscopy, we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton leakage and degradation of the cell wall. Between the corresponding genes, we found that two extensively nested open-reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR endolysin, spanin, and Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress.
Lysis of bacteria can have multiple consequences, such as the release of host DNA to foster robust biofilm. Phage-induced lysis of Gram-negative cells requires the disruption of three layers, the outer and inner membranes and the cell wall. In most cases, the lysis systems of phages infecting Gram-negative cells comprise holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membranes, completing cell envelope disruption and allowing phage egress. Here, we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages, further factors may be required.