Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene.
Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene.
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DOI：
10.1038/srep29495
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发表时间：
2016-07-12
影响因子：
4.6
通讯作者：
Hutchings MI
中科院分区：
文献类型：
--
作者：
Munnoch JT;Widdick DA;Chandra G;Sutcliffe IC;Palmer T;Hutchings MI
细菌脂蛋白是通过共价附着的脂质附着在细胞膜上的细胞外蛋白。脂蛋白信号肽酶（lsp）基因的缺失会导致链霉菌的生长发育缺陷，而这种缺陷无法通过重新引入lsp来恢复。这使我们假设lsp是必不可少的，并且我们先前分离的lsp突变体已经获得了代偿性次生突变。在这里，我们报告了野生型M145和顺式互补的∆lsp突变体（BJT1004）的基因组重测序，以绘制和鉴定这些次生突变，但我们表明它们不会增加破坏lsp的效率，也不是lsp抑制子。我们提供的证据表明，它们是通过引入cosmid St4A10∆lsp诱导的，作为ReDirect PCR诱变方案的一部分，该方案短暂地复制了许多重要的细胞分裂基因。使用自杀载体破坏lsp（不导致基因复制）仍然会导致生长和发育延迟，我们得出结论，lsp功能的丧失会导致发育缺陷，这是由于细胞膜上所有脂蛋白的丢失。值得注意的是，我们的结果还表明，使用cosmid文库对细菌进行遗传操作可以导致不一定与感兴趣的基因相关的表型。
Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10∆lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest.