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
复制标题
基于粘粒的诱变导致天蓝色链霉菌遗传不稳定，如脂蛋白信号肽酶基因靶向所示。
DOI：
10.1101/049320
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Munnoch J
中科院分区：
文献类型：
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作者：
Munnoch J
细菌脂蛋白是通过共价连接的脂质与细胞膜相连的细胞外蛋白。天蓝色链霉菌脂蛋白信号肽酶（lsp）基因缺失导致生长和发育缺陷，不能通过重新引入lsp来恢复。这使我们假设lsp是必需的，我们以前分离的突变体获得了补偿性的二次突变。在这里，我们报告了野生型M145和互补的lsp突变体（BJT 1004）的基因组重测序，以定位和鉴定这些二级突变，但我们表明，它们不能增加破坏lsp的效率，也不是lsp抑制因子。我们提供的证据表明，它们是通过引入粘粒St4A10 Δ lsp诱导的，作为ReDirect PCR诱变方案的一部分，其瞬时复制了许多重要的细胞分裂基因。使用自杀载体的破坏（其不导致基因复制）仍然导致生长和发育延迟，并且我们得出结论，Lsp功能的丧失由于细胞膜上所有脂蛋白的丧失而导致发育缺陷。值得注意的是，我们的结果还表明，使用粘粒文库进行细菌的遗传操作可能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 inStreptomyces coelicolorresults in growth and developmental defects that cannot be restored by reintroducinglsp. This led us to hypothesise thatlspis essential and that thelspmutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and thecis-complemented ∆lspmutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disruptinglspand are notlspsuppressors. 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 oflspusing 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.