Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus.
Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus.
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DOI：
10.1099/mgen.0.000669
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发表时间：
2021-11
影响因子：
3.9
通讯作者：
Wilkinson B
中科院分区：
文献类型：
--
作者：
Gomez-Escribano JP;Algora Gallardo L;Bozhüyük KAJ;Kendrew SG;Huckle BD;Crowhurst NA;Bibb MJ;Collis AJ;Micklefield J;Herron PR;Wilkinson B
棒状链霉菌是一种工业上重要的放线菌，其遗传操作受到转化和接合效率低、引入的DNA重组水平低以及难以获得一致的孢子形成的限制。我们描述了用于该菌株 Cas9 介导的基因组编辑的多功能载体的构建和应用。为了充满信心地设计间隔序列，我们为典型菌株（ATCC 27064）的分离株获得了高度准确的基因组组装。这产生了染色体组装体 (6.75 Mb) 以及 pSCL4 (1795 kb) 和 pSCL2 (149 kb) 的组装体。该菌株还携带 pSCL1 (12kb)，但其尺寸较小，导致仅覆盖部分序列。该分离株中不存在先前描述的 pSCL3 (444kb)。使用我们的 Cas9 载体，我们通过靶向质粒的 parB 基因，高效地治愈了 pSCL4。对所得 pSCL4 治愈的分离株中的 5 个进行了表征，所有分离株都显示出孢子形成受损。对这些衍生物的鸟枪基因组测序显示，所有这些衍生物的染色体末端都有大量缺失，并且对于两个克隆，获得了足够的序列数据来表明染色体已经环化。总而言之，这些数据表明 pSCL4 对于线性染色体的结构稳定性至关重要。
Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.
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BAMVIEW：在参考序列的上下文中查看映射的读取对齐数据。
影响因子：
5.8
作者：
Carver, Tim;Bohme, Ulrike;Otto, Thomas D.;Parkhill, Julian;Berriman, Matthew
通讯作者：
Berriman, Matthew
影响因子：
4.6
作者：
Fernández-Martínez LT;Bibb MJ
通讯作者：
Bibb MJ
影响因子：
5
作者：
Funnell BE
通讯作者：
Funnell BE
DOI：
10.1128/genomea.01020-16
发表时间：
2016-09-22
期刊：
Genome announcements
影响因子：
--
作者：
Cao G;Zhong C;Zong G;Fu J;Liu Z;Zhang G;Qin R
通讯作者：
Qin R
影响因子：
4.7
作者：
Cobb, Ryan E.;Wang, Yajie;Zhao, Huimin
通讯作者：
Zhao, Huimin