The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation.
The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation.
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DOI：
10.1371/journal.pone.0111252
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
BRaDP1T Consortium
中科院分区：
文献类型：
--
作者：
Bossé JT;Soares-Bazzolli DM;Li Y;Wren BW;Tucker AW;Maskell DJ;Rycroft AN;Langford PR;BRaDP1T Consortium
我们已经开发了一种简单的方法，通过利用这种细菌进行自然转化的能力，在胸膜肺炎放线杆菌中产生无瘢痕，无标记的突变，并且不需要引入编码重组酶或解离酶的质粒。该方法涉及使用线性DNA的连续两轮天然转化：第一轮引入携带cat（其允许通过氯霉素选择）和sacB（其允许使用蔗糖进行反选择）的盒，所述盒的侧翼序列位于靶基因的任一侧;第二次转化利用直接彼此连接的侧翼序列以去除cat-sacB盒。为了确保转化过程中靶DNA的有效摄取，A.将胸膜肺炎菌摄取序列加入到两轮转化中使用的构建体中。该方法可用于产生多个连续缺失，也可用于将靶点突变或异源基因插入A.用于开发减毒活疫苗株的胸膜肺炎染色体。到目前为止，我们已经将这种方法应用于血清型8（MIDG 2331）和15（HS 143）的高度可转化分离株，血清型8在英国最流行。通过筛选其他血清型的临床分离株，应该有可能鉴定出其他适合的菌株。
We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.
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