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首页> 外文期刊>Infection and immunity >Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.
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Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.

机译：放线放线杆菌的定向基因组整合：确定的白细胞毒素阴性突变体的产生。

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摘要

To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.
机译：为了在牙周病原体放线放线杆菌中发展靶向基因整合，将含有白细胞毒素基因片段的基于ColE1的抗壮观霉素质粒电穿孔到菌株JP2中。在所有出现的对壮观霉素抗性稳定的转化子中，由于基于ColE1的载体无法在放线杆菌中进行染色体外复制，因此该质粒已重组入基因组白细胞毒素基因座。然后，通过用含有白细胞毒素基因内部片段的基于ColE1的质粒转化白细胞毒素产生菌株JP2，直接进行基因组整合来构建白细胞毒素阴性菌株。细胞毒性试验证明，这些转化体的亲本菌株白细胞毒素活性<0.1％。这些结果表明，基于整合的方法可用于在放线放线杆菌的特定毒力基因中产生等基因突变体。

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