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首页> 外文期刊>Infection and immunity >Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.
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Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.

机译：鼠伤寒沙门氏菌100碱基碱基质粒的毒力基因的克隆和转座子诱变。

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摘要

We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.
机译：我们已经克隆了鼠伤寒沙门氏菌SR-11的100碱基对（kb）质粒pStSR100的区域，该区域赋予了质粒固化鼠伤寒沙门氏菌的毒力。携带赋予毒性的重组质粒的细胞是通过在重组质粒中经鼠瘟鼠伤寒沙门氏菌口服接种小鼠并收集感染脾脏的分离株来选择的。用粘粒载体pCVD305构建的三个质粒pYA401，pYA402和pYA403赋予质粒固化鼠伤寒沙门氏菌以野生型水平，并具有共同的14-kb DNA插入序列。用载体pACYC184构建的另一种重组质粒pYA422使质粒固化的鼠伤寒沙门氏菌具有野生型50％致死剂量（LD50）的水平，但小鼠的死亡比感染野生型鼠伤寒沙门氏菌的情况更慢。此外，pYA422赋予了在质粒固化的鼠伤寒沙门氏菌上引起较高水平但不是野生型水平的脾感染的能力。 pYA422具有一个3.2kb的插入序列，该序列映射到粘粒克隆的14kb共同序列的中心。 pYA403中的转座子Tn5插入突变在不同程度上抑制了毒力，当转入鼠伤寒沙门氏菌的天然毒力质粒中时，这些Tn5插入将毒力降低到类似于pYA403中存在Tn5插入时观察到的程度。相对于未诱变的毒力质粒，pStSR100中的vir-22 :: Tn5大大降低了脾脏的感染，而vir-26 :: Tn5和vir-27 :: Tn5则将脾脏感染的感染程度降低了一些。至少23个蛋白质由包含23 kb插入序列的pYA403和亚克隆pYA420编码，亚克隆pYA420包含所有粘粒克隆中存在的14 kb共同插入序列。这些蛋白质中的一种，其表观分子量为28,000，也由pYA422编码。 Tn5插入最弱毒力，vir-22 :: Tn5，抑制28,000分子量蛋白质的合成。 vir-22 :: Tn5插入被仅编码28,000分子量蛋白的重组质粒所补充，表明该蛋白在毒力中的作用。然而，仅编码28,000分子量蛋白质的重组质粒，例如pYA422，没有赋予完全的毒力。

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