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首页> 外文期刊>Infection and immunity >Genetic Characterization of a Tn5-Disrupted Glycosyltransferase Gene Homolog in Brucella abortus and Its Effect on Lipopolysaccharide Composition and Virulence
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Genetic Characterization of a Tn5-Disrupted Glycosyltransferase Gene Homolog in Brucella abortus and Its Effect on Lipopolysaccharide Composition and Virulence

机译：流产布鲁氏菌中Tn5中断的糖基转移酶基因同源物的遗传特征及其对脂多糖组成和毒力的影响。

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We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kbXbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kbXbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been namedwboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype.B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.
机译：我们通过转座子（Tn 5 ）诱变构建了流产布鲁氏菌 2308的粗糙突变体。全细胞或从该突变体RA1中提取的脂多糖（LPS）均未与 Brucella O侧链特异性单克隆抗体（MAb）Bru-38反应，表明不存在O-侧链合成。菌株RA1的LPS组成分析表明，相对于亲本野生型菌株2308中的水平，喹喔胺和甘露糖水平降低。我们通过克隆a分离了菌株RA1中Tn 5 插入的DNA将25kb的 Xba I基因组片段插入pGEM-3Z中以产生质粒pJM6。 B基因组DNA的等位基因交换。 pJM6电穿孔介导的流产2308产生了抗卡那霉素的克隆，这些克隆不与MAb Bru-38反应。这些粗糙克隆的基因组DNA的Southern印迹分析揭示了25kb Xba I基因组片段中的Tn 5 。通过推导的Tn 5 破坏了开放阅读框的推导氨基酸序列进行同源搜索，发现与各种糖基转移酶的同源性有限。这个 B。流产基因已被命名为 wboA 。用带有野生型 B的宽宿主范围的质粒转化RA1菌株。流产的wboA 基因导致O-侧链合成的恢复和平滑表型。流产 RA1在小鼠体内的存活力减弱。然而，品系RA1在小鼠脾脏中的存活时间比 B更长。流产疫苗菌株RB51，但不出所料，这两种菌株均未诱导出对O侧链具有特异性的抗体。

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