Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence
Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence
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DOI：
10.1128/iai.67.8.3830-3835.1999
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发表时间：
1999-08-01
影响因子：
3.1
通讯作者：
Boyle, SM
中科院分区：
文献类型：
--
作者：
McQuiston, JR;Vemulapalli, R;Boyle, SM
我们通过转座子（Tn5）诱变构建了流产布鲁氏菌2308的粗突变体。该突变体（命名为 RA1）的全细胞和提取的脂多糖 (LPS) 均未与布鲁氏菌 O 侧链特异性单克隆抗体 (MAb) Bru-38 发生反应，表明不存在 O 侧链合成。菌株 RA1 的 LPS 组成分析显示，与亲本野生型菌株 2308 相比，喹诺糖胺和甘露糖的水平降低。我们通过将 25-kb XbaI 基因组片段克隆到 pGEM-3Z 中以创建质粒 pJM6，分离了菌株 RA1 中 Tn5 插入侧翼的 DNA。 pJM6 电穿孔介导的流产芽孢杆菌 2308 中基因组 DNA 的等位基因交换产生了卡那霉素抗性：不与 MAb Bru-38 反应的克隆。对这些粗克隆的基因组 DNA 进行 Southern 印迹分析，揭示了 25 kb XbaI 基因组片段中的 Tn5。对被 Tn5 破坏的开放阅读框推导的氨基酸序列进行的同源性搜索显示与各种糖基转移酶的同源性有限。该流产布鲁氏菌基因被命名为 wboA。用带有野生型流产布鲁氏菌 wboA 基因的广宿主范围质粒转化菌株 RA1，导致 O-侧链合成和平滑表型的恢复。 B. abortus RA1 被减毒以保证小鼠的存活。然而，RA1 菌株在小鼠脾脏中的存留时间比流产布鲁氏菌疫苗菌株 RB51 更长，但正如预期的那样，两种菌株均未诱导 O 侧链特异性抗体。
We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) ham 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-kb XbaI 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-kb XbaI 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 named wboA. 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.