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首页> 外文期刊>Infection and immunity >Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization.
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Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization.

机译：霍乱弧菌ToxR regulon的表征：鉴定涉及肠道菌落的新基因。

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A gene fusion library of Vibrio cholerae classical strain O395 was generated by using a broad host range vector for delivery of the transposon TnphoA. The insertion library was screened for colonies expressing alkaline phosphatase-positive (PhoA+) fusion proteins on LB agar at 30 degrees C in the presence of 0.2% glucose. Over 600 PhoA+ strains were isolated and then tested for regulation of their gene fusions in broth media that permitted high or low expression of cholera toxin. This strategy resulted in the isolation of 60 TnphoA (Tn5 IS50L::phoA) fusions to genes encoding secreted proteins that are apparently coordinately regulated with cholera toxin. Introduction of a toxR null mutation into 10 of these fusion strains confirmed that these TnphoA gene fusions are controlled either directly or indirectly by the cholera toxin transcriptional activator encoded by toxR. A combination of Southern and immunoblot analysis identified 17 distinct ToxR-regulated genes in V. cholerae O395. Many of these insertions were located in one of the two cholera toxin operon copies of strain O395, as well as a large gene cluster involved in the biogenesis of the toxin-coregulated pilus colonization factor. In addition, insertions were identified in genes that had no effect on either cholera toxin or toxin-coregulated pilus expression. Several of these insertions were localized to a cluster of four genes, the disruption of any of which by TnphoA reduced the ability of strain O395 to colonize the intestines of suckling mice. The product encoded by this second gene cluster was named accessory colonization factor to describe its possible role in cholera pathogenesis. These studies reinforce the contribution of ToxR-regulated genes to the virulence properties of V. cholerae. This report also demonstrates a new approach for the identification of bacterial virulence factors, based on the characterization of genes that are regulated by the same environmental signals that control the expression of a known virulence factor.
机译：霍乱弧菌经典菌株O395的基因融合文库是通过使用广泛的宿主载体传递转座子TnphoA产生的。在0.2％葡萄糖存在下，于30℃在LB琼脂上筛选表达文库中表达碱性磷酸酶阳性（PhoA +）融合蛋白的菌落。分离出600多种PhoA +菌株，然后在允许高或低表达霍乱毒素的肉汤培养基中测试其基因融合的调控。该策略导致了60个TnphoA（Tn5 IS50L :: phoA）融合蛋白与编码分泌型蛋白的基因的分离，这些蛋白显然受到霍乱毒素的调控。将toxR无效突变引入这些融合菌株中的10种中，证实了这些TnphoA基因融合体直接或间接受toxR编码的霍乱毒素转录激活因子控制。 Southern和免疫印迹分析的组合在霍乱弧菌O395中鉴定了17个不同的ToxR调控基因。这些插入物中的许多位于菌株O395的两个霍乱毒素操纵子拷贝之一中，以及一个与毒素结合的菌毛定殖因子的生物发生有关的大基因簇中。另外，在对霍乱毒素或毒素结合的菌毛表达均无影响的基因中鉴定出插入。这些插入中的几个被定位在四个基因的簇中，其中任何一个被TnphoA破坏都会降低O395菌株在乳鼠肠道中定植的能力。第二个基因簇编码的产物被称为辅助定居因子，以描述其在霍乱发病中的可能作用。这些研究加强了ToxR调控基因对霍乱弧菌毒力特性的贡献。该报告还展示了一种鉴定细菌毒力因子的新方法，该方法基于对基因进行表征的基因，这些基因受控制已知毒力因子表达的相同环境信号调控。

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