Peterson K M, Mekalanos J J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Infect Immun. 1988 Nov;56(11):2822-9. doi: 10.1128/iai.56.11.2822-2829.1988.
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.
利用一个广泛宿主范围的载体来递送转座子TnphoA，构建了霍乱弧菌经典菌株O395的基因融合文库。在含有0.2%葡萄糖的LB琼脂平板上，于30℃筛选该插入文库中表达碱性磷酸酶阳性（PhoA+）融合蛋白的菌落。分离出600多个PhoA+菌株，然后在允许霍乱毒素高表达或低表达的肉汤培养基中测试它们基因融合的调控情况。这一策略导致分离出60个与编码分泌蛋白的基因的TnphoA（Tn5 IS50L::phoA）融合体，这些分泌蛋白显然与霍乱毒素协同调控。将toxR无效突变引入其中10个融合菌株，证实这些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的控制。Southern分析和免疫印迹分析相结合，确定了霍乱弧菌O395中17个不同的受ToxR调控的基因。这些插入中有许多位于O395菌株的两个霍乱毒素操纵子拷贝之一中，以及一个参与毒素共调节菌毛定植因子生物合成的大基因簇中。此外，在对霍乱毒素或毒素共调节菌毛表达均无影响的基因中也发现了插入。其中一些插入定位于一个由四个基因组成的簇中，TnphoA对其中任何一个基因的破坏都会降低O395菌株在乳鼠肠道中定植的能力。这个第二个基因簇编码的产物被命名为辅助定植因子，以描述其在霍乱发病机制中的可能作用。这些研究强化了受ToxR调控的基因对霍乱弧菌毒力特性的贡献。本报告还展示了一种鉴定细菌毒力因子的新方法，该方法基于对受控制已知毒力因子表达的相同环境信号调控的基因的表征。
