Institut Pasteur, Unité de Biologie des Spirochètes, Paris, France.
Appl Environ Microbiol. 2010 Dec;76(24):8135-42. doi: 10.1128/AEM.02199-10. Epub 2010 Oct 29.
Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.
与其他细菌物种相比，我们对钩端螺旋体发病机制的遗传和分子基础的了解非常有限。要深入了解发病机制，就需要可靠的遗传工具来进行功能基因分析。在这里，我们报告了 GFP 和 mRFP1 基因在腐生和致病性钩端螺旋体菌株中受组成型螺旋体启动子控制的表达情况。我们能够通过荧光显微镜和基于荧光酶标仪的检测方法可靠地测量钩端螺旋体的荧光。结果表明，GFP 基因的表达对 L. interrogans 的体内生长和致病性没有显著影响。我们构建了一个包含 lacI 阻遏物、诱导型 lac 启动子和 GFP 作为报告基因的 L. biflexa 表达载体，证明了 lac 系统在钩端螺旋体中是功能性的。在携带表达载体的 L. biflexa 转化体中加入异丙基-β-D-硫代半乳糖苷（IPTG）可诱导 GFP 表达。最后，我们表明 GFP 可用于评估不同环境条件下启动子活性。这些结果可能有助于进一步研究钩端螺旋体属的遗传学。
