Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin
Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin
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DOI：
10.1016/j.mimet.2007.04.015
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发表时间：
2007-07-01
影响因子：
2.2
通讯作者：
Stein, Torsten
中科院分区：
文献类型：
--
作者：
Burkard, Michael;Entian, Karl-Dieter;Stein, Torsten
枯草芽孢杆菌ATCC 6633中lantibiotic枯草菌素的产生在群体感应样机制中被调节，其中枯草菌素作为自诱导物并通过枯草菌素特异性双组分调节系统SpaRK进行信号转导。在这里，我们报告了枯草杆菌素报告菌株的构建和应用，其中枯草杆菌素诱导lacZ基因在B中的表达。在显色平板测定中，通过β-半乳糖苷酶监测和可视化枯草杆菌ATCC 6633 spas基因缺失突变体。建立并优化了微量滴定板枯草杆菌素生物测定方法。在含有300 mM NaCl的培养基中，将报告菌株与枯草杆菌素一起孵育6 h后，系统达到最大灵敏度。将该方法应用于枯草杆菌素产生菌B的鉴定。枯草杆菌野生型菌株，培养物收集和土壤样品。B。与枯草菌素相比，具有四个氨基酸交换的枯草杆菌羊毛硫抗生素ericin S诱导枯草菌素报告菌株，与结构上密切相关的乳酸乳球菌羊毛硫抗生素乳链菌肽相反。这些观察结果表明组氨酸激酶SpaK具有一定的底物特异性，然而，这也将允许鉴定枯草杆菌蛋白同工型生产微生物。(C)2007 Elsevier B. V.保留所有权利。
Production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633 is regulated in a quorum sensing-like mechanism with subtilin acting as autoinducer and signal transduction via the subtilin-specific two-component regulation system SpaRK. Here, we report the construction and application of a subtilin reporter strain in which subtilin induced lacZ gene expression in a B. subtilis ATCC 6633 spas gene deletion mutant is monitored and visualized by the beta-galactosidase in a chromogenic plate assay. A quantitative microtiter plate subtilin bioassay was developed and optimized. Maximal sensitivity of the system was achieved after 6 h of incubation of the reporter strain together with subtilin in a medium containing 300 mM NaCl. This sensitive and unsusceptible method was applied to identify subtilin producing B. subtilis wild type strains from both, culture collections and soil samples. The B. subtilis lantibiotic ericin S with four amino acid exchanges compared to subtilin induces the subtilin reporter strain, in contrast to the structurally closely related Lactococcus lactis lantibiotic nisin. These observations suggest a certain substrate specificity of the histidine kinase SpaK, which however, also would allow the identification of subtilin-isoform producing microorganisms. (C) 2007 Elsevier B.V. All rights reserved.