A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron.
A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron.
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DOI：
10.3389/fmicb.2016.01080
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发表时间：
2016
影响因子：
5.2
通讯作者：
Stentz R
中科院分区：
文献类型：
--
作者：
Horn N;Carvalho AL;Overweg K;Wegmann U;Carding SR;Stentz R
人们对研究驻留在人类胃肠道（GI）中的拟杆菌属物种的功能及其对宿主健康的贡献非常感兴趣。反向遗传学和蛋白质表达技术，如那些开发的充分表征大肠杆菌不能应用于拟杆菌属物种，因为他们和其他成员的拟杆菌门有独特的启动子结构。因此，有用的类杆菌特异性遗传工具的可用性有限。本文描述了多形拟杆菌甘露聚糖调控基因表达系统的构建，该系统包含α-1，2-甘露糖苷酶基因（BT_3784）的甘露聚糖诱导型启动子区、一个用于调控表达的核糖体结合位点、一个便于克隆目的基因的多克隆位点和一个转录终止子。使用乳杆菌pepI作为报告基因，甘露聚糖诱导导致报告活性以时间和浓度依赖性方式增加，具有广泛的活性范围。内源性BtcepA头孢菌素酶基因被用来证明这种新的表达系统的适用性，使一个组氨酸标记的BtCepA的版本的分离。我们还通过在小鼠中进行的实验表明，该系统可以在存在外源性甘露聚糖源的情况下在体内诱导。通过使内源和外源基因在B中的受控表达成为可能。这种新的诱导物依赖性表达系统将有助于确定单个基因的生理作用及其产物的功能分析。
There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI)-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterized Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter–region of an α-1,2-mannosidase gene (BT_3784), a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products.