Tetracycline-inducible gene regulation in mycobacteria.
Tetracycline-inducible gene regulation in mycobacteria.
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DOI：
10.1093/nar/gni023
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发表时间：
2005-02-01
影响因子：
14.9
通讯作者：
Robertson BD
中科院分区：
文献类型：
--
作者：
Blokpoel MC;Murphy HN;O'Toole R;Wiles S;Runn ES;Stewart GR;Young DB;Robertson BD
四环素诱导的分枝杆菌基因表达调控系统已经开发出来。我们已经将谷氨酸棒杆菌TetZ基因座的tetRO区域亚克隆到分枝杆菌穿梭质粒中，使tetRO下游克隆的基因表达对四环素有反应。使用来自哈氏弧菌的luxAB编码的荧光素酶作为报告基因（pMind-Lx），我们观察到在添加20 ng ml−1四环素后2 h，耻垢分枝杆菌培养物的光输出增加了40倍。类似地，暴露于药物导致携带报告构建体的牛分枝杆菌BCG的相对光单位增加高达20倍，结核分枝杆菌增加10倍。在对数期和稳定期培养物中证实了四环素诱导。为了评估该系统是否适合在体内使用，用牛分枝杆菌BCG[pMind-Lx]感染J774巨噬细胞，用阿米卡星处理以杀死细胞外细菌，然后用四环素孵育。24小时后测量到光输出增加10倍，表明细胞内细菌可接近并响应于外源添加的四环素。为了测试四环素诱导系统用于条件性基因沉默的用途，用具有tetRO的pMind构建体转化分枝杆菌，所述tetRO驱动ftsZ基因的反义RNA的表达。含有反义构建体的细菌细胞在暴露于四环素24小时后形成细丝。这些结果证明了这种四环素调控系统在细胞内外操纵分枝杆菌基因表达的潜力。
A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml−1 of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.
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影响因子：
2.1
作者：
Triccas, JA;Parish, T;Gicquel, B
通讯作者：
Gicquel, B
影响因子：
120.7
作者：
Dye, C;Scheele, S;Raviglione, RC
通讯作者：
Raviglione, RC
影响因子：
3.1
作者：
Snewin, VA;Gares, MP;Young, DB
通讯作者：
Young, DB
DOI：
10.1099/00221287-139-3-575
发表时间：
1993-03-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
--
作者：
MAHENTHIRALINGAM, E;DRAPER, P;COLSTON, MJ
通讯作者：
COLSTON, MJ
影响因子：
2.1
作者：
Narayanan, S;Selvakumar, S;Narayanan, PR
通讯作者：
Narayanan, PR