Identification and characterization of the dps promoter of Mycobacterium smegmatis:: Promoter recognition by stress-specific extracytoplasmic function sigma factors σH and σH
Identification and characterization of the dps promoter of Mycobacterium smegmatis:: Promoter recognition by stress-specific extracytoplasmic function sigma factors σH and σH
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DOI：
10.1128/jb.01222-07
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发表时间：
2007-12-01
影响因子：
3.2
通讯作者：
Chatterji, Dipankar
中科院分区：
文献类型：
--
作者：
Chowdhury, Rakhi Pait;Gupta, Surbhi;Chatterji, Dipankar
在氧气或营养供应耗尽的情况下，细菌的生存对其在压力条件下在宿主体内的长期生存非常重要。我们研究了耻垢分枝杆菌的DPS基因，它编码一种蛋白质DPS(饥饿细胞的DNA结合蛋白)，它在氧化和营养压力下过表达，并对细菌DNA提供双峰保护。因此，在体内鉴定DPS启动子是很重要的。我们克隆了耻垢分枝杆菌DPS基因1 kb的启动子区域，并将其克隆到位于lacZ基因上游的大肠杆菌-分枝杆菌穿梭载体pSD5B中。通过定量测定β-半乳糖苷酶活性，在固体培养和液体培养中检测启动子的活性。为了确定最小的启动子区域，在同一载体中进一步克隆了全长为200-kb的片段，并以同样的方法定量测定了β-半乳糖苷酶的活性。通过引物延伸分析确定该基因的+1转录起始点。在可能的启动子序列的-10和-20区域插入点突变，并确认了启动子序列。在多轮和单轮体外转录实验中，纯化的耻垢分枝杆菌核心RNA聚合酶与纯化的结核分枝杆菌sigma(A)或sigma(B)重组后不识别启动子。用含有DPS启动子的1-kb DNA片段进行的体内启动子特异性下拉分析证实，细胞外功能西格玛因子与这个饥饿诱导的启动子有关。DPS启动子的单轮转录进一步支持只有与sigma(H)或sigma(H)重组的核心RNA聚合酶才能产生正确的转录本的想法。
The survival of a bacterium with a depleted oxygen or nutrient supply is important for its long-term persistence inside the host under stressful conditions. We studied a gene, dps, from Mycobacterium smegmatis, encoding a protein, Dps (for DNA binding protein from starved cells), which is overexpressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the dps promoter in vivo is therefore important. We cloned a 1-kb putative promoter region of the dps gene of M. smegmatis in an Escherichia coli-Mycobacterium shuttle vector, pSD5B, immediately upstream of the lacZ gene. Promoter activities were assayed in vivo both in solid medium and in liquid cultures by quantitative P-galactosidase activity measurements. To characterize the minimal promoter region, a 200-bp fragment from the whole 1-kb sequence was further cloned in the same vector, and in a similar way, beta-galactosidase activity was quantitated. Primer extension analysis was performed to determine the +1 transcription start site of the gene. Point mutations were inserted in the putative promoter sequences in the -10 and -20 regions, and the promoter sequence was confirmed. The promoter was not recognized by purified M. smegmatis core RNA polymerase reconstituted with purified Mycobacterium tuberculosis sigma(A) or sigma(B) during multiple- and single-round in vitro transcription assays. Promoter-specific in vivo pull-down assays with an immobilized 1-kb DNA fragment containing the dps promoter established that extracellular function sigma factors were associated with this starvation-inducible promoter. Single-round transcription at the dps promoter further supported the idea that only core RNA polymerase reconstituted with sigma(H) or sigma(H) can generate proper transcripts.