• 主页
  • 查找结果
  • MtrA, an essential response regulator of the MtrAB two-component system, regulates the transcription of resuscitation-promoting factor B of Mycobacterium tuberculosis.

MtrA, an essential response regulator of the MtrAB two-component system, regulates the transcription of resuscitation-promoting factor B of Mycobacterium tuberculosis.

Microbiology (Reading, England) (2015-04-03)
Arun Kumar Sharma, Ayan Chatterjee, Shamba Gupta, Rajdeep Banerjee, Sukhendu Mandal, Jayanta Mukhopadhyay, Joyoti Basu, Manikuntala Kundu
摘要

The resuscitation-promoting factors of Mycobacterium tuberculosis are hydrolytic enzymes, which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of M. tuberculosis from chronic infection in vivo, underscoring the need to understand its transcriptional regulation. Here, we identified the transcriptional and translational start points of rpfB, and suggested from rpf promoter-driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrated that rpfB transcription is regulated by MtrA - the response regulator of the essential two-component system MtrAB. Association of MtrA with the rpfB promoter region in vivo was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the MtrA-binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter. Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the MtrA-binding site, and repression of rpfB expression, upon overexpression of MtrA, suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated by EMSAs showing diminished association of RNA polymerase (RNAP) with the rpfB promoter in the presence of MtrA. In vitro transcription assays confirmed that MtrA inhibits RNAP-driven rpfB transcription.

材料
货号
品牌
产品描述

Sigma-Aldrich
甘油, for molecular biology, ≥99.0%
Sigma-Aldrich
羟乙基哌嗪乙硫磺酸, ≥99.5% (titration)
Sigma-Aldrich
2-巯基乙醇, ≥99.0%
Sigma-Aldrich
甲醛 溶液, ACS reagent, 37 wt. % in H2O, contains 10-15% Methanol as stabilizer (to prevent polymerization)
Sigma-Aldrich
2-巯基乙醇, for molecular biology, suitable for electrophoresis, suitable for cell culture, BioReagent, 99% (GC/titration)
Sigma-Aldrich
氯化钠, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
十二烷基硫酸钠, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
羟乙基哌嗪乙硫磺酸, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
甲醛 溶液, for molecular biology, 36.5-38% in H2O
Sigma-Aldrich
脱氧胆酸钠, ≥97% (titration)
Sigma-Aldrich
甘油, ≥99.5%
Sigma-Aldrich
十二烷基硫酸钠, ACS reagent, ≥99.0%
Sigma-Aldrich
氯化钠, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Sigma-Aldrich
乙二胺四乙酸, ACS reagent, 99.4-100.6%, powder
Sigma-Aldrich
十二烷基硫酸钠, ReagentPlus®, ≥98.5% (GC)
Sigma-Aldrich
氯化钠, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
氯化钠 溶液, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
氯化钠 溶液, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
乙二胺四乙酸, anhydrous, BioUltra, ≥99% (titration)
Sigma-Aldrich
十二烷基硫酸钠 溶液, BioUltra, for molecular biology, 10% in H2O
Sigma-Aldrich
HEPES缓冲溶液, 1 M in H2O
Sigma-Aldrich
甘油, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
Sigma-Aldrich
氯化钠 溶液, 5 M
Sigma-Aldrich
脱氧胆酸钠, BioXtra, ≥98.0% (dry matter, NT)
Sigma-Aldrich
十二烷基硫酸钠, ≥99.0% (GC), dust-free pellets
Sigma-Aldrich
十二烷基硫酸钠, BioUltra, for molecular biology, ≥99.0% (GC)
Sigma-Aldrich
甲醛 溶液, for molecular biology, BioReagent, ≥36.0% in H2O (T)
SAFC
氯化钠 溶液, 5 M
Sigma-Aldrich
溴酚蓝