Walker S L, Hiremath L S, Galloway D R
Department of Microbiology, Ohio State University, Columbus 43210-1292.
Gene. 1995 Feb 27;154(1):15-21. doi: 10.1016/0378-1119(94)00870-x.
The Pseudomonas aeruginosa (Pa) structural gene (toxA), which encodes the exotoxin A protein has been shown to be regulated at the transcriptional level by a protein designated ToxR (also known as RegA). We have previously reported that ToxR directly enhances toxA transcription in vitro; however, in the absence of ToxR, Pa RNA polymerase (RNAP) transcribes toxA with low efficiency. In the present study, we have examined the ability of ToxR to initiate toxA transcription using the heterologous Escherichia coli (Ec) RNAP and found that ToxR can function with Ec RNAP to efficiently transcribe toxA both in vitro and in vivo. Antibodies produced against the sigma 70 subunit of Ec RNAP inhibit ToxR-mediated enhancement of toxA transcription, suggesting that the RNAP holoenzyme (E sigma 70) is required for transcriptional activation of toxA. We further demonstrate that ToxR is required for open-complex formation at the toxA promoter. By selectively deleting toxA upstream sequences, we have localized at 214-bp region containing both the toxA promoter and a putative ToxR-binding site sufficient for ToxR-mediated transcription of toxA.
编码外毒素A蛋白的铜绿假单胞菌（Pa）结构基因（toxA）已被证明在转录水平上受一种名为ToxR（也称为RegA）的蛋白质调控。我们之前报道过，ToxR在体外可直接增强toxA转录；然而，在没有ToxR的情况下，Pa RNA聚合酶（RNAP）转录toxA的效率很低。在本研究中，我们使用异源大肠杆菌（Ec）RNAP检测了ToxR启动toxA转录的能力，发现ToxR可与Ec RNAP共同作用，在体外和体内高效转录toxA。针对Ec RNAP的σ70亚基产生的抗体可抑制ToxR介导的toxA转录增强，这表明RNAP全酶（Eσ70）是toxA转录激活所必需的。我们进一步证明，ToxR是toxA启动子开放复合物形成所必需的。通过选择性删除toxA上游序列，我们将一个214 bp的区域定位在其中，该区域包含toxA启动子和一个足以实现ToxR介导的toxA转录的假定ToxR结合位点。
