Leoni L, Orsi N, de Lorenzo V, Visca P
"Istituto Pasteur - Fondazione Cenci Bolognetti" - Istituto di Microbiologia, Università di Roma "La Sapienza", 00100 Rome, Italy.
J Bacteriol. 2000 Mar;182(6):1481-91. doi: 10.1128/JB.182.6.1481-1491.2000.
In Pseudomonas aeruginosa, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes, pvdS, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and FecI from Escherichia coli, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of P. aeruginosa is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, alpha(2)betabeta' subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the pvdA gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (pvd) promoters was significantly lower in E. coli overexpressing the multicopy pvdS gene than in wild-type P. aeruginosa PAO1 carrying the single gene copy, and pvd::lacZ transcriptional fusions were silent in both pfrI (the pvdS homologue) and pfrA (a positive regulator of pseudobactin biosynthetic genes) mutants of P. putida WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from E. coli lacked the ability to generate in vitro transcripts from the pvdA promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.
在铜绿假单胞菌中，铁通过一系列负调控和正调控蛋白来调节基因表达。主要调节因子Fur参与了多个基因的铁依赖性抑制。其中一个基因pvdS，预计编码一种假定的σ因子，负责Fur调控子中一部分基因的转录。PvdS似乎属于胞外功能家族替代σ因子中一个结构和功能独特的亚组。这个亚组的成员，还包括荧光假单胞菌的PbrA、恶臭假单胞菌的PfrI和PupI，以及大肠杆菌的FecI，都受Fur阻遏物的控制，并且它们激活铁载体生物合成或摄取相关基因的转录。有证据表明，铜绿假单胞菌的PvdS蛋白具有真细菌σ因子的生化特性，因为它能与RNA聚合酶（RNAP，α(2)ββ'亚基）的核心部分自发形成1:1复合物，从而促进PvdS - RNAP全酶在体外与pvdA基因的启动子区域结合。PvdS的这些功能特性与预测参与核心RNAP结合、启动子识别和开放复合物形成的结构域的存在是一致的。在过量表达多拷贝pvdS基因的大肠杆菌中，绿脓菌素生物合成（pvd）启动子的活性显著低于携带单基因拷贝野生型铜绿假单胞菌PAO1中的活性，并且pvd::lacZ转录融合在恶臭假单胞菌WCS358的pfrI（pvdS同源物）和pfrA（假菌素生物合成基因的正调控因子）突变体中均不表达，而在野生型WCS358中它们以PAO1水平表达。此外，从大肠杆菌中纯化的PvdS - RNAP全酶缺乏从pvdA启动子产生体外转录本的能力。这些观察结果表明，在体内和体外，PvdS依赖性转录复合物的完全活性可能至少还需要一个额外的正调控因子。这与荧光假单胞菌的铁饥饿σ因子PvdS、PfrI和PbrA所控制的启动子转录起始位点不同距离处存在一个假定的激活剂结合位点（铁饥饿盒）是一致的。
