Kim I, Stiefel A, Plantör S, Angerer A, Braun V
Mikrobiologie II, Universität Tübingen, Germany.
Mol Microbiol. 1997 Jan;23(2):333-44. doi: 10.1046/j.1365-2958.1997.2401593.x.
Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE without entering the cells of Escherichia coli K-12. Point mutants of the outer membrane-receptor protein FecA are affected in induction independent of the FecA transport activity, suggesting that FecA is directly involved in induction. Alignment of FecA with the other ferric siderophore receptors of E. coli reveals an N-terminal extension in FecA that is not found in the receptors whose synthesis is not induced by their cognate ferric siderophores. In this study, we show that excision of the N-terminal region abolished the inducing activity of FecA, but retained its transport activity. Overproduction of the N-terminal FecA fragment inhibited FecA-dependent induction, but not transport. Constitutive expression caused by C-terminally truncated FecR derivatives was not inhibited by the N-terminal FecA fragment. The N-terminal region of FecA was localized in the periplasm, which indicates that FecA probably interacts with FecR, which is involved in signal transduction across the cytoplasmic membrane. Transcription initiation of the fec transport genes required the Ton system, consisting of TonB, ExbB, and ExbD, and was inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), which dissipate the electrochemical potential of the cytoplasmic membrane. fec transcription of mutant fecA4, which displays constitutive fec transcription in the absence of TonB, was not affected by CCCP. The data support a model that proposes initiation of fec transport gene transcription by binding of ferric citrate to FecA. The transcription initiation signal is transferred across the outer membrane through the activity of the Ton system at the expense of the electrochemical potential of the cytoplasmic membrane. The N-terminus of FecA interacts in the periplasm with the C-terminus of FecR, through which the signal is transferred across the cytoplasmic membrane into the cytoplasm, where it increases the activity of the sigma factor Fecl, which then directs the RNA polymerase to the fec promoter upstream of fecA.
柠檬酸铁可诱导柠檬酸铁转运基因fecABCDE的转录，而无需进入大肠杆菌K-12细胞。外膜受体蛋白FecA的点突变体在诱导过程中受到影响，且与FecA的转运活性无关，这表明FecA直接参与诱导过程。将FecA与大肠杆菌的其他铁载体受体进行比对，发现FecA存在一个N端延伸，而在那些合成不受其同源铁载体诱导的受体中未发现该延伸。在本研究中，我们发现切除N端区域会消除FecA的诱导活性，但保留其转运活性。N端FecA片段的过量表达会抑制FecA依赖的诱导，但不影响转运。由C端截短的FecR衍生物引起的组成型表达不受N端FecA片段的抑制。FecA的N端区域定位于周质，这表明FecA可能与FecR相互作用，FecR参与跨细胞质膜的信号转导。fec转运基因的转录起始需要由TonB、ExbB和ExbD组成的Ton系统，并且受到羰基氰化物间氯苯腙（CCCP）和2,4-二硝基苯酚（DNP）的抑制，这两种物质会消耗细胞质膜的电化学势。在没有TonB的情况下表现出组成型fec转录的突变体fecA4的fec转录不受CCCP的影响。这些数据支持了一个模型，该模型提出柠檬酸铁与FecA结合启动fec转运基因的转录。转录起始信号通过Ton系统的活性跨外膜传递，代价是细胞质膜的电化学势。FecA的N端在周质中与FecR的C端相互作用，信号通过该相互作用跨细胞质膜传递到细胞质中，在那里它增加了σ因子Fecl的活性，然后Fecl引导RNA聚合酶到fecA上游的fec启动子处。
