Welz D, Braun V
Mikrobiologie/Membranphysiologie, Universität Tübingen, Germany.
J Bacteriol. 1998 May;180(9):2387-94. doi: 10.1128/JB.180.9.2387-2394.1998.
Transcription of the ferric citrate transport genes of Escherichia coli is induced by ferric citrate bound to the outer membrane receptor FecA. Additional ferric citrate-specific regulatory proteins are FecR in the cytoplasmic membrane and the FecI sigma factor in the cytoplasm. To further understand the assumed FecR-mediated signal transduction across the cytoplasmic membrane, the transmembrane topology of FecR (317 amino acids) was determined with hybrid proteins containing portions of FecR and mature BlaM beta-lactamase. BlaM fused to FecR regions extending from residues 107 to 149 and residues 230 to 259 conferred high ampicillin resistance to cells, while BlaM fused to sites between residues 159 and 210 and between residues 265 and 301 conferred low resistance. Cells that synthesized FecR'-BlaM with fusion joints between residues 8 and 81 of FecR were fully sensitive to ampicillin. The ampicillin resistance of the low-resistance FecR'-BlaM hybrids was increased 2- to 10-fold by cosynthesis of plasmid-encoded GroEL GroES and SecB chaperones and in degP and ompT protease mutants, which suggested that the decreased ampicillin resistance level of these hybrids was caused by the formation of inclusion bodies and proteolytic degradation. Replacement of glycine by aspartate residues in the only hydrophobic FecR sequence (residues 85 to 100) abolished the beta-lactamase activity of high-resistance FecR'-BlaM proteins, indicating that there are no other transmembrane regions in FecR that translocate BlaM into the periplasm independent of the hydrophobic sequence. All FecR'-BlaM proteins with at least 61 FecR residues complemented a fecR mutant such that it could grow on ferric citrate as the sole iron source and induced fecA-lacZ transcription independent of ferric citrate. The low resistance mediated by two FecR'-BlaM proteins in a fecA deletion mutant was increased 20-fold by transformation with a fecA-encoding plasmid. We propose that FecR spans the cytoplasmic membrane once, interacts in the periplasm with its C-terminal region with FecA occupied by ferric citrate, and transmits the information through the cytoplasmic membrane into the cytoplasm, where it converts FecI into an active sigma factor.
大肠杆菌柠檬酸铁转运基因的转录由与外膜受体FecA结合的柠檬酸铁诱导。其他柠檬酸铁特异性调节蛋白包括位于细胞质膜的FecR和位于细胞质的FecI σ因子。为了进一步了解假定的FecR介导的跨细胞质膜的信号转导，利用含有FecR部分和成熟BlaMβ-内酰胺酶的杂合蛋白确定了FecR（317个氨基酸）的跨膜拓扑结构。与FecR从第107位到149位以及从第230位到259位残基延伸区域融合的BlaM赋予细胞高氨苄青霉素抗性，而与第159位到210位以及第265位到301位残基之间位点融合的BlaM赋予低抗性。合成FecR'-BlaM且融合接头位于FecR第8位到81位残基之间的细胞对氨苄青霉素完全敏感。通过共合成质粒编码的GroEL GroES和SecB伴侣蛋白以及在degP和ompT蛋白酶突变体中，低抗性FecR'-BlaM杂合体的氨苄青霉素抗性提高了2至10倍，这表明这些杂合体氨苄青霉素抗性水平降低是由包涵体形成和蛋白水解降解导致的。在唯一的疏水FecR序列（第85位到100位残基）中用天冬氨酸残基取代甘氨酸消除了高抗性FecR'-BlaM蛋白的β-内酰胺酶活性，表明FecR中不存在其他独立于疏水序列将BlaM转运到周质中的跨膜区域。所有具有至少61个FecR残基的FecR'-BlaM蛋白都能互补fecR突变体，使其能够在柠檬酸铁作为唯一铁源的条件下生长，并诱导fecA-lacZ转录，且不依赖于柠檬酸铁。在fecA缺失突变体中由两种FecR'-BlaM蛋白介导的低抗性通过用编码fecA的质粒转化提高了20倍。我们提出FecR跨细胞质膜一次，在周质中其C末端区域与被柠檬酸铁占据的FecA相互作用，并通过细胞质膜将信息传递到细胞质中，在那里它将FecI转化为活性σ因子。
