Ogierman Monica, Braun Volkmar
Mikrobiologie/Membranphysiologie, Universität Tübingen, D-72076 Tübingen, Germany.
J Bacteriol. 2003 Mar;185(6):1870-85. doi: 10.1128/JB.185.6.1870-1885.2003.
Both induction of transcription of the ferric citrate transport genes and transport of ferric citrate by the Escherichia coli outer membrane receptor FecA require energy derived from the proton motive force (PMF) of the inner membrane. The energy is transduced to FecA by the inner membrane complex, TonB, ExbB, and ExbD. Region 160 of TonB and the conserved TonB box of other TonB-dependent receptors are implicated as sites of interaction. In the present study, the postulated TonB box (D(80)A(81)L(82)T(83)V(84)) of FecA was deleted in frame, with a subsequent loss of both FecA functions. DALTV of FecA could be functionally replaced with the core TonB boxes of FhuA (DTITV) and FepA (DTIVV). Each residue of the TonB box of FecA was sequentially replaced with cysteine residues, and only the D80C replacement showed a loss (reduction) of both FecA functions. A physical interaction between TonB and FecA was demonstrated using both in vivo site-specific disulfide bond cross-linking and nonspecific formaldehyde (FA) cross-linking. Pairwise combinations of FecA (DALTV)/Cys substitutions were cross-linked via disulfide bond formation with TonBQ160C, TonBQ162C, and TonBY163C. Unexpectedly, this cross-linking was not enhanced by substrate (ferric citrate). In contrast, the TonB-FecA interaction was enhanced by ferric citrate in the FA-cross-linking assay. Energy derived from the PMF was not required for the TonB-FecA interaction in either the disulfide- or FA-cross-linking assay. TonB/CysExbB/ExbD(D25N) was still able to cross-link with the FecA (DALTV)/Cys derivatives in a tonB tolQ background, even though ExbD25N renders the TonB/ExbBD complex nonfunctional (V. Braun, S. Gaisser, C. Herrmann, K. Kampfenkel, H. Killmann, and I. Traub, J. Bacteriol. 178:2836-2845, 1996). TonB cross-linked to FecA via FA was not inhibited by either carbonylcyanide-m-chlorophenylhydrazone or 1 mM 2,4-dinitrophenol, which dissipate the electrochemical potential of the cytoplasmic membrane and disrupt both FecA functions. The studies shown here demonstrate the significance of the TonB box for FecA functions and are consistent with the view that it is the structure and not the sequence of the TonB box that is important for activity. Demonstrated here for the first time is the physical interaction of TonB and FecA, which is enhanced by ferric citrate.
柠檬酸铁转运基因转录的诱导以及大肠杆菌外膜受体FecA对柠檬酸铁的转运都需要来自内膜质子动力（PMF）的能量。该能量通过内膜复合物TonB、ExbB和ExbD传递给FecA。TonB的第160位区域以及其他TonB依赖性受体的保守TonB框被认为是相互作用位点。在本研究中，FecA假定的TonB框（D(80)A(81)L(82)T(83)V(84)）被框内缺失，随后FecA的两种功能均丧失。FecA的DALTV可被FhuA（DTITV）和FepA（DTIVV）的核心TonB框功能性取代。FecA的TonB框的每个残基依次被半胱氨酸残基取代，只有D80C取代导致FecA的两种功能丧失（降低）。使用体内位点特异性二硫键交联和非特异性甲醛（FA）交联都证明了TonB与FecA之间存在物理相互作用。FecA（DALTV）/半胱氨酸取代的成对组合通过二硫键形成与TonBQ160C、TonBQ162C和TonBY163C交联。出乎意料的是，底物（柠檬酸铁）并未增强这种交联。相比之下，在FA交联试验中，柠檬酸铁增强了TonB - FecA的相互作用。在二硫键或FA交联试验中，PMF产生的能量对于TonB - FecA的相互作用并非必需。即使ExbD25N使TonB/ExbBD复合物失去功能（V. Braun、S. Gaisser、C. Herrmann、K. Kampfenkel、H. Killmann和I. Traub，《细菌学杂志》178:2836 - 2845，1996），在tonB tolQ背景下，TonB/CysExbB/ExbD(D25N)仍能与FecA（DALTV）/半胱氨酸衍生物交联。通过FA与FecA交联的TonB不受羰基氰化物 - m - 氯苯腙或1 mM 2,4 - 二硝基苯酚的抑制，这两种物质会消除细胞质膜的电化学势并破坏FecA的两种功能。此处所示的研究证明了TonB框对FecA功能的重要性，并且与认为对于活性重要的是TonB框的结构而非序列这一观点一致。首次证明了TonB与FecA的物理相互作用，该相互作用会被柠檬酸铁增强。
