ABSTRACT
In most Gram-negative bacteria, the TonB system is required to actively transport vital nutrients across their unenergized outer-membrane barriers. The cytoplasmic membrane proteins of the system ExbB, ExbD, and TonB work together to transduce the energy from the protonmotive force (PMF) of the inner membrane to the ligand-specific, outer membrane TonB-dependent transporters. However, the precise coordination and interactions between ExbB, ExbD, and TonB during energy transduction is unclear. Previously, deletions within a periplasmic disordered domain of ExbD had been shown to be important for TonB system activity and the essential ExbD-TonB PMF-dependent interaction. In this study, we have discovered a conserved motif, ΨXΨXLP (Ψ = hydrophobic-branched residues; X = non-hydrophobic residues) specifically within this domain that was required for both TonB system function and the ExbD-TonB PMF-dependent interaction. Alanine scanning mutagenesis found that the only functionally important residues within the ExbD disordered domain were those located in the motif. In addition, in vivo photo-cross-linking captured and identified five ExbD complexes from pBpa substitutions within the ExbD disordered domain: 2-ExbB-ExbD complexes, the ExbD homodimer, the PMF-dependent ExbD-TonB, and the first captured, ExbD-TonB independent interaction. Interestingly, many of these complexes were captured from single pBpa substitutions, which suggests that these residues are involved in multiple interactions. The data presented in this study indicate that the ExbD disordered domain is a highly dynamic region in vivo and a conserved motif within this domain is required for the TonB system to respond to protonmotive force.
Importance Within the context of pathogenicity, the TonB system is a virulence factor in many Gram-negative pathogens including E-S-K-A-P-E pathogenic species Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Because the TonB system is unique to Gram-negative bacteria and is a periplasmically localized virulence factor, it is an appealing target for novel antibiotics. However, there is no current antibiotic against this system. Understanding the mechanism by which the TonB system functions will provide valuable information to design potential inhibitors targeting the system.