Evidence for a TonB‐dependent energy transduction complex in Escherichia coli

Escherichia coli TonB protein is required for the active transport of vitamin B₁₂ and Fe(III)‐siderophore complexes across the outer membrane, infection by bacteriophages T1 and φ80, and sensitivity to B‐group colicins. TonB appears to function as an energy transducer in these processes, coupling cytoplasmic membrane electrochemical potential to receptors in the outer membrane. Previous reports have demonstrated that chromosomally encoded TonB is functionally unstable in the absence of protein synthesis (half‐life ∼15–30 minutes) and have shown that plasmid‐encoded, overexpressed TonB is chemically unstable (half‐life ∼5 minutes). In contrast, this study has shown that chromosomally encoded TonB was chemically stable for greater than 90 minutes while maintaining its functional instability. These data suggest that proteolytic degradation of TonB protein is not the basis of its functional instability. Auxiliary proteins such as ExbB also play a role in TonB‐dependent energy transduction. In this study, we have shown that the chemical half‐life of chromosomally encoded TonB in an exbB::Tn10 mutant was reduced at least 18‐fold, suggesting that TonB is a part of a cytoplasmic membrane complex that includes, at the minimum, ExbB. These results also suggest that the chemical instability of plasmid‐encoded TonB resulted when the TonB ExbB ratio was too high and are consistent with previous observations that plasmid‐encoded ExbB can stabilize plasmid‐encoded TonB. The exbB mutation also resulted in a significant decrease in TonB function as measured by the ability of cells to adsorb bacterio‐phage Φ80. In a previous study, strains carrying a tolQ nonsense mutation in combination with an exbB mutation were observed to mimic a tonB phenotype, suggesting that either ExbB or TolQ can alternatively activate TonB. In contrast, we have shown that neither the chemical half‐life nor the function of TonB are affected by the tolQ mutation, and thus, unlike ExbB, TolQ plays a minimal role in TonB‐dependent processes.