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 phi 80, 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 approximately 15-30 minutes) and have shown that plasmid-encoded, overexpressed TonB is chemically unstable (half-life approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)