CHARACTERIZATION OF THE EXBBD OPERON OF ESCHERICHIA-COLI AND THE ROLEOF EXBB AND EXBD IN TONB FUNCTION AND STABILITY

TonB protein appears to couple the electrochemical potential of the cy toplasmic membrane to active transport across the essentially unenergi zed outer membrane of gram-negative bacteria. ExbB protein has been id entified as an auxiliary protein in this process. In this paper we sho w that ExbD protein, encoded by an adjacent gene in the exb cluster at 65', was also required for TonB-dependent energy transduction and, li ke ExbB, was required for the stability of TonB. The phenotypes of exb B exbD(+) strains were essentially indistinguishable from the phenotyp es of exbB(+) exbD strains. Mutations in either gene resulted in the d egradation of TonB protein and in decreased, but not entirely absent, sensitivities to colicins B and Ia and to bacteriophage phi 80. Eviden ce that the absence of ExbB or ExbD differentially affected the half-l ives of newly synthesized and steady-state TonB was obtained. In the a bsence of ExbB or ExbD, newly synthesized TonB was degraded with a hal f-life of 5 to 10 min, while the half-life of TonB under steady-state conditions was significantly longer, approximately 30 min. These resul ts were consistent with the idea that ExbB and ExbD play roles in the assembly of TonB into an energy-transducing complex. While interaction between TonB and ExbD was suggested by the effect of ExbD on TonB sta bility, interaction of ExbD with TonB was detected by neither in vivo cross-linking assays nor genetic tests for competition. Assays of a ch romosomally encoded exbD::phoA fusion showed that exbB and exbD were t ranscribed as an operon, such that ExbD-PhoA levels in an exbB::Tn10 s train were reduced to 4% of the levels observed in an exbB(+) strain u nder iron-limiting conditions. Residual ExbD-PhoA expression in an exb B::Tn10 strain was not iron regulated and may have originated from wit hin the Tn10 element in exbB.