9408006 Postle Energy for the active transport of iron-bearing siderophores, vitamin B-12, and other ligands across the outer membrane of Gram-negative bacteria is transduced from the cytoplasmic membrane by a cytoplasmic membrane protein complex consisting of TonB and a set of auxiliary proteins. Recent results from this laboratory using in vivo crosslinking have demonstrated that TonB protein can interact directly with high affinity receptors in the outer membrane to transduce energy. In this project, both genetic and biochemical experiments will be performed to dissect the interactions between TonB and the outer membrane receptors. Preliminary data suggest that the ability of TonB to fractionate with the M-band (a sucrose density gradient fraction proposed to consist of attached cytoplasmic and outer membranes) is determined by its level of activity. Thus the ability of TonB to form stable associations with the outer membrane --- via outer membrane receptors --- should be influenced by the several parameters to be tested: iron availability; protonophores; effects of TonB mutants; etc. The sites through which TonB interacts the the outer membrane receptors will be mapped by crosslinking interactions and genetic tests for dominant negativity. %%% Gram-negative eubacteria are shielded from their environment by a multilayered external structure, consisting of an inner (cytoplasmic) membrane, a rigid wall, and an outer membrane. The outer membrane serves as a permeability barrier which selectively allows for transport of needed metabolites. Some metabolites (small hydrophilic molecules, such as sugars and amino acids) can traverse the outer membrane freely. Others, such as larger molecules or those which are relatively rare in the environment and thus need to be transported actively into the cell, are brought in via energy-dependent, high-affinity specific mechanisms. In the model bacterium, Escherichia coli, and other Gram-negatives, this transport mechanism is mediated by a mul ticomponent system that couples cytoplasmic membrane energy to active transport across the outer membrane. The central component of this system in E. coli is a small (26 Kilodalton) protein which is the product of the tonB genetic locus. This bacterial gene was originally discovered through mutational phenotypes in 1943, and its vital significance to the life of the bacterial cell was documented heavily in the 1970's. However, it is only relatively recently that biochemical studies of the protein have made possible the formulation of plausible hypotheses about how this protein works. This project addresses the question of TonB structure and mechanism of action. The results of this work will increase our understanding of the biochemical mechanisms underlying bacterial assimilation of essential nutrients from the environment. This kind of information has key implications for understanding microbial biodiversity. ***
