title: Performance of standard phenotypic assays for TonB activity, as evaluated by varying the level of functional, wild-type TonB.
abstract: The ability of gram-negative bacterial cells to transport cobalamin and iron-siderophore complexes and their susceptibility to killing by some bacteriophages and colicins are characteristics routinely used to assay mutations of proteins in the TonB-dependent energy transduction system. These assays vary greatly in sensitivity and are subject to perturbation by overexpression of TonB and, perhaps, other proteins that contribute to the process. Thus, the choice of assay and the means by which a potential mutant is expressed can greatly influence the interpretation and recognition of a given mutant. In the present study, we expressed TonB at several different quantified levels in cells that were then subjected to a panel of assays. Our results suggest that it is reasonable to regard the assays as having windows of sensitivity. Thus, while no single assay satisfactorily spans the potential range of TonB activity, it is evident that certain assays are better suited for resolving small deviations from wild-type levels of activity, with others most useful when activity levels are very low. It is apparent from the results that the application of all possible assays to the characterization of new mutants will yield the most meaningful results.
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234, USA.
authors.forename: Ray A
authors.initials: RA
authors.lastname: Larsen
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Chen
authors.forename: Kathleen
authors.initials: K
authors.lastname: Postle
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D010582:Bacteriophage lambda; D003087:Colicins / Q000494:pharmacology; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000821:virology; D029968:Escherichia coli Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010641:Phenotype; D017262:Siderophores / Q000378:metabolism; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003087:Colicins; D003470:Culture Media; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D017262:Siderophores; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D014805:Vitamin B 12