Analysis of glutathione gated potassium efflux systems of bacteria
The KefB and KefC systems of E. coli are two separate potassium efflux systems that are regulated by glutathione and specific glutathione metabolites. The aims of this study were to clone and sequence the E. coli kefB structural gene and to investigate the subunit stoichiometry of the E. coli KefC protein. A further objective was to determine the distribution of the KefC class of transport system in a range of bacterial species, with the ultimate aim of cloning the kefC homologue from a highly divergent organism. Attempts to clone the kefB gene by various strategies proved unsuccessful; possible explanations for this are discussed. Analysis of the suppression of a kefC leaky mutation by plasmids bearing varying lengths of the wild-type kefC gene indicated that the mutant and wild-type KefC proteins interact in the membrane to form a structure that has intermediate properties. These observations are consistent with KefC functioning as an oligomer. Using a variety of techniques, it was demonstrated that the KefC class of transport system is widely distributed among Gram-negativespecies but is absent from all the Gram-positive bacteria tested with the exception of Staph. aureus. In addition the kefC homologue from Er. carotovora was cloned and DNA sequencing initiated. The cloned Er. carotovora kefC gene product was able to suppress an E. coli kefC leaky mutation, thus providing further evidence that KefC exists as an oligomer.