Methionine Importers in Soil Bacteria: Potential for Transporter-Component Crosstalk

Abstract

ABC transporters are widely distributed amongst kingdoms and are responsible for nutrient transport across cellular membranes. Certain soil bacteria contain a higher than average percentage of ABC transporters, representing 40-70% of encoded transporters. While studies of SBPs have indicated that permeases can interact with multiple SBPs with varying affinity and specificity, limited information exists regarding permease specificity for ATPases. The potential for permeases to interact with multiple ATPases was suggested given the high genetic redundancy of homologous types of ABC transporters in plant growth promoting bacteria (PGPB). Genomic analyses of four Pseudomas fluorescens strains identified a tractable set of ABC-type amino acid importers with high sequence similarity to the structurally characterized E. coli methionine importer MetNI, sharing the presence of a C-terminal C2 regulatory domain on the ATPase. This domain inhibits methionine uptake by preventing ATP hydrolysis when intracellular methionine is bound.Using a dual-expression-vector strategy, both native complexes and ‘hybrid’ complexes were stably isolated. Native complexes utilize permease and ATPase components from an individual operon whereas hybrid complexes partner a permease domain from one operon with an ATPase domain from another. Interestingly, three target ATPases from strain PF-5 were able to form stable complexes with a single PF-5 permease target, suggesting this strain has high potential for hybrid interactions. These PF-5 hybrid complexes are shown to be dimers by gel filtration and functional by ATPase assays.The transinhibition mechanism of the C2 domain was also investigated, showing that D- and L-methionine differentially inhibit ATP hydrolysis of PF-5 hybrid complexes. Initial studies of SBPs associated with these complexes show a correlation between SBP ligands and compounds allosterically inhibiting ATPases. The ability for crosstalk could be especially significant for PGPB, allowing them to provide rapid and specific nutrient exchange with symbiotic species.