The proU locus of Escherichia coli encodes a high-affinity, binding-protein-dependent transport system (ProU) for the osmoprotectant glycine betaine. We cloned this locus into both low-copy-number lambda vectors and multicopy plasmids and demonstrated that these clones restore osmotically controlled synthesis of the periplasmic glycine betaine binding protein (GBBP) and the transport of glycine betaine in a delta (proU) strain. These clones allowed us to investigate the influence of osmolarity on ProU transport activity independent of the osmotically controlled expression of proU. ProU activity was strongly stimulated by a moderate increase in osmolarity and was partially inhibited by high osmolarity. This activity profile differs from the profile of the osmotically regulated proU expression. The proU locus is organized in an operon and the position of the structural gene (proV) for GBBP is defined using a minicell system. We determined that at least three proteins (in addition to GBBP) are encoded by the proU locus. We also investigated the permeation of glycine betaine across the outer membrane. At low substrate concentration (0.7 microM), permeation of glycine betaine was entirely dependent on the OmpF and OmpC porins.