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The Escherichia coli proU operon encodes a high-affinity, binding-protein-dependent transport system for the osmoprotectant glycine betaine. Expression of proU is osmoregulated, and transcription of this operon is greatly increased In cells grown at high osmolarity. Characterization of the proU operon and its promoter provided results similar to those published elsewhere (Gowrishankar, 1989; Stirling et al., 1989). The previously identified proU601 mutation, which leads to increased proU expression both at low- and high osmolarity, is a G to A transition in the Pribnow box of the proU promoter, which increases the homology of the -10 region to the consensus sequence of E. coli promoters. Using an antiserum raised against a ProV-β-gaiactosidase hybrid protein, we have identified ProV as a protein associated with the cytoplasmic membrane. This cellular location is consistent with its proposed role as the energy-coupling component of the ProU transport system. [ABSTRACT FROM AUTHOR]

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 Δ(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 μM), permeation of glycine betaine was entirely dependent on the OmpF and OmpC porins. [ABSTRACT FROM AUTHOR]