The ion coupling and organic substrate specificities of osmoregulatory transporter ProP in Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily, mediates osmoprotective proline or glycine betaine accumulation by bacteria exposed to high osmolality environments. Morpholinopropane sulfonic acid, a common constituent of microbiological media, accumulates in osmoadapting E. coli cells but it is not osmoprotective and it did not influence proP transcription or ProP activity. The apparent K(m) for proline uptake via ProP increased with decreasing pH in the range 7.5-4. ProP-dependent proline uptake by de-energized bacteria was associated with alkalinization of the external medium. Thus ProP mediates cotransport of H(+) and zwitterionic proline and a transporter functional group with a pK(a) of 5-6 is implicated in catalysis. Exogenous proline or glycine betaine elicits K(+) release from osmoadapting E. coli cells and ProP activity is stimulated by exogenous K(+). However, uptake of proline or glycine betaine stimulated K(+) efflux from K(+)-loaded bacteria which expressed either ProP or alternative, osmoregulatory transporter ProU. This indicated that ProP was unlikely to mediate K(+) efflux. Zwitterions ectoine, pipecolate, proline betaine, N,N-dimethylglycine, carnitine and 1-carboxymethylpyridinium were identified as alternative ProP substrates. Choline, a cation and a structural analogue of glycine betaine, was a low affinity inhibitor but not a substrate of ProP.

Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts.

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.