Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.The osmotic stress response of Escherichia coli and Salmonella typhimurium includes accumulation of K+ (17), amino acid derivatives, such as glycine betaine (18), and the amino acids glutamate and proline (3,20). Sugar transport is reduced by osmotic stress (31, 32). Proline is a particularly effective osmoprotectant; wild-type bacteria accumulate proline in response to osmotic stress if they are grown in complex media (3,20), but proline does not accumulate when S. typhimurium is grown in high-osmotic-strength, defined medium lacking proline (11). Proline-overproducing mutants show enhanced osmotic stress tolerance (11), and exogenously added proline is osmoprotective (8,9,11,12).Proline accumulation in E. coli and S. typhimurium is mediated by two transport systems, proline porter I (PPI) and PPII (1,21,34). These bacteria can utilize L-proline as a sole source of carbon and nitrogen by expressing the put genes (30,36). Mutations in putP eliminate the active accumulation of proline via PPI, which mediates Na+-proline symport (4, 7). The putA gene product catalyzes proline oxidation (22,33) and also serves as a repressor that controls the transcription of putP and putA (19,23,36). Strains defective in putP retain PPII, an active transport system that is induced during amino acid-limited growth and is inactivated by mutations in proP (1,21,34). A third porter with weak proline uptake activity, which is induced in S. typhimurium by osmotic stress, is inactivated by mutations in proU (12, 13).The observations described above suggest that increased proline transport is required to generate or maintain an osmoprotective transmembrane proline gradient. In this paper we describe a biochemical analysis of proline uptake by E. coli K-12, in which we examined the responses of the proline porters to changes in carbon source, to tryptophanlimited growth (a nutritional stress), and to high-osmo...