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...

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.