Genes encoding osmoregulatory proline/glycine betaine transporters and the proline catabolic system are present and expressed in diverse clinical Escherichia coli isolates.

Sixty-three clinical isolates identified as Escherichia coli, 30 from the human urinary tract and 33 derived from other human origins, were screened for proline/glycine betaine transporters similar to those that support proline catabolism and proline- or glycine betaine-based osmoregulation in E. coli K-12. Both molecular (DNA- and protein-based) analyses and physiological tests were performed. All tests were calibrated with E. coli K-12 derivatives from which genetic loci putP (encoding a proline transporter required for proline catabolism), proP, and (or) proU (loci encoding osmoregulatory proline/glycine betaine transporters) had been deleted. All clinical isolates showed both enhanced sensitivity to the toxic proline analogue azetidine-2-carboxylate on media of high osmolality and growth stimulation by glycine betaine in an artificial urine preparation of high osmolality. DNA sequences similar to the putP, proP, and proU loci of E. coli K-12 were detected by DNA amplification and (or) hybridization and protein specifically reactive with antibodies raised against the ProX protein of E. coli K-12 (a ProU constituent) was detected by western blotting in over 95% of the isolates. Two anomalous isolates were reclassified as non-E. coli on the basis of the API 20E series of tests. A protein immunochemically cross-reactive with the ProP protein of E. coli K-12 was also expressed by the clinical isolates. Since all three transporters were ubiquitous, no particular correlation between clinical origin and PutP, ProP, or ProU activity was observed. These data suggest that the transporters encoded in loci putP, proP, and proU perform housekeeping functions essential for the survival of E. coli cells in diverse habitats.

Hydrolysis of proline dipeptides completely fulfills the proline requirement in a proline-auxotrophic Chinese hamster ovary cell line.

Proline- and hydroxyproline-containing oligopeptides may be important in protein nutrition because intestinal hydrolases are incapable of recognizing their imido bonds. Peripheral tissues have a cytosolic enzyme prolidase that cleaves dipeptides containing C-terminal proline (X-Pro) or hydroxyproline. The role of dipeptides in intracellular metabolism is uncertain. This study examined the ability of X-Pro to provide proline to the proline-auxotrophic cell line, CHO-K1. The action of prolidase on exogenously supplied Gly-Pro, the most abundant dipeptide product of digestion, provided adequate proline to support normal cell growth of CHO-K1 cells in a dose-dependent manner. The growth curve generated by addition of Gly-Pro to CHO-K1 cells was similar to that due to proline. Two other structurally unrelated X-Pro also supported growth indistinguishably from Gly-Pro. Gly-Hyp was completely ineffective for growth. The ability of X-Pro to sustain cultures of a proline-auxotrophic cell line may be important in elucidating intracellular nutritional and physiological functions for those dipeptides.