Regulation of cytoplasmic proline levels in Salmonella typhimurium: effect of osmotic stress on synthesis, degradation, and cellular retention of proline

Abstract

I investigated the effects of osmotic stress on the synthesis and catabolism of proline in Salmonella typhimurium by measuring the intracellular and extracellular proline levels in various strains. In the wild-type strain, exposure to 0.8 M NaCl did not cause a significant change in the intracellular proline level; however, it brought about a 6.5-fold increase in the intracellular glutamate pool size. These results indicate that 'y-glutamyl kinase is inhibited by proline in wild-type cells in media of normal or elevated osmolarity. I also tested whether proline is subject to turnover in cells wild type with respect to the enzymes of the proline degradation pathway. In strains that were wild type for proline biosynthesis, the loss of the proline catabolic enzymes, due to put4 mutations, did not result in a statistically significant increase in the intracellular proline levels. Therefore, in the wild-type strain, proline turnover does not seem to be important for control of the intracellular proline levels. However, in a proline-overproducing mutant, a putA lesion caused a threefold increase in the intracellular proline level and a 6.5-fold increase in the extraceilular proline level, indicating that proline is subject to turnover in the overproducing mutant. The proline-overproducing mutants excreted large quantities of the proline into the culture medium; osmotic stress altered the partitioning of proline such that the ratio of intracellular to extracellular levels of proline increased with increased osmotic stress. The increased cellular retention of proline in media of high osmolarity is probably due to the functioning of the ProP and ProU proline transport systems, which are stimulated under conditions of osmotic stress.In bacteria, proline is synthesized from glutamate by four reactions: (i) glutamate + ATP --y-glutamyl phosphate + ADP, (ii) -y-glutamyl phosphate + NADPH --y-glutamyl semialdehyde + NADP, (iii) -y-glutamyl semialdehyde <-+ A'-pyrroline-5-carboxylate + H20, and (iv) A1-pyrroline-5-carboxylate + NADPH -* proline + NADP (7, 12). The first, second, and fourth reactions are catalyzed by the enzymes -y-glutamyl kinase, -y-glutamyl phosphate reductase, and A'-pyrroline-5-carboxylate reductase, which are specified in members of the family Enterobactericeae by the proB+, proA+, and proC+ genes, respectively. The third reaction, the cyclization of y-glutamyl semialdehyde to Al-pyrroline-5-carboxylate, is spontaneous. The catabolism of proline entails two oxidation steps to glutamate via 17). In enteric bacteria, these reactions are mediated by a bifunctional protein with proline dehydrogenase (oxidase) and A1-pyrroline-5-carboxylate dehydrogenase activities, encoded in the putA+ gene (17).

Regulation of compatible solute accumulation in Salmonella typhimurium: evidence for a glycine betaine efflux system

“…4). The data presented by Csonka (1988) raise the interesting possibility that the efflux system for glycine betaine and proline is active at low osmolarity and that its activity is diminished at high osmotic pressure. The combination of activation of the uptake system and inhibition of the efflux system by high osmotic pressure would control the accumulation of the compatible solutes glycine betaine and proline.…”

“…However, early experiments to demonstrate activation of K+ efflux by high turgor created by downshock were similarly unsuccessful (Meury et al, 1985). It has been reported that osmotic pressure controls the amount of proline that is excreted into the medium by mutants of S. typhimurium that are osmotolerant due to overproduction of proline (Csonka, 1988). At low osmolarity excretion is high but at higher osmolarity the proline is retained by the cells.…”

Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

“…During anaerobic growth, proline (71.9 nmol ml Ϫ1 OD 550 unit Ϫ1 ) was the only osmolyte above our limit of detection (Յ4.0 nmol ml Ϫ1 OD 550 unit Ϫ1 ). Since allosteric regulation prevents the biosynthesis of proline as an osmoprotectant (12,41,42), it is likely that corn steep liquor is also the source of intracellular proline. Supplementing CSL medium with additional proline (17 mM) had no effect on cell growth or volumetric productivity despite a 14% increase in the intracellular pool.…”

The Chemical Chaperone Proline Relieves the Thermosensitivity of a dnaK Deletion Mutant at 42°C

“…Proline transport, however, does not result, even under osmotic stress conditions, in high intracellular proline pools (14). Proline is endogenously synthesized from glutamate by the proA, proB, and proC gene products, which are synthesized at similar levels regardless of the availability of proline (7). Proline synthesis is negatively regulated through feedback inhibition of the first biosynthetic enzyme of the pathway, the proB gene product ␥-glutamylkinase (2).…”