Glycine Betaine Uptake Research Articles

Fate of compatible solutes during dilution stress inEctothiorhodospira halochloris

Ectothiorhodospira halochloris reacts upon enhancement of the water activity in the environment by excreting its major compatible solute, glycine betaine, thus decreasing the osmotic pressure inside the cell. A suddenly induced dilution stress leads to an overshoot of this reaction, so that more glycine betaine than necessary to compensate the external osmotic change is released. Subsequently the cells take up glycine betaine until they reach osmotic balance with the medium. E. halochloris possesses an active transport system that allows an uptake of glycine betaine against a concentration gradient. Glycine betaine is not metabolized in E. halochloris. Ectoine, a minor compatible solute of E. halochloris, is excreted in a similar manner to that of glycine betaine during dilution stress, whereas no excretion of the third compatible solute, trehalose, was detected.

Fate of compatible solutes during dilution stress in Ectothiorhodospira halochloris

Ectothiorhodospira halochloris reacts upon enhancement of the water activity in the environment by excreting its major compatible solute, glycine betaine, thus decreasing the osmotic pressure inside the cell. A suddenly induced dilution stress leads to an overshoot of this reaction, so that more glycine betaine than necessary to compensate the external osmotic change is released. Subsequently the cells take up glycine betaine until they reach osmotic balance with the medium. E. halochloris possesses an active transport system that allows an uptake of glycine betaine against a concentration gradient. Glycine betaine is not metabolized in E. halochloris. Ectoine, a minor compatible solute of E. halochloris, is excreted in a similar manner to that of glycine betaine during dilution stress, whereas no excretion of the third compatible solute, trehalose, was detected.

Uptake of glycine betaine and its analogues by bacteroids of Rhizobium meliloti

Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent Km for glycine betaine uptake was 8.3 microM and V was about 0.84 nmol min-1 (mg protein)-1 in the presence of 200 mM-NaCl which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, gamma-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.

Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems.

The proU loci of Salmonella typhimurium and Escherichia coli encode high-affinity glycine betaine transport systems which play an important role in survival under osmotic stress. Transcription of the proU locus is tightly regulated by osmolarity and this regulation appears to be mediated by osmotically induced changes in DNA supercoiling. In order to study the regulatory mechanisms involved we have cloned and characterized the proU locus of S. typhimurium by an in vivo transductional procedure. The locus is shown to consist of at least three genes, designated proVWX, cotranscribed as a single operon. The first gene in the operon encodes a protein sharing considerable sequence identity with ATP-binding proteins from other periplasmic transport systems. Unexpectedly, the highly expressed periplasmic glycine betaine binding protein was found to be encoded by a distal gene, proX, in the operon. The operon has no significant internal promoters but is expressed from a single osmoregulated promoter whose transcription start site has been mapped. The proU promoter of E. coli has also been sequenced and the transcription start site shown to be similar to that of S. typhimurium. Evidence is presented which suggests that, besides de novo glycine betaine uptake, an important function of ProU may be the recapture and recycling of other osmolytes that leak from the cell.

A glycine betaine transport system in Aphanothece halophytica and other glycine betaine-synthesising cyanobacteria

Uptake of exogenous 14C-glycine betaine has been followed in the cyanobacterium Aphanothece halophytica and other species able to synthesise glycine betaine in response to osmotic stress. At 1 mmol dm−3 uptake was rapid (flux rate=29.50 nmol m−2 s−1), equilibrating at an internal concentration of 120 mmol dm−3 within 30 min. This rapid uptake, coupled with high internal accumulation, was characteristic of glycine betaine-synthesising cyanobacteria only. The 14C-glycine betaine transported was not catabolised. Kinetic studies indicated a Michaelis-Menten type relationship (K m=2.0 μmol dm−3, V max=45 nmol min−1 mm−3 cell volume), with a pH optimum of 8.0–8.5. Darkness dramatically decreased the flux rate. Higher 14C-glycine betaine levels occurred in cells growth in medium of elevated osmotic strength, and glycine betaine uptake was sensitive to changes in external salinity. A relationship between Na+ availability and glycine betaine uptake was observed, with >80 mmol dm−3 Na+ required for optimal stimulation of uptake in seawater-grown cells. Severe hyperosmotic stress (1000 mmol dm−3 NaCl) reduced the rate of glycine betaine uptake but increased internal glycine betaine concentration at equilibrium. Hypo-osmotic stress caused a decline in the internal glycine betaine concentration due to an increased rate of loss, indicating that the efflux system was also sensitive to ambient salinity changes. It is envisaged that this active transport system may be an adaptive mechanism in halophilic glycine betaine-synthesising cyanobacteria.

Genetics of osmoregulation in Escherichia coli: Uptake and biosynthesis of organic osmolytes

Escherichia coli cells growing under osmotic stress activate systems for the transport or synthesis of several organic osmolytes. Glutamate and trehalose syntheses seem to represent mechanisms for achieving a low level of osmotic tolerance. The uptake and synthesis of betaines represent mechanisms of achieving a high level of osmotic tolerance. The osmotically-controlled ProP and ProU systems are involved in the uptake of glycine betaine and proline. However, glycine betaine synthesis occurs only in the presence of the precursor molecule choline. The osmotically controlled genes governing the high-affinity uptake of choline are located in close proximity to those encoding the dehydrogenases involved in the oxidation of choline to glycine betaine, but represent a different transcriptional unit. It is not known if each of these systems has its own osmotic sensor or whether a common osmotic sensor regulates all cell osmolytes.

Genetics of osmoregulation inEscherichia coli: Uptake and biosynthesis of organic osmolytes

Escherichia coli cells growing under osmotic stress activate systems for the transport or synthesis of several organic osmolytes. Glutamate and trehalose syntheses seem to represent mechanisms for achieving a low level of osmotic tolerance. The uptake and synthesis of betaines represent mechanisms of achieving a high level of osmotic tolerance. The osmotically-controlled ProP and ProU systems are involved in the uptake of glycine betaine and proline. However, glycine betaine synthesis occurs only in the presence of the precursor molecule choline. The osmotically controlled genes governing the high-affinity uptake of choline are located in close proximity to those encoding the dehydrogenases involved in the oxidation of choline to glycine betaine, but represent a different transcriptional unit. It is not known if each of these systems has its own osmotic sensor or whether a common osmotic sensor regulates all cell osmolytes.

Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaine by oxidation of choline through glycine betaine aldehyde (choline----glycine betaine aldehyde----glycine betaine; B. Landfald and A.R. Strøm, J. Bacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codAB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aldehyde dehydrogenase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betaine uptake or low-affinity choline uptake, both of which were osmotically regulated.

Variations in the response of salt-stressed Rhizobium strains to betaines

A total of 15 rhizobial strains representing Rhizobium meliloti, Rhizobium japonicum, Rhizobium trifolii, Rhizobium leguminosarum, Rhizobium sp. (Sesbania rostrata) and Rhizobium sp. (Hedysarum coronarium), were studied with regard to growth rate under salt stress in defined liquid media. In the presence of inhibitory concentrations of NaCl, enhancement of growth resulting from added glycine betaine was observed for R. meliloti strains and Rhizobium sp. (Hedysarum coronarium) but not for other Rhizobium species. The concentration of glycine betaine required for maximal growth stimulation was very low (1 mM) in comparison with the osmolarity of the medium. The stimulation was shown to be independent of any specific solutes. Other related compounds like proline betaine, carnitine, choline, γ-butyrobetaine and pipecolate betaine were also effective compounds in restoring the growth rate of cells grown in medium of elevated osmolarity. High rate of glycine betaine uptake was demonstrated in R. meliloti cells grown in media of increased osmotic strength. The intracellular concentration of this solute was found to be 308 mM in 0.3 M NaCl-grown cells and 17 times lower in minimal medium-grown cells. Glycine betaine was used for growth under conditions of low osmolarity but could not serve as sole carbon or nitrogen source in medium of increased osmotic strength. Experiments with [14C]glycine betaine showed that this molecule was not metabolized by cells subjected to osmotic stress, whereas it was rapidly converted to dimethylglycine, sarcosine and glycine in minimal medium-grown cells.

Glycine betaine transport in Escherichia coli: osmotic modulation.

Exogenous glycine betaine highly stimulates the growth rate of various members of the Enterobacteriaceae, including Escherichia coli, in media with high salt concentrations (D. Le Rudulier and L. Bouillard, Appl. Environ. Microbiol. 46:152-159, 1983). In a nitrogen- and carbon-free medium, glycine betaine did not support the growth of E. coli either on low-salt or high-salt media. This molecule was taken up by the cells but was not catabolized. High levels of glycine betaine transport occurred when the cells were grown in media of elevated osmotic strength, whereas relatively low activity was found when the cells were grown in minimal medium. A variety of electrolytes, such as NaCl, KCl, NaH2PO4, K2HPO4, K2SO4, and nonelectrolytes like sucrose, raffinose, and inositol triggered the uptake of glycine betaine. Furthermore, in cells subjected to a sudden osmotic upshock, glycine betaine uptake showed a sixfold stimulation 30 min after the addition of NaCl. Part of this stimulation might be a consequence of protein synthesis. The transport of glycine betaine was energy dependent and occurred against a concentration gradient. 2,4-Dinitrophenol almost totally abolished the glycine betaine uptake. Azide and arsenate exerted only a small inhibition. In addition, N,N'-dicyclohexylcarbodiimide had a very low inhibitory effect at 1 mM. These results indicated that glycine betaine transport is driven by the electrochemical proton gradient. The kinetics of glycine betaine entry followed the Michaelis-Menten relationship, yielding a Km of 35 microM and a Vmax of 42 nmol min-1 mg of protein-1. Glycine betaine transport showed considerable structural specificity. The only potent competitor was proline betaine when added to the assay mixtures at 20-fold the glycine betaine concentration. From these results, it is proposed that E. coli possesses an active and specific glycine betaine transport system which is regulated by the osmotic strength of the growth medium.