Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.     

Taste receptor stimulation and feeding behaviour in the puffer, Fugu pardalis. II. Effects produced by mixtures of constituents of clam extracts

The effects of extracts of short-necked clam, Tapes japonica,as feeding stimulants on the puffer, Fugu pardalis, were studiedby applying the constituents of the extracts in starch pellets.As observed elsewhere (Hidaka et al., 1978), the starch pelletcontaining all the constituents found in the clam extracts (Konosuet al., 1965), except homarine and choline, at the same concentrationratios as in the clam tissue was taken up by the fish. Omissiontests on the chemical constituents suggested that the palatabilityof the clam pellet might be largely produced by some amino acidsand betaine: Pellets containing alanine, glycine, proline, serineplus betaine were nearly as effective as those containing allthe extractive compounds except homarine and choline. Mixturesfree of either the above amino acids or betaine tested wereall less effective. The nucleotides and related substances andorganic acids detected in the clam extracts had no appreciableeffect except that a mixture of all of them was weakly acceptedby adding betaine to it. The other amino acids found in theextracts appear to be ineffective.     

Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish

Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also possibly cysteine. The specificities of these olfactory transduction processes in the catfish are similar to those for the biochemically determined receptor sites for amino acids in other species of fishes and to amino acid transport specificities in tissues of a variety of organisms.     

The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos

Betaine and proline protect preimplantation mouse embryos against increased osmolarity and decreased cell volume, implying that they may function as organic osmolytes. However, the transport system(s) that mediates their accumulation in fertilized eggs and early embryos was unknown, and previously identified mammalian organic osmolyte transporters could not account for their transport. Here, we report that there is a single saturable transport component shared by betaine and proline in 1-cell mouse embryos. A series of inhibitors had nearly identical effects on both betaine and proline transport by this system. In addition, K(i) values for reciprocal inhibition of betaine and proline transport were approximately 100-300 microM, similar to K(m) values ( approximately 200-300 microM) for their transport, and both had similar maximal transport rates (V(max)). The K(i) values for inhibition of betaine and proline transport by dimethylglycine were similar ( approximately 2 mM), further supporting transport of both substrates by a single transport system. Finally, betaine and proline transport each required Na(+)- and Cl(-). These data were consistent with a single, Na(+)- and Cl(-)-requiring, betaine/proline transport system in 1-cell mouse embryos. While betaine was only transported by a single saturable system, we found an additional, less conspicuous proline transport route that was betaine-insensitive, Na(+)-sensitive, and inhibited by alanine, leucine, cysteine, and methionine. Furthermore, we showed that betaine, like proline, is present in the mouse oviduct and thus could serve as a physiological substrate. Finally, accumulation of both betaine and proline increased with increasing osmolarity, consistent with a possible role as organic osmolytes in early embryos.     

Hyperosmotic volume regulation in the gills of the ribbed mussel, Geukensia demissa: rapid accumulation of betaine and alanine

The content of betaine and alanine in gills of the ribbed mussel Geukensia demissa increases rapidly following transfer of the tissues from 250 to 1000 mOsm seawater (SW). Increases in alanine, proline and glycine account for most of the increase in the amino acid pool. The betaine content increases from 45 to 150 μmol/g dry weight within 12 h. Transfer of isolated gills from 250 to 1000 mOsm SW results in a temporary cessation of all ciliary activity. Within 20–40 min following transfer, ciliary activity has recovered. Recovery of ciliary activity precedes recovery of tissue hydration. The uric acid content of gills is unchanged by exposure to hyperosmotic media, suggesting that uric acid is not a store of nitrogen for alanine synthesis from pyruvate. In other organisms, the accumulation of betaine in response to hyperosmotic stress is a slow (days to weeks) process that probably involves changes in gene expression. The rapid, large increases in betaine reported here suggest that gene expression is not a factor in volume recovery by euryhaline bivalve tissues exposed to acute hyperosmotic stress.     

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, -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.Abbreviations LAS lactate-aspartate-salts - LGS lactate-glutamate-salts - LS lactate-succinate - MSY mannitol-salts-yeast - YLS yeast-lactate-succinate     

Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [(14)C]betaine uptake. The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.     

Gustatory responses of eel palatine receptors to amino acids and carboxylic acids

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.     

Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica

Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. One of gene products (ORF1) catalyzed the methylation reactions of glycine and sarcosine with S-adenosylmethionine acting as the methyl donor. The other one (ORF2) specifically catalyzed the methylation of dimethylglycine to betaine. Both enzymes are active as monomers. Betaine, a final product, did not show the feed back inhibition for the methyltransferases even in the presence of 2 m. A reaction product, S-adenosyl homocysteine, inhibited the methylation reactions with relatively low affinities. The co-expressing of two enzymes in Escherichia coli increased the betaine level and enhanced the growth rates. Immunoblot analysis revealed that the accumulation levels of both enzymes in A. halophytica cells increased with increasing the salinity. These results indicate that A. halophytica cells synthesize betaine from glycine by a three-step methylation. The changes of amino acids Arg-169 to Lys or Glu in ORF1 and Pro-171 to Gln and/or Met-172 to Arg in ORF2 significantly decreased V(max) and increased K(m) for methyl acceptors (glycine, sarcosine, and dimethylglycine) but modestly affected K(m) for S-adenosylmethionine, indicating the importance of these amino acids for the binding of methyl acceptors. Physiological and functional properties of methyltransferases were discussed.     

Osmoregulation in Klebsiella pneumoniae: enhancement of anaerobic growth and nitrogen fixation under stress by proline betaine, gamma-butyrobetaine, and other related compounds

Exogenous proline betaine ( stachydrine or N- dimethylproline ) or gamma-butyrobetaine (gamma-trimethylaminobutyrate), at a concentration as low as 1 mM, were found to stimulate the growth rate of Klebsiella pneumoniae, wild type M5A1 , in media of inhibitory osmotic strength (0.8 M NaC1). Simultaneously, nitrogen fixation by whole cells, a process particularly sensitive to osmotic stress, was strongly enhanced by these compounds. However, in the absence of sodium chloride, both the growth and nitrogen fixation were not affected by the addition of the methylammonium derivatives in the medium. The sensitivity of the nitrogen fixation to osmotic stress was used as a bioassay to evaluate the potentiality of osmoprotective compound in relation to the number of methyl groups on the nitrogen atom of glycine, proline, and gamma-aminobutyrate. Experiments with sarcosine ( monomethylglycine ), dimethylglycine, and glycine betaine ( trimethylglycine ), or experiments with mono- and di- methylproline or gamma-mono-, gamma-di, gamma-tri- methylaminobutyrate , indicated that the greatest stress tolerance was always obtained with the more N-methylated compounds.     

Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic Methanoarchaeon Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate β-glutamine, N-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance ¹³C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of N-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis in M. portucalensis.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Accumulation of proline and glycine betaine in Ipomoea pes-caprae induced by NaCl

The present study pertains to the effect of different concentration of NaCl on the contents of proteins, free amino acids, proline and glycine betaine in leaves, stems and roots of Ipomoea pes-caprae. The protein content of the tissues increased in response to salinity upto 200 mM NaCl; the free amino acids content showed a reversal trend. The proline and glycine betaine contents increased with increasing salinity upto 500 mM NaCl. The accumulation of proline and glycine betaine might play a role in the alleviation of salt stress. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of glycine betaine,an osmoprotective compound on the growth of Brevibacterium lactofermentum

Summary Osmoregulation of Brevibacterium lactofermentum was examined. Exogenous glycine betaine was found to stimulate the growth rate of the bacterium in media of inhibitory osmotic strength. The stimulation was independent of any specific solute, electrolyte, or non-electrolyte. The bacterium did not utilize glycine betaine as a sole carbon source or nitrogen source, or degrade it even in complete medium. The changes in intracellular proline and glycine betaine concentrations were measured in media of different osmolarity. Brevibacterium lactofermentum grown in media without glycine betaine did not accumulate it, but synthesized several hyndred millimoles of proline inside the cells. On the other hand, when glycine betaine was added to the growth media, it accumulated in the cell instead of proline. These data indicate that glycine betaine is an osmoprotective compound for B. lactofermentum. Offprint requests to: Yoshio Kawahara     

Relationship between osmoprotection and the structure and intracellular accumulation of betaines by Escherichia coli

Abstract Naturally occuring betaines, especially glycine betaine and proline betaine, were accumulated by Escherichia coli from urine. In synthetic hyperosmotic medium, with an homologous series of added betaines, (CH₃)₃N⁺-(CH₂) _n -COO⁻, osmoprotective activity and intracellular accumulation decreased monotonically as n increased from 1 to 5. In contrast, α -substituted glycine betaines were accumulated in a similar manner to glycine betaine, but with different osmoprotective activities. Arsenobetaine, with a quaternary arsonium group, was also accumulated but amino acids which can become negatively charged in a chemically basic environment were not.     

Betaine aldehyde dehydrogenase in sorghum.

The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa.     

Electro-olfactogram and multiunit olfactory receptor responses to binary and trinary mixtures of amino acids in the channel catfish, Ictalurus punctatus

In vivo electrophysiological recordings from populations of olfactory receptor neurons in the channel catfish, Ictalurus punctatus, clearly showed that responses to binary and trinary mixtures of amino acids were predictable with knowledge obtained from previous cross-adaptation studies of the relative independence of the respective binding sites of the component stimuli. All component stimuli, from which equal aliquots were drawn to form the mixtures, were adjusted in concentration to provide for approximately equal response magnitudes. The magnitude of the response to a mixture whose component amino acids showed significant cross-reactivity was equivalent to the response to any single component used to form that mixture. A mixture whose component amino acids showed minimal cross-adaptation produced a significantly larger relative response than a mixture whose components exhibited considerable cross-reactivity. This larger response approached the sum of the responses to the individual component amino acids tested at the resulting concentrations in the mixture, even though olfactory receptor dose-response functions for amino acids in this species are characterized by extreme sensory compression (i.e., successive concentration increments produce progressively smaller physiological responses). Thus, the present study indicates that the response to sensory stimulation of olfactory receptor sites is more enhanced by the activation of different receptor site types than by stimulus interaction at a single site type.     

Amino acid receptor sites in the facial taste system of the sea catfish Arius felis

1. The amino acid sensitivity and specificity of the facial taste system of the marine catfish, Arius felis, is characterized electrophysiologically.
2. The facial taste system of Arius felis responded to all 28 amino acids tested, but was highly sensitive to only a few. In general, acidic amino acids and neutral amino acids with short side chains were more effective than imino, basic and neutral amino acids with long side chains.
3. A reciprocal cross-adaptation protocol used to characterize the receptor sites identified at least some relatively independent receptor sites for L-arginine, L-histidine, L-proline, L-alanine, glycine, D-alanine and L-glutamate.
4. Of the 7 amino acids that were indicated to have relatively independent receptor sites, the median electrophysiological threshold for L-alanine, the most stimulatory, and L-proline, the least stimulatory compounds, were 10 nM and 10,000 nM, respectively. The integrated facial taste response did not saturate at test amino acid concentrations up to 10 mM.
5. The generalized depression in responsiveness to test stimuli observed during amino acid adaptation is proposed to be a result of the co-distribution of sensitivity at the level of single taste cells rather than high cross-reactivity of the respective amino acid receptor sites for the test stimuli.

     

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

The regulation of glycine betaine accumulation has been investigated in Salmonella typhimurium. The size of the glycine betaine pool in the cells is determined by the external osmotic pressure and is largely independent of the external glycine betaine concentration. Analysis of the activity of the ProP and ProU transport systems suggests that other systems must be active in the regulation of the glycine betaine pool. Addition of p-chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulphonate (PCMBS) to cells that have accumulated glycine betaine provokes rapid loss of glycine betaine. The route of glycine betaine efflux under the influence of PCMB is independent of either the ProP or ProU transport systems. Rapid loss of the accumulated pool of glycine betaine in the presence of PCMB is specific to glycine betaine and proline; accumulated pools of serine and lysine are not significantly affected by the -SH reagent. A specific glycine betaine/proline efflux system is postulated on the basis of these data and its role in the regulation of glycine betaine and proline accumulation is discussed.