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.     

Hyperosmotic hbridoma cell cultures: Increased monoclonal antibody production with addition of glycine betaine

When mouse hybridoma cells were grown in culture media which were made hyperosmotic through the addition of NaCl or sucrose, the specific rate of antibody production increased with medium osmolality, reaching approx. 1.9 times the level obtained at physiological osmolality. However, due to a simultaneous reduction of the maximal cell density in the hyperosmotic media, the effect of the increased production rate did not give significant increases in the maximum antibody titer obtained in the cultures. When the osmoprotective compound, glycine betaine, was included in the NaCl- or sucrose-stressed cultures, the specific antibody production rate wasincreased up to 2.6-fold and maximum antibody titer up to twofold over that obtained in the control culture (physiological osmolality). A similar pattern of response was observed when other osmoprotective compounds (sarcosine, proline, glycine) were added to NaCl-stressed hybridoma cell cultures. For the present experiments, the results suggest that medium osmolality, rather than growth rate, will determine the specific antibody production rate by hybridoma cell line 6H11 growing in hyperosmotic culture media. (c) 1994 John Wiley & Sons, Inc.     

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.     

Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12

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.     

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.     

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.     

Choline transport activity in Staphylococcus aureus induced by osmotic stress and low phosphate concentrations.

Uptake of [14C]choline upon hyperosmotic stress of exponential-phase Staphylococcus aureus cultures in a complex medium occurred after a delay of 2.5 to 3.5 h. This uptake could be prevented by chloramphenicol, suggesting that it occurred via an inducible transport system. Radioactivity from [14C]choline was accumulated as [14C]glycine betaine. However, neither choline nor glycine betaine could act as the major carbon and energy source for the organism, suggesting that choline was not metabolized beyond glycine betaine. Assay of choline transport activity in cells grown under different conditions in defined media revealed that osmotic stress was mainly responsible for the induction, but choline gave a further increase in induction. The system was not induced in anaerobically grown cells. Choline transport activity was repressed by glycine betaine and proline betaine, suggesting that these compounds are corepressors. Choline transport activity was not induced in cells osmotically stressed by 1 M potassium phosphate or 0.5 M sodium phosphate, but was induced in cells grown in low-phosphate medium in the absence of osmotic stress. This suggests that there is a connection between the phosphate and osmotic stress regulons. Choline transport was energy and Na+ dependent and had a Km of 46 microM and a maximum rate of transport (Vmax) of 54 nmol/min/mg (dry weight). The results of competition studies suggested that N-methyl and an alcohol group or aldehyde groups at the ends of the molecule were important in its recognition by the system. Glycine betaine was not a highly effective competitor, suggesting that its transport system and the choline transport system were distinct from each other. Choline transport was highly susceptible to a variety of inhibitors, which may be related to the greater dependence on respiratory metabolism of cells grown in the presence of high NaC1 concentrations.     

Osmoprotective effect of glycine betaine on thrombopoietin production in hyperosmotic Chinese hamster ovary cell culture: clonal variations

When 23 recombinant Chinese hamster ovary (rCHO) cell clones were cultivated in hyperosmolar medium resulting from NaCl addition (533 mOsm/kg), their specific thrombopoietin (TPO) productivity (q(TPO)) was increased. However, due to depressed cell growth at elevated osmolality, no enhancement in the maximum TPO titer was made in batch cultures of all 23 clones. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved TPO production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures of 23 clones were performed in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all 23 clones. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled 22 clones to grow at 542 mOsm/kg, where most clones could not grow in the absence of glycine betaine, but at a cost of reduced q(TPO). However, the relative decrease in q(TPO) varied significantly among clones. Thus, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among clones. Six out of 23 clones displayed more than a 40% increase in the maximum TPO titer in the hyperosmolar medium containing glycine betaine, compared with that in the standard medium with a physiological osmolality. Taken together, the results obtained here emphasize the importance of selection of clones for the successful use of hyperosmotic pressure and glycine betaine as an economical means to improve TPO production.     

Effect of novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), on the osmoprotectant activity of glycine betaine,cholien and l-proline in Escherichia coli

A novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), was found to block the osmoprotectant activity of choline and L-proline, but not glycine betaine in Escherichia coli. MPMS was more active against salt-sensitive than salt-resistant strains, but had no effect on the salt tolerance of a mutant which was unable to transport choline, glycine betaine and proline. Growth of E. coli in NaCl was inhibited by MPMS and restored by glycine betaine, but not by choline or L-proline. Uptake of radiolabeled glycine betaine, choline or L-proline by cells grown at high osmolarity was not inhibited when MPMS and the radioactive substrates were added simultaneously. Preincubation for 5 min with MPMS reduced the uptake of choline and L-proline, but not glycine betaine. Similar incubation with MPMS had no effect on the uptake of radiolabeled glucose or succinate. The toxicity of MPMS was much lower than that of the L-proline analogues L-azetidine-2-carboxylic acid and 3,4-dehydro-DL-proline. The exact mechanism by which MPMS exerts its effect is not entirely clear. MPMS or a metabolite may interfere with the activity of several independent permeases involved in the uptake of osmoprotective compounds, or the conversion of choline to glycine betaine, or effect the expression of some of the osmoregulatory genes.Abbreviations MPMS 1-methyl-1-piperidino-methane sulfonate     

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a K_D of 5.2 μM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (K_D of 1.4 μM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.     

The methanogenic archaeon Methanosarcina thermophila TM-1 possesses a high-affinity glycine betaine transporter involved in osmotic adaptation.

Methanogenic Archaea are found in a wide range of environments and use several strategies to adjust to changes in extracellular solute concentrations. One methanogenic archaeon, Methanosarcina thermophila TM-1, can adapt to various osmotic conditions by synthesis of alpha-glutamate and a newly discovered compatible solute, Ne-acetyl-beta-lysine, or by accumulation of glycine betaine (betaine) and potassium ions from the environment. Since betaine transport has not been characterized for any of the methanogenic Archaea, we examined the uptake of this solute by M. thermophila TM-1. When cells were grown in mineral salts media containing from 0.1 to 0.8 M NaC1, M. thermophila accumulated betaine in concentrations up to 140 times those of a concentration gradient within 10 min of exposure to the solute. The betaine uptake system consisted of a single, high-affinity transporter with an apparent K3 of 10 microM and an apparent maximum transport velocity of 1.15 nmol/min/mg of protein. The transporter appeared to be specific for betaine, since potential substrates, including glycine, sarcosine, dimethyl glycine, choline, and proline, did not significantly inhibit betaine uptake. M. thermophila TM-1 cells can also regulate the capacity for betaine accumulation, since the rate of betaine transport was reduced in cells pregrown in a high-osmolarity medium when 500 microM betaine was present. Betaine transport appears to be H+ and/or Na+ driven, since betaine transport was inhibited by several types of protonophores and sodium ionophores.     

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a K_D of 5.2 M for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (K_D of 1.4 M). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.     

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.     

Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics.

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid, identified as a compatible solute in moderately halophilic bacteria. Exogenously provided ectoine was found to stimulate growth of Escherichia coli in media of inhibitory osmotic strength. The stimulation was independent of any specific solute, electrolyte or nonelectrolyte. It is accumulated in E. coli cells proportionally to the osmotic strength of the medium, and it is not metabolized. Its osmoprotective ability was as potent as that of glycine betaine. The ProP and ProU systems are both involved in ectoine uptake and accumulation in E. coli. ProP being the main system for ectoine transport. The intracellular ectoine pool is regulated by both influx and efflux systems.     

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     

BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.