Osmoregulation in Agrobacterium tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength and glycine betaine.

We have investigated the mechanism of osmotic stress adaptation (osmoregulation) in Agrobacterium tumefaciens biotype I (salt-tolerant) and biotype II (salt-sensitive) strains. Using natural-abundance 13C nuclear magnetic resonance spectroscopy, we identified all organic solutes that accumulated to significant levels in osmotically stressed cultures. When stressed, biotype I strains (C58, NT1, and A348) accumulated glutamate and a novel disaccharide, beta-fructofuranosyl-alpha-mannopyranoside, commonly known as mannosucrose. In the salt-sensitive biotype II strain K84, glutamate was observed but mannosucrose was not. We speculate that mannosucrose confers the extra osmotic tolerance observed in the biotype I strains. In addition to identifying the osmoregulated solutes that this species synthesizes, we investigated the ability of A. tumefaciens to utilize the powerful osmotic stress protectant glycine betaine when it is supplied in the medium. Results from growth experiments, nuclear magnetic resonance spectroscopy, and a 14C labeling experiment demonstrated that in the absence of osmotic stress, glycine betaine was metabolized, while in stressed cultures, glycine betaine accumulated intracellularly and conferred enhanced osmotic stress tolerance. Furthermore, when glycine betaine was taken up in stressed cells, its accumulation caused the intracellular concentration of mannosucrose to drop significantly. The possible role of osmoregulation of A. tumefaciens in the transformation of plants is discussed.     

Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli.

Glycine betaine and its precursors choline and glycine betaine aldehyde have been found to confer a high level of osmotic tolerance when added exogenously to cultures of Escherichia coli at an inhibitory osmotic strength. In this paper, the following findings are described. Choline works as an osmoprotectant only under aerobic conditions, whereas glycine betaine aldehyde and glycine betaine function both aerobically and anaerobically. No endogenous glycine betaine accumulation was detectable in osmotically stressed cells grown in the absence of the osmoprotectant itself or the precursors. A membrane-bound, O2-dependent, and electron transfer-linked dehydrogenase was found which oxidized choline to glycine betaine aldehyde and aldehyde to glycine betaine at nearly the same rate. It displayed Michaelis-Menten kinetics; the apparent Km values for choline and glycine betaine aldehyde were 1.5 and 1.6 mM, respectively. Also, a soluble, NAD-dependent dehydrogenase oxidized glycine betaine aldehyde. It displayed Michaelis-Menten kinetics; the apparent Km values for the aldehyde, NAD, and NADP were 0.13, 0.06, and 0.5 mM, respectively. The choline-glycine betaine pathway was osmotically regulated, i.e., full enzymic activities were found only in cells grown aerobically in choline-containing medium at an elevated osmotic strength. Chloramphenicol inhibited the formation of the pathway in osmotically stressed cells.     

Role of Glycine Betaine and Related Osmolytes in Osmotic Stress Adaptation in Yersinia enterocolitica ATCC 9610

Yersinia enterocolitica is a gram-negative, food-borne pathogen that can grow in 5% NaCl and at refrigerator temperatures. In this report, the compatible solutes (osmolytes) which accumulate intracellularly and confer the observed osmotic tolerance to this pathogen were identified. In minimal medium, glutamate was the only detectable osmolyte that accumulated in osmotically stressed cells. However, when the growth medium was supplemented with glycine betaine, dimethylglycine, or carnitine, the respective osmolyte accumulated intracellularly to high levels and the growth rates of the osmotically stressed cultures improved from 2.4- to 3.5-fold. Chill stress also stimulated the intracellular accumulation of glycine betaine, but the growth rate was only slightly improved by this osmolyte. Both osmotic upshock and temperature downshock stimulated the rate of uptake of [(sup14)C]glycine betaine by more than 30-fold, consistent with other data indicating that the osmolytes are accumulated from the growth medium via transport.     

Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti.

Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti. In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R. meliloti. Results from growth experiments, 14C labeling of intermediates, and enzyme activity assays are presented. The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway. High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline. Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells. This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant. The mechanisms of osmoregulation in R. meliloti and Escherichia coli are compared.     

Hypersaline stress induces the turnover of phosphatidylcholine and results in the synthesis of the renal osmoprotectant glycerophosphocholine in Saccharomyces cerevisiae

The role of phosphatidylcholine turnover during hypersaline stress is investigated in Saccharomyces cerevisiae. In the wild-type strain, 2180-1A hypersaline stress induced the rapid turnover of phosphatidylcholine, a major membrane lipid. Yeast cells were grown in the presence of [14C]-choline to label phosphatidylcholine. Upon shifting the cells to medium with 0.8 M NaCl, phosphatidylcholine levels were diminished by c. 30% within 20 min to yield glycerophosphocholine, a methylamine osmoprotectant that has been previously identified in renal cells. High-performance liquid chromatography studies showed that osmotically mediated glycerophosphocholine production was enhanced if 10 mM choline was added as a supplement to synthetic dextrose medium with 1.6 M NaCl, but glycine betaine was not detected. Enhanced glycerophosphocholine production also correlated with improved growth in media containing 1.6 M NaCl and choline. Enhanced growth is specific to methylamines: salt-stressed cells supplemented with 10 mM choline or glycine betaine showed enhanced growth relative to unsupplemented control cultures, but other additives had no effect on growth or adversely affected it. Nutritional effects are ruled out because yeast cannot use choline or glycine betaine as carbon or nitrogen sources in normal or high-salt medium. Finally, enhanced growth in hypersaline media with choline or glycine betaine is dependent on the choline permease Hnm1. These results in yeast highlight a similarity with mammalian renal cells, namely that phosphatidylcholine turnover contributes to osmotic adaptation via synthesis of the osmoprotectant glycerophosphocholine.     

Staphylococcus aureus osmoregulation: roles for choline, glycine betaine, proline, and taurine.

Choline, glycine betaine, and L-proline enhanced the growth of Staphylococcus aureus at high osmolarity (i.e., they acted as osmoprotectants) on various liquid and solid defined media, while an osmoprotective effect of taurine was shown only for cells growing on high-NaCl solid medium that lacked other osmoprotectants. Potassium pool levels were high, and there was little difference in levels in cells grown at different osmolarities. Glycine betaine accumulated to high levels in osmotically stressed cells, and choline was converted to glycine betaine. Proline and taurine also accumulated in response to osmotic stress but to lower levels than glycine betaine.     

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.     

Characterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na(+)-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12 degrees C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and gamma-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

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.     

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.     

Characterization of Glycine Betaine Porter I from Listeria monocytogenes and Its Roles in Salt and Chill Tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na⁺-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12°C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and γ-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli.

It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in the flbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of beta-galactosidase in ots-lacZ fusion mutants.     

Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress

Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40–50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.     

Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes.

Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerator temperatures. These two characteristics make it an insidious threat to public health. Like several other organisms, L. monocytogenes accumulates glycine betaine, a ubiquitous and effective osmolyte, intracellularly when grown under osmotic stress. However, it also accumulates glycine betaine when grown under chill stress at refrigerator temperatures. Exogenously added glycine betaine enhances the growth rate of stressed but not unstressed cells, i.e., it confers both osmotolerance and cryotolerance. Both salt-stimulated and cold-stimulated accumulation of glycine betaine occur by transport from the medium rather than by biosynthesis. Direct measurement of glycine betaine uptake shows that cells transport betaine 200-fold faster at high salt concentration (4% NaCl) than without added salt and 15-fold faster at 7 than at 30 degrees C. The kinetics of glycine betaine transport suggest that the two transport systems are indistinguishable in terms of affinity for betaine and may be the same. Hyperosmotic shock and cold shock experiments suggest the transport system(s) to be constitutive; activation was not blocked by chloramphenicol. A cold-activated transport system is a novel observation and has intriguing implications concerning the physical state of the cell membrane at low temperature.     

Myceloid cell formation in Arthrobacter globiformis during osmotic stress

C.E. DEUTCH AND G.S. PERERA. 1992. Arthrobacter globiformis was grown in a semi-defined liquid medium containing added solutes to determine the effects of osmotic stress on its reproduction and cell morphology. There was a progressive reduction in the specific growth rate during exponential phase as the concentration of NaCl was increased, although the final yields of the cultures during stationary phase were not affected. Clusters of branching myceloid cells rather than the typical bacillary forms predominated during exponential phase. These myceloids did not undergo complete septation and persisted into stationary phase. Similar responses were observed with potassium sulphate as the exogenous solute but less dramatic morphological effects were found with added polyethylene glycol or sucrose. The myceloids formed in response to osmotic stress could not be disrupted mechanically but were more sensitive than normal cells to lysozyme, particularly during stationary phase. Addition of osmoprotective compounds such as proline, glutamate, glycine betaine, or trehalose to the growth medium did not significantly relieve the effects of osmotic stress on growth rate or morphology. A. simplex also formed myceloid cells during osmotic stress but A. crystallopoietes did not. These results indicate that arthrobacters exhibit characteristic responses to osmotic stress and suggest these bacteria may contain novel osmoprotective compounds.     

Survival of Salmonella paratyphi B and Pseudomonas aeruginosa in seawater after incubation or washing in the presence of osmolytes]

The authors have compared the survival in seawater of Salmonella paratyphi B and Pseudomonas aeruginosa cells grown at low or high osmolarity, in the presence of organic osmolytes: glycine betaine, choline, proline, and glutamate. The four substrates enhanced the survival potential of S. paratyphi B while only glycine betaine protected P. aeruginosa. In addition only S. paratyphi B cells were more resistant after a preliminary growth at high osmolarity. Both bacteria were sensitive to osmotic down-shock, sensitization of S. paratyphi B being inversely proportional (p greater than or equal to 0.01) to the osmolarity of the medium used to wash cells. The transit in wastewater, at low osmolarity, can therefore modify the behavior of these pathogens in the marine environment.     

Three transporters mediate uptake of glycine betaine and carnitine by Listeria monocytogenes in response to hyperosmotic stress

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.     

Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.