Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a _w) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a _w?=?0.96 or 0.99 were then recovered by centrifugation, added to a sucrose–phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a _w?=?0.96. Growing the cells in complex media adjusted at a _w?=?0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose–phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.     

Impact of osmotic stress on volume regulation,cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B

The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.     

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.     

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.     

Roles of N-acetylglutaminylglutamine amide and glycine betaine in adaptation of Pseudomonas aeruginosa to osmotic stress.

The mechanism of osmotic stress adaptation in Pseudomonas aeruginosa PAO1 was investigated. By using natural abundance 13C nuclear magnetic resonance spectroscopy, osmotically stressed cultures were found to accumulate glutamate, trehalose, and N-acetylglutaminylglutamine amide, an unusual dipeptide previously reported only in osmotically stressed Rhizobium meliloti and Pseudomonas fluorescens. The intracellular levels of these osmolytes were dependent on the chemical composition and the osmolality of the growth medium. It was also demonstrated that glycine betaine, a powerful osmotic stress protectant, participates in osmoregulation in this organism. When glycine betaine or its precursors, phosphorylcholine or choline, were added to the growth medium, growth rates of cultures in 0.7 M NaCl were increased more than threefold. Furthermore, enhancement of growth could be observed with as little as 10 microM glycine betaine or precursor added to the medium. Finally, the mechanism of osmotic stress adaptation of two clinical isolates of P. aeruginosa was found to be nearly identical to that of the laboratory strain PAO1 in all aspects studied.     

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     

Characterization of an NaCl-sensitive Staphylococcus aureus mutant and rescue of the NaCl-sensitive phenotype by glycine betaine but not by other compatible solutes.

To further study mechanisms of coping with osmotic stress-low water activity, mutants of Staphylococcus aureus with transposon Tn917-lacZ-induced NaCl sensitivity were selected for impaired ability to grow on solid defined medium containing 2 M NaCl. Southern hybridization experiments showed that NaCl-sensitive mutants had a single copy of the transposon inserted into a DNA fragment of the same size in each mutant. These NaCl-sensitive mutants had an extremely long lag phase (60 to 70 h) in defined medium containing 2.5 M NaCl. The osmoprotectants glycine betaine and choline (which is oxidized to glycine betaine) dramatically shortened the lag phase, whereas L-proline and proline betaine, which are effective osmoprotectants for the wild type, were ineffective. Electron microscopic observations of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudomulticellular cells similar to those observed previously in the wild type under the same conditions. Glycine betaine, but not L-proline, corrected the morphological abnormalities. Studies of the uptake of L-[14C]proline and [14C]glycine betaine upon osmotic upshock revealed that the mutant was not defective in the uptake of either osmoprotectant. Comparison of pool K+, amino acid, and glycine betaine levels under NaCl stress conditions in the mutant and the wild type revealed no striking differences. Glycine betaine appears to have additional beneficial effects on NaCl-stressed cells beyond those of other osmoprotectants. The NaCl stress protein responses of the wild type and the NaCl-sensitive mutant were characterized and compared by labeling with L-[35 S]methionine and two-dimensional gel electrophoresis. The synthesis of 10 proteins increased in the wild type in response to NaCl stress, whereas the synthesis of these 10 proteins plus 2 others increased in response to NaCl stress in the NaCl-sensitive mutant. Five proteins, three of which were NaCl stress proteins, were produced in elevated amounts in the NaCl-sensitive mutant under unstressed conditions compared to the wild type. The presence of glycine betaine during NaCl stress decreased the production of three NaCl stress proteins in the mutant versus one in the wild type.     

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.     

Osmotically induced intracellular trehalose, but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli

Trehalose considerably increased the tolerance of Escherichia coli to air drying, whether added as an excipient prior to drying or accumulated as a compatible solute in response to osmotic stress. The protective effect of exogenously added trehalose was concentration dependent, up to a threshold value of 350 mM. However, trehalose alone cannot explain the intrinsically greater desiccation tolerance of stationary compared to exponential phase E. coli cells, although their tolerance was also enhanced by exogenous or endogenously accumulated trehalose. In contrast, glycine betaine whether added as an excipient or accumulated intracellularly had no influence on desiccation tolerance. These data demonstrate that the protection provided by compatible solutes to cells subjected to desiccation differs from that during osmotic stress, due to the much greater reduction in available cell water. The protective effects of trehalose during desiccation appear to be due to its stabilising influence on membrane structure, its chemically inert nature and the propensity of trehalose solutions to form glasses upon drying, properties which are not shared by glycine betaine.     

Fate of compatible solutes during dilution stress in Ectothiorhodospira halochloris

Abstract 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.     

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.     

Betaine-induced stimulation of respiration at high osmolarities in a halotolerant bacterium.

It was shown that non-penetrating solutes at high concentrations inhibit the respiration of the halotolerant bacterium Ba1. Betaine relieved the inhibition caused by osmotic stress and exhibited in this respect a considerable structural specificity. The rate of oxidation of various substrates was stimulated to different extents. It stimulated the rates of both respiration and growth to a similar extent, leaving the energy yield essentially unchanged. In cells pre-loaded with labelled glutamate, betaine also stimulated the rate of oxidation of this intracellular substrate. Betaine was accumulated by respiring cells, and the maximum amount taken up was correlated with the osmolarity of the medium. As judged by chromatography, accumulated intracellular betaine underwent no chemical modification, and this accumulated betaine could not be exchanged with the betaine in the medium or released by passive efflux when respiration was inhibited. Intracellular betaine caused no stimulation of respiration, whereas betaine added to the medium increased the respiratory rate to the same extent in cells pre-loaded with betaine as that in the nonloaded cells. The above observations suggest that iso-economic adjustment is not involved in the anti-osmotic effect of betaine, and that betaine exerts its action on the cellular membrane from the outside.     

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.     

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 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.     

甜菜碱提高植物抗寒性的机理及其应用

甜菜碱是植物重要的渗透调节物质,在低温等逆境条件下,许多植物细胞中迅速积累甜菜碱以维持细胞的渗透平衡.对近几年来甜菜碱提高植物抗寒性的机理研究及其应用,包括甜菜碱的生物合成途径、低温胁迫下甜菜碱对植物的保护机理、甜菜碱合成酶基因的转化及外源甜菜碱在植物抗寒中的应用进行了综述.     

Physiological response of Pseudomonas putida S12 subjected to reduced water activity

Abstract The effect of osmotic stress, given as decreased water activity (a_w), on growth and the accumulation of potassium and the compatible solute betaine by Pseudomonas putida S12 was investigated. Reduced a_w was imposed by addition of sodium chloride, sucrose, glycerol or polyethylene glycol to the growth medium. Accumulation of potassium and betaine was established when sodium chloride and sucrose were used to cause osmotic stress. No accumulation of these solutes was found in the presence of glycerol. Addition of polyethylene glycol to the medium strongly decreased the growth rate in comparison with the other osmolytes tested at the corresponding a_w. Although polyethylene glycol did decrease the a_w, neither potassium nor betaine was accumulated by the cells.     

外源甜菜碱提高恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的研究

研究了外源甜菜碱对恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的影响并对其渗透保护机制进行了初步的探讨;结果表明培养基中添加甜菜碱可以改善DLL-1细胞在高盐培养基中的生长情况,添加150mg/L的甜菜碱可以使DLL-1在1.2mol/L NaCl的基础盐培养基中生长,添加10mg/L的甜菜碱就足以显著缩短渗透胁迫条件下DLL-1细胞的延滞期和代时,增加生长量;和不添加对照相比,延滞期由24h缩短到6h,代时由60min缩短到35.7min,最大生长量OD610由1.29增长到1.57。在渗透胁迫条件下,细胞从外界快速吸收外源甜菜碱来代替自身相容性溶质的合成。     

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.