Potential osmoprotectants for the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila

The physiological responses of the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila (formely known as P. halophila), subjected to osmotic stress in the presence of molecules known to act as osmoprotectants for other bacteria were studied. In a defined medium, glycine betaine, dimethylsulfonioacetate, choline, proline and L-carnitine were able to relieve inhibition of growth at 0.8 M NaCl. The five compounds were shown to efficiently compete with glycine betaine transport, suggesting the existence of common transporter(s) for these molecules. T. halophila, the most tolerant strain, exhibited a larger spectrum of compatible solutes including dimethylsulfonioacetate, dimethylsulfoniopropionate and ectoine. Preliminary data suggest that restoration of growth by ectoine under osmotic constraint seems specific to the genus Tetragenococcus.     

Growth of Escherichia coli ATCC 9637 through the uptake of compatible solutes at high osmolarity

The growth rate of Escherichia coli ATCC 9637 was determined in a chemically defined (CD) medium with high osmolarity, 1-1.2 M. The addition of ectoine or glycine betaine to the medium resulted in a significant stimulation of growth rate for this strain. In the presence of ectoine derivatives, hydroxyectoine and homoectoine, cell growth was not stimulated to the same extent as when ectoine was added, but it was improved slightly. The acceleration of growth rate of E. coli ATCC 9637 at elevated osmolarity was ascribed to the accumulation in the cells of ectoine or glycine betaine added to the medium, both of which were proved to be genuine osmolytes in cells. Rapid uptake of ectoine by cells was confirmed when ectoine was available in the CD medium with high osmolarity. Since strain ATCC 9637 did not accumulate ectoine in the absence of an energy source, ectoine uptake might take place not only through cellular sensing of the external high osmolarity but through cellular functioning via energization.     

Efficient utilization of ectoine by halophilic Brevibacterium species and Escherichia coli subjected to osmotic downshock

Halophilic and non-halophilic bacteria subjected to osmotic downshock, from 0.7 M NaCl to deionized water, were examined for their survival, with the uptake and utilization of the cyclic amino acid ectoine, one of the representative compatible solutes, being taken into account. The uptake of ectoine added externally and survival of the cells were monitored as a function of incubation time in the presence and absence of NaCl. The halophilic Brevibacterium sp. JCM 6894 and B. epidermidis JCM 2593 actively accumulated ectoine regardless of the presence of NaCl, which led to cell survival. Brevibacterium casei JCM 2594 belonging to the same Brevibacterium species, however, revealed Na+-dependence of its uptake activity of ectoine. Non-halophilic Escherichia coli K-12 did not accumulate ectoine, and thereby this strain failed to survive irrespective of whether NaCl was present. The physiological meanings of the downshock procedure are discussed in connection with the uptake and the subsequent utilization of ectoine.     

Humectant permeability influences growth and compatible solute uptake by Staphylococcus aureus subjected to osmotic stress

The effects of different humectants (sodium chloride, sucrose, and glycerol) on the growth of and compatible solute (glycine betaine, proline, and carnitine) uptake by the osmotolerant foodborne pathogen Staphylococcus aureus were investigated. While growth in the presence of the impermeant humectants sodium chloride and sucrose induced the accumulation of proline and glycine betaine by cells, growth in the presence of the permeant humectant glycerol did not. When compatible solutes were omitted from low-water-activity media, growth was very poor in the presence of impermeant humectants. In contrast, the addition of compatible solutes had essentially no effect on growth when cells were grown in low-water-activity media containing glycerol as the humectant. Carnitine was found to accumulate to high intracellular levels in osmotically stressed cells when proline and glycine betaine were absent, making it a potentially important compatible solute for this organism.     

Uptake of choline from salmon flesh and its conversion to glycine betaine in response to salt stress in Shewanella putrefaciens

When cultured in M63 minimal medium plus 0.6 M NaCl, the growth of Shewanella putrefaciens was strongly inhibited. The addition of an extract from smoked salmon to this medium restored the growth almost to the unstressed level. A comparison of the 13C NMR spectra of intracellular solutes extracted from S. putrefaciens cells cultured in both conditions revealed the accumulation of glycine betaine (GB) from the smoked salmon extract (SSE). Analysis of the osmoprotective properties of this extract for several strains of Escherichia coli (which differ from each other in their ability to accumulate GB (i) from the surrounding environment, and (ii) from its hydroxylated precursor choline), demonstrated the absence of GB in the SSE. From the overall results, we inferred that salt-stressed S. putrefaciens cells accumulated GB from choline present in the SSE. Furthermore, the use of [14C]-labeled betaines gave evidence that S. putrefaciens (i) oxidised choline to GB, (ii) accumulated GB as a non-metabolisable osmolyte (up to 1300 nmol (mg dw)(-1) when cultured in a medium containing 0.5 M NaCl and either 1 mM choline or 1 mM GB), and (iii) both choline and GB uptake activities were osmotically upregulated (both activities were increased more than 50-fold in media containing 0.4 to 0.6 M NaCl). In all, our results suggest that in salted smoked salmon, S. putrefaciens imports and oxidises choline, leading to the intracellular accumulation of GB.     

Growth of Lactococcus lactis strains at low water activity: correlation with the ability to accumulate glycine betaine

Lactococcus lactis strains were divided into two groups based on their ability to grow in the presence of an upper limit of either 2% w/v NaCl (sensitive) or 4% w/v NaCl (tolerant). Growth inhibition of NaCl tolerant strains was substantially relieved by glycine betaine which was accumulated in significant amounts when growing at low water activities (a(w)). Very little accumulation of glycine betaine occurred during growth of the NaCl sensitive strains. The NaCl tolerant strains had substantial levels of glycine betaine transport activity in vitro, whereas the NaCl sensitive strains had little or no such activity. A low a(w) sensitive mutant of L. lactis subsp. cremoris MG1363 (NaCl tolerant) was isolated following ISS1 insertional mutagenesis. This mutant was inhibited at an a(w) of 0.988 produced by addition of 2% w/v NaCl or the equivalent glucose concentration (0.58 M). The mutant did not accumulate glycine betaine when growing at low a(w), and did not transport glycine betaine when assayed in vitro.     

甘氨酸甜菜碱和分子伴侣dnaK在嗜盐四联球菌耐盐机制中的作用

能耐高盐的嗜盐四联球菌CICC 10469是从传统的酱料中分离得到.为研究耐盐特性与相容性溶质积累和分子伴侣dnaK特性的关系,使用核磁共振来检测其主要的相容性溶质.不同盐浓度对耐盐的嗜盐四联球菌和不耐盐的乳酸乳球菌MG 1363的生长的影响通过测量OD600来获得.盐浓度对分子伴侣dnaK表达的影响通过实时荧光定量RT-PCR来检测.相容性溶质和可溶性蛋白随盐浓度的变化通过HPLC和酶标仪来检测.结果显示嗜盐四联球菌以甘氨酸甜菜碱作为主要的相容性溶质.适合的盐浓度能够促进菌体的生长,在一定盐浓度范围内,甘氨酸甜菜碱,可溶性蛋白和分子伴侣dnaK随盐浓度的增加而升高,然而超过一定盐浓度范围后,表达量将会减少.这表明细菌能够根据外部环境做出适当的调整以维持细胞的正常生理功能.而乳酸乳球菌MG1363很少有相容性溶质和分子伴侣dnaK以耐受外部盐浓度.这表明相容性溶质和分子伴侣dnak在嗜盐四联球菌生长中起重要的作用.总之,中度嗜盐菌的主要耐盐机制是通过快速合成和释放甘氨酸甜菜碱及分子伴侣dnaK高活性表达来完成.     

Effects of exogenous compatible solutes on growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Six known compatible solutes as well as twenty L-amino acids were individually added to a glucose minimal medium and their effects on the growth of Sulfolobus solfataricus (DSM 1617) were examined. Among the compatible solutes tested, putrescine, trehalose, and l-glutamate enhanced the growth of S. solfataricus. On the other hand, glycine betaine, choline, and L-proline showed little or no influence on cell growth. When cells were grown in the glucose medium supplemented with trehalose or L-glutamate, S. solfataricus preferentially utilized the compatible solute over glucose. The growth-enhancement effect of L-glutamate was also observed to be dependent on the glucose concentration in the medium: growth enhancement was higher when the concentration of glucose was low and gradually decreased with increasing glucose concentration. Interestingly, the effects of amino acids on cell growth differed markedly depending on the chemical nature of the amino acid added. While acidic amino acids-L-glutamate and L-aspartate-enhanced the growth rate, almost no growth was observed in the presence of glycine, L-leucine, L-valine, L-phenylalanine, L-threonine, L-methionine, or L-cysteine. Among all the low-molecular-weight solutes tested in this study, the growth-stimulation effect was most profound in the presence of L-glutamate. When S. solfataricus cells were grown in a glucose (1.0 g/l) medium supplemented with 3.0 g/l L-glutamate, the maximal cell density and growth rate were about 3.2- and 2.3-fold higher than those obtained without L-glutamate.     

甘氨酸甜菜碱及复合纤维素酶对酱油发酵的影响

将甘氨酸甜菜碱作为渗透压保护剂,于酱醪上罐时(0d)或酱醪发酵30d后添加到高盐稀态酱油发酵的酱醪中,同时添加外源复合纤维素酶,观察其对酱醪发酵的影响。结果表明：在酱醪上罐时(0d)或酱醪发酵30d后同时添加不同质量分数的甘氨酸甜菜碱(0.20%、0.30%)及质量分数0.15%的复合纤维素酶(105EGu/100g酱醪),均能有效提高发酵酱油中蛋白质转化率,达2.26%～7.92%。添加甘氨酸甜菜碱能显著改善高盐稀态发酵酱油头油品质,平均缩短发酵时间15d左右;另外,在酱醪发酵30d后再添加甘氨酸甜菜碱和外源复合纤维素酶其效果更佳,同0d时添加相比,蛋白质转化率平均提高了3.17%。     

Effect of duration of osmotic downshock and coexisting glutamate on survival and uptake of ectoine in halotolerant Brevibacterium sp. JCM 6894

Halotolerant Brevibacterium sp. JCM 6894 that was subjected to an osmotic downshock (0.7 M NaCl to 0 M) was examined for its survival and uptake of ectoine in the presence of ectoine and/or carbon sources. In the presence of ectoine alone, the rates of ectoine uptake by the 1 h-downshocked cells were low and high in the absence and presence of 0.7 M NaCl, respectively, which were in parallel with the rates of cell growth. The presence of glutamate or amino acids together with ectoine exerted a stimulative effect on the survival of downshocked cells. The incubation time of the cells subjected to osmotic downshock strongly affected ectoine uptake as well as the cell growth of this strain, suggesting that the transporter of ectoine in the strain JCM 6894 was stimulated during the osmotic downshock for about 1 h. Different downshock strengths had marked effects on the rate of ectoine uptake when the downshocked cells were incubated in the presence of NaCl.     

Tolerance to high osmolality of the lactic acid bacterium Oenococcus oeni and identification of potential osmoprotectants

Growth of the lactic acid bacterium Oenococcus oeni under hyperosmotic constraint was investigated in a chemically defined medium. The bacterium could grow on media with an elevated osmolality, preferably below 1.5 Osm kg(-)(1) H(2)O. At osmolalities comprised between 0.6 and 1.5 Osm kg(-)(1) H(2)O, the growth deficit elicited by the sugars glucose and fructose was slightly more severe than with salts (NaCl or KCl). In contrast to what was observed in other lactic acid bacteria, proline, glycine betaine and related molecules were unable to relieve inhibition of growth of O. oeni under osmotic constraint. This was correlated to the absence of sequences homologous to the genes coding for glycine betaine and/or proline transporters described in Lactococcus lactis and Lactobacillus plantarum. The amino acid aspartate proved to be osmoprotective under electrolyte and non-electrolyte stress. Examination of the role of peptides during osmoregulation showed that proline- and glutamate-containing peptides were protective under salt-induced stress, and not under sugar-induced stress. Under high salt, PepQ a cytoplasmic prolidase that specifically liberated proline from di-peptides increased activity, while PepX (X-prolyl-dipeptidyl aminopeptidase) and PepI (iminopeptidase) activities were unaffected. Our data suggest that proline- and glutamate-containing peptides may contribute to the adaptation of O. oeni to high salt through their intracellular hydrolysis and/or direct accumulation.     

Biosynthesis of exopolysaccharide by a Bacillus licheniformis strain isolated from ropy cider

Listeria monocytogenes accumulates low molecular weight compounds (osmolytes, or compatible solutes) in response to chill stress. This response has been shown to be responsible, in part, for the chill tolerance of the species. Among the osmolytes tested to date, glycine betaine, gamma-butyrobetaine and carnitine display the strongest cryoprotective effect. These osmolytes are not synthesized in the cell and must be transported from the medium. In this study, the compatible solute accumulation profile of the food-borne pathogen L. monocytogenes was determined in balanced growth and stationary phase cultures grown in milk whey at 7 and 30 degrees C. In balanced growth cultures at 7 degrees C, glycine betaine (720 nmol/10(10) cfu) and carnitine (130 nmol/10(10) cfu) were the major osmolytes accumulated by wild-type L. monocytogenes 10403S, whereas carnitine (490 nmol/10(10) cfu) was the dominant osmolyte and glycine betaine was present in smaller amounts (270 nmol/10(10) cfu) in a mutant (L. monocytogenes LTG59) blocked in the major glycine betaine uptake system, glycine betaine porter II. In strain 10403S, glycine betaine and carnitine were present in eightfold and twofold excess at 7 degrees C compared to 30 degrees C; the respective ratios for strain LTG59 were 6 and 8. The intracellular concentration of osmolytes in stationary phase cultures at 7 degrees C was markedly reduced compared to that during balanced growth. Furthermore, at 4 degrees C, small but highly significant differences in growth were observed between strains. Strain LTG59 grew with a lag phase that was significantly longer, a generation time that was significantly greater and reached a final cell yield that was significantly lower than that of strain 10403S. The elevated accumulation of carnitine in the absence of glycine betaine porter II was insufficient to confer the magnitude of the cryoprotective effect displayed by the wild type.     

Low Temperature Growth of Type E Clostridium botulinum Spores. 2. Effects of Solutes and Incubation Temperature

SUMMARY— The inhibition of outgrowth of spores of Clostridium botulinum Type E by the addition of different concentrations of various solutes to a trypticase-peptone-sucrose-yeast extract medium (TPSY) was studied at different incubation temperatures. Solutes added to TPSY medium were sodium chloride (NaCl), sodium formate (NaCOOH), potassium chloride (KCI), sucrose and glucose. Incubation was at 30, 21.1, 15.6, 10 and 7.2°C, and growth was confirmed by mouse toxin assay. The effect of the solutes on the equilibrium relative humidities (ERHI of the various media was measured using the graphical interpolation method and expressed as water activity (a_w). The possibility of an osmotic effect was also investigated. The molar (M) concentrations and osmotic pressures of the different solutes were calculated and related to the approximate inhibitory concentrations of 5.0% NaCl (a_w 0.975), 6.0% KCI (a_w 0.974), 5.5% NeCOOH (a_w 0.9711, 38.5% sucrose (a_w 0.976) and 22.5%. glucose (a_w 0.970).
The inhibitory salt concentrations were approximately 0.80 M and produced osmotic pressures of about 36.0 atmospheres. The approximate osmotic pressure of the inhibitory 1.12 and 1.24 M sucrose and glucose solutions were 25.08 and 27.97 atmospheres respectively.
Growth response decreased as the incubation temperature decreased as measured by delayed outgrowth times and in decreased amount of cellular material produced by the Type E strains tested.     

Supplementation effect of ectoine on thermostability of phytase

In this study, we elucidated the supplementation effect of compatible solutes on the thermostability of phytase, designated as PHYA II, which was encoded by the phytase gene phyA I (GeneBank AY013315) from Aspergillus ficuum As3.324 and expressed in Pichia pastoris GS115. When PHYA II in acetate buffer was heated at 90 degrees C for 15 min, more than 80% of the residual activity was retained by adding the cyclic amino acid ectoine, a representative compatible solute. Furthermore, the presence of ectoine led to an increase in the relative hydrolytic rate of sodium phytate by 15.7% with heating at 80 degrees C for 15 min. Among the compatible solutes examined, ectoine was confirmed to be the most efficient thermoprotectant for PHYA II.     

Uptake and utilization of ectoine by halotolerant Brevibacterium sp. JCM 6894 subjected to osmotic downshock

The concentration changes of the cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyridine carboxylic acid) in Brevibacterium sp. JCM 6894 cells subjected to an osmotic downshock were investigated. When the cells grown in the presence of 2 M NaCl were suspended in deionized water, they immediately released about 60% of the ectoine synthesized intracellularly. During the subsequent incubation, we observed that both the extra- and intracellular concentrations of ectoine were reduced almost linearly with the incubation time. When ectoine was provided externally to the downshocked cells, a similar reduction in both intra- and extracellular ectoine concentrations was recognized. In addition, we observed an increase in ectoine accumulation at about 10 h of incubation, which indicates that ectoine was taken up by such downshocked cells in the absence of external Na+. Furthermore, the downshocked cells showed higher levels of survival, respiration, and growth in the presence of ectoine than in its absence. The ability to take ectoine up was induced in the cells grown in the presence of >0.25 M NaCl for >12 h. Thus, we conclude that even under the lower osmotic condition ectoine might be taken up and subsequently utilized by strain JCM 6894 subjected to the osmotic downshock, indicating that the uptake of ectoine by such cells occurred for the survival and growth of the cell itself rather than for cellular osmoregulation.     

Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli

Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli.     

Nanofiltration of glucose solution containing salts: Effects of membrane characteristics,organic component and salts on retention

The understanding on nanofiltration especially the rejection of uncharged solutes, charged solutes and mixture of solutes is crucial for its application in food industry. This is because process streams in food industry usually contain organic components without charge and salts. In this work, DK and CK membranes were utilized for the separation of multi-component feeds containing glucose, NaCl and multivalent salts (MgCl₂ or Na₂SO₄). Glucose rejection is slightly affected by salt concentration which may due to pore swelling of CK membranes at high concentration of NaCl. Meanwhile, rejection of NaCl is reduced by the increment of glucose and NaCl concentration which causes concentration polarization. The addition of multivalent salts even induces negative rejection of NaCl in DK membranes. However, both membranes show high rejection of multivalent salts with or without the presence of glucose. The rejection behavior follows the pattern expected from the structural and electrical properties of the membranes.     

Induction of salt tolerance in Bacillus subtilis IFO 3025

Salt tolerance was induced in Bacillus subtilis IFO 3025 cells when a moderate osmotic stress was imposed by incubation in the presence of compatible solutes and 0.5 M NaCl or 0.8 M sorbitol. The optimum condition for inducing salt tolerance was observed when the cells were incubated in a solution containing 5 mM glutamate, 2.5 mM KCl, and 0.5 M NaCl or 0.8 M sorbitol. After 60 min incubation, the cells were able to form colonies on an agar plate with high salinity. It was confirmed that the cells acquired salt tolerance by accumulating glutamate and potassium ions in the cytoplasm as the main solute and ion, respectively.     

Characterization of the induction of increased thermotolerance by high osmolarity inSalmonella

Factors limiting the growth ofSalmonellaat high temperature were analyzed in minimal medium. The growth ofS. typhimuriumwas curtailed at 44°C by high temperature-dependent block in methionine synthesis. At 45°C, the organism could not grow in glucose-mini- mal medium even with methionine, but growth could be restored at this temperature by the addition of NaCl in the final concentration range of 0.15–0.3m. Similar results were obtained with twoS. enteritidisstrains. The growth stimulatory effect of high osmolarity at 45°C was not altered by the osmoprotectant compound glycine betaine. Exposure to 0.3mNaCl also increased the resistance ofS. typhimuriumto thermal death at 50°C and to the oxidizing agent H₂O₂. However, the high osmolarity-dependent induction of enhanced resistance to 50°C and to H₂O₂was completely abolished by glycine betaine. The finding that glycine betaine blocks the induction of resistance to lethal high temperature by high osmolarity could provide a means for enhancing the bactericidal efficacy of heating, because it may be possible to increase the heat sensitivity of contaminating bacteria by supplementing food products with this naturally occurring plant metabolite, prior to thermal processing.