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.     

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.     

Effect of compatible solutes on the respiratory activity and growth of Escherichia coli K-12 under NaCl stress

The respiratory activity of Escherichia coli K-12 was inhibited by high NaCl concentrations. The addition of compatible solutes such as proline and ectoine led to the recovery of the respiration of E. coli K-12 inhibited by 1 M NaCl to a similar extent as did the addition of glycine betaine. Glucose, an exogenous substrate for the stimulation of respiratory activity, was not taken up by the cells in the presence of 1 M NaCl, but active uptake of glucose was observed following the addition of compatible solutes. As a result, we obtained a good correlation between respiratory activities and glucose uptake rates, suggesting that the glucose uptake activity inhibited by high NaCl concentrations was facilitated by these solutes. The addition of proline did not lead to cell proliferation in the minimal medium containing 1 M NaCl, although the cells took up proline quite efficiently under high osmolarity. On the other hand, cell growth occurred after a lag time in the medium containing glycine betaine or ectoine in place of proline. Similar actions of the compatible solutes mentioned above were observed for E. coli ATCC 9637.     

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.     

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.     

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%。     

Reconstitution and function of Tetragenococcus halophila chaperonin 60 tetradecamer

Tetragenococcus halophila originally isolated from soy sauce is a halophilic lactic acid bacterium which can grow under 4 M sodium chloride. T. halophila chaperonin composed of a core moiety of chaperonin 60 (cpn60) and a lid moiety of chaperonin 10 (cpn10), is thought to contribute to host halotolerant capability. In this report, we reconstituted and characterized the core complex of T. halophila chaperonin by using a recombinant T. halophila cpn60 (Tcpn60) overexpressed in Escherichia coli. The reconstitution of Tcpn60 was performed in the presence of 10 mM MgCl2, 2 mM ATP and 0.8 M (NH4)2SO4 and the resultant oligomer was purified by gel filtration chromatography. Electron microscopy of the reconstituted Tcpn60 revealed a double toroidal tetradecameric structure that is characteristic of bacterial cpn60. The T. halophila tetradecamer cpn60 exhibited an ATPase activity and a refolding activity of both chemically and thermally denatured enolases under wide range of salt concentrations. Furthermore, we demonstrated that heterologous expression of Tcpn60 allowed the normal growth of host Escherichia coli cells under salt stress conditions and this effect was further enhanced by co-expression with Tcpn10. These results suggested that Tcpn60 contributes to the halotolerance property of T. halophila cell as a tetradecamer complex, probably associated with the Tcpn10 complex.     

Ectoine as a natural component of food: detection in red smear cheeses

Ectoine is a compatible solute accumulated in halophilic bacteria in response to high salt concentrations and offers protection from osmotic stress. The occurrence of compatible solutes is widespread among bacteria, yet ectoine has never been detected in foods. The use of an ectoine producing microorganism (Brevibacterium linens) in the surface ripening of red smear cheeses led to the question whether ectoine can be found in cheese. Therefore we examined samples from a variety of cheese manufacturers and different types of red smear cheeses for the presence of ectoine using HPLC and HPLC/MS analysis. Ectoine solely appears in the rind and was detected up to 178 mg/200 g red smear cheese, depending on several factors like ripening status and conditions throughout the cheese production process (e.g. salt concentrations of used brine baths).     

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.     

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.     

Production and characterization of ectoine by Marinococcus sp. ECT1 isolated from a high-salinity environment

A halophilic bacterium isolated from a salt environment in southern Taiwan was identified as a Marinococcus sp. ECT1. This bacterium could synthesize and accumulate intracellular ectoine as a compatible solute capable of resisting osmotic stress in a hyper-osmotic environment. This study also developed a semi-synthesized medium (YAMS medium), capable of facilitating the growth of this Marinococcus sp. ECT1 with 600 mg/L crude ectoine production. Moreover, Marinococcus sp. ECT1 was grown on YAMS medium containing different initial yeast extract concentrations (C(YE)) (0 to 60 g/L) to demonstrate how C(YE) affects crude ectoine production. While the maximum cell concentration was increased by 23-fold when the C(YE) was 40 g/L, the maximum crude ectoine production reached 2.5 g/L when C(YE) was 40 g/L. In addition to demonstrating the success of the fermentation strategy of ectoine in increasing the production and production yield, experimental results further demonstrated that the fermentation medium of ectoine is highly promising for commercialization. Furthermore, the molecular weight and chemical structure of ectoine were identified and characterized by FAB-MS and (1)H-NMR.     

甜菜碱对单针藻Monoraphidium sp.QLY-1油脂积累的影响

为提高单针藻Monoraphidium sp.QLY-1的生物量和油脂产量,本文结合两阶段策略,即异养-光诱导培养方法,研究甜菜碱（GB）对单针藻QLY-1生长和油脂积累的影响。结果表明,异养、自养的微藻生物量分别为（5.54±0.22）、（0.878±0.12）g/L,且异养比自养提高了5.3倍。此外,在光胁迫下,添加5 mmol/L甜菜碱时可有效提高微藻的油脂含量,其油脂含量（47.37%±2.93%）相比对照（36.68%±1.34%）提高了0.29倍（p<0.05）。进一步的研究表明,与对照相比,添加5 mmol/L甜菜碱时,藻细胞中性脂增加了12.83%±0.75%,对藻细胞内脂肪酸组成无显著性影响。研究表明,甜菜碱作为一种外源诱导子可有效促进微藻细胞中油脂积累,甜菜碱结合两阶段法可作为促进微藻细胞油脂积累的另一策略。      

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.     

Role of choline and glycine betaine in the formation of N,N-dimethylpiperidinium (mepiquat) under Maillard reaction conditions

This study is the first to examine the role of choline and glycine betaine, naturally present in some foods, in particular in cereal grains, to generate N,N-dimethylpiperidinium (mepiquat) under Maillard conditions via transmethylation reactions involving the nucleophile piperidine. The formation of mepiquat and its intermediates piperidine – formed by cyclisation of free lysine in the presence of reducing sugars – and N-methylpiperidine were monitored over time (240°C, up to 180 min) using high-resolution mass spectrometry in a model system comprised of a ternary mixture of lysine/fructose/alkylating agent (choline or betaine). The reaction yield was compared with data recently determined for trigonelline, a known methylation agent present naturally in coffee beans. The role of choline and glycine betaine in nucleophilic displacement reactions was further supported by experiments carried out with stable isotope-labelled precursors (¹³C- and deuterium-labelled). The results unequivocally demonstrated that the piperidine ring of mepiquat originates from the carbon chain of lysine, and that either choline or glycine betaine furnishes the N-methyl groups. The kinetics of formation of the corresponding demethylated products of both choline and glycine betaine, N,N-demethyl-2-aminoethanol and N,N-dimethylglycine, respectively, were also determined using high-resolution mass spectrometry.     

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.