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.     

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.     

Supplementation effects of hydroxyectoine on proline uptake of downshocked Brevibacterium sp. JCM 6894

Downshock treatment of the halotolerant Brevibacterium sp. JCM 6894 was a prerequisite for proline uptake which is a function for cell survival. Hydroxyectoine served as an effective stimulator for the proline uptake and cell survival of the downshocked cells of this strain. Duration of osmotic downshock, downshock strength, and the kinds of osmolyte affected the efficient rate of growth (ERG) and the uptake of proline. A shorter duration of osmotic downshock, that is 相似文献   

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.     

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.     

Characterization of lytic enzyme activities of Lactobacillus gasseri with special reference to autolysis

Lactobacillus gasseri JCM 1130 and JCM 1131(T) exhibited autolytic activity in agar containing autoclaved cells of each strain as substrate. By zymogram analysis of JCM 1131(T), two lytic bands with apparent molecular masses of 54.5 and 35 kDa, were detected. Similarly, JCM 1130 yielded two lytic bands with apparent molecular masses of 35 and 33.5 kDa. In simple buffers as well, JCM 1131(T) suffered a drastic decrease in cell turbidity, but JCM 1130 did not undergo the decrease. The optimal pH for autolysis of JCM 1131(T) was in the range of 6.0-7.0, and the lysis was completely inhibited at pH 4-5. The lysis of JCM 1131(T) was suppressed by NaCl, in a concentration-dependent way. When subjected to UV irradiation or mitomycin C (MMC) treatment, cultures of both strains elicited conspicuous turbidity decrease after 2-4 h of growth, suggesting the occurrence of prophage induction. The 35-kDa lytic band of JCM 1131(T) and the 33.5-kDa protein of JCM 1130 were considerably increased by UV irradiation.     

Role of cellulose in protecting Shiga toxin-producing Escherichia coli against osmotic and chlorine stress

This study was undertaken to determine the role of cellulose in protecting Shiga toxin-producing Escherichia coli (STEC) against osmotic and chlorine treatments. STEC cells producing cellulose (19B and 49B) and their respective cellulose-deficient counterparts (19D or 49D) were subjected to osmotic (1, 2, and 3 M NaCl) or chlorine (25, 50, and 100 μg/ml sodium hypochlorite) treatments. The survival of STEC cells was determined at different treatment intervals. Populations of 19B cells were significantly higher (P < 0.05) than those of 19D cells at all sampling intervals for the chlorine treatments, at 24- to 48-h intervals for the 1 M NaCl treatment, and at 9- to 48-h intervals for the 2 M NaCl treatment. Significant differences in populations of 49B and 49D cells were observed after 9, 36, and 48 h of treatment with 2 M NaCl and after 3, 12, 36, and 48 h of treatment with 3 M NaCl (P < 0.05). Populations of 49B cells were higher than those of 49D cells (P < 0.05) also after 5 to 10 min of treatment with 50 μg/ml sodium hypochlorite and 3 to 10 min of treatment with 100 μg/ml sodium hypochlorite. The protective effects conferred by cellulose may explain the greater survival of cellulose-producing STEC under adverse environmental conditions.     

The influence of attachment to beef surfaces on the survival of cells of Salmonella enterica serovar Typhimurium DT104, at different a(w) values and at low storage temperatures

This study investigated the influence of attachment to beef surfaces on the survival, injury and death of stationary phase cells of Salmonella enterica serovar Typhimurium DT104, compared to cells free in solution. The effects on cells are considered at different a(w) values and low temperatures in relation to osmotic and cold temperature shock effects. Attachment of cells to meat surfaces prevented cell injury and death from hyperosmosis and low temperatures, compared to meat solutions. Storage of cells for 72h resulted in higher levels of cell death on cells attached to meat surfaces. The improved survival of cells in solutions was considered to be related to adaptation to osmotic stress as a result of exposure to a previous hyperosmotic shock and the ability of the cells to produce cold shock proteins. Pathogen cell growth at low temperatures is discussed in relation to the presence of low levels of NaCl. Finally the data is discussed in relation to pathogen survival on beef carcass surfaces during refrigeration.     

Roles of outer membrane protein W (OmpW) on survival,morphology, and biofilm formation under NaCl stresses in Cronobacter sakazakii

Cronobacter sakazakii is an important foodborne pathogen associated with rare but severe infections through consumption of powdered infant formula. Tolerance to osmotic stress in Cronobacter has been described. However, the detailed factors involved in tolerance to osmotic stress in C. sakazakii are poorly understood. In this study, roles of outer membrane protein W (OmpW) on survival rates, morphologic changes of cells, and biofilm formation in C. sakazakii under different NaCl concentrations between wild type (WT) and OmpW mutant (ΔOmpW) were determined. The survival rates of ΔOmpW in Luria-Bertani medium with 3.5% or 5.5% NaCl were reduced significantly, and morphological injury of ΔOmpW was significantly increased compared with survival and morphology of WT. Compared with biofilm formation of the WT strain, biofilms in ΔOmpW were significantly increased in Luria-Bertani with 3.5% or 5.5% NaCl using crystal violet staining assay after 48 and 72 h of incubation. Detection of biofilms using confocal laser scanning microscopy and scanning electron microscopy further confirmed the changes of biofilm formation under different NaCl stresses. This study demonstrates that OmpW contributes to survival of cells in planktonic mode under NaCl stresses, and biofilm formation is increased in ΔOmpW in response to NaCl stress.     

Difference between Escherichia coli O157:H7 and non-pathogenic E. coli: survival and growth in seasonings

We examined the survival and growth of Escherichia coli O157:H7 cells incubated with several seasonings, in comparison with those of non-pathogenic E. coli. The cells were incubated at 25 degrees C for 24 h with several concentrations of NaCl, sucrose, soy sauce, worcester sauce and tomato ketchup, and their survival ratios were determined. The E. coli O157:H7 strains showed relatively higher survival ratios in 0.5-1.0 M sucrose, 25% soy sauce and 12.5-50% worcester sauce than the non-pathogenic strains, but slightly lower survival ratios in 0.5-2.0 M NaCl. A noteworthy difference between E. coli O157:H7 and the non-pathogenic strains was that incubation in the presence of 12.5% soy sauce allowed the growth of E. coli O157:H7 strains but reduced the viable cell numbers of non-pathogenic E. coli strains.     

Effect of osmotic stabilizers on protoplast generation of Chlorella ellipsoidea yellow/white color mutants

The effect of osmotic stabilizers on protoplast formation of yellow/white color mutants of Chlorella ellipsoidea was studied. The UF-1 strain, one of the mutants, had the same growth rate as the parent C. ellipsoidea at 4.02 microE/m2/s and no reversion to normal cells at 0-26.8 microE/m2/s. Therefore, protoplast formation from UF-1 was carried out, and it was found that the yield of protoplasts was 93% in the medium containing 1.0 M NaCl as an osmotic stabilizer.     

The Osmotic Hypersensitivity of the Yeast Saccharomyces cerevisiae is Strain and Growth Media Dependent: Quantitative Aspects of the Phenomenon

Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth. Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory and industrial strains of Saccharomyces cerevisiae. During respiro-fermentative growth in rich medium with glucose as energy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value of about 1 m-NaCl; most strains fell within a 300 mm range in LD₁₀ values (lethal dose yielding 10% survival). Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e. ten-fold decreased viability for every 250 mm increase in salinity. Addition of small amounts of salt to the growth medium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations. This salt-instigated tolerance could partly be reversed by washing in water. The washing procedure depleted cells of the glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improved tolerance was about 50% in the two strains examined. Growth on derepressing carbon sources like galactose, ethanol or glycerol gave strain-dependent responses. The laboratory strain X2180–1A drastically improved tolerance while the bakers' yeast strain Y41 did so only marginally. It was concluded that all strains of S. cerevisiae display the osmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ. It was also proposed that growth rate does not dictate the level of osmotic hypersensitivity of S. cerevisiae. © 1997 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Acid and NaCl limits to growth of Listeria monocytogenes and influence of sequence of inimical acid and NaCl levels on inactivation kinetics

Variability in growth limits of Listeria monocytogenes in response to low pH (adjusted with HCl) or high salinity (NaCl) was evaluated for 127 strains in brain heart infusion broth at 25 degrees C. Over 95% of strains habituated at pH 5.0 grew subsequently at pH 4.2, while 25% were able to grow at pH 4.1. More than 85% of strains preadapted to growth at 8.5% NaCl (wt/vol) subsequently grew in the presence of 11.3% NaCl, while 25% were able to grow at 13% NaCl, and 4.7% grew in the presence of 13.9% NaCl. The results extend the generally accepted growth limits for L. monocytogenes in response to these hurdles. Two strains, one of which was relatively tolerant of both hurdles, and another that was less tolerant of both hurdles, were subjected to different sequences of lethal acid (pH 3.5) and NaCl (14%, wt/vol) stresses to determine whether survival was affected by growth limits, or by sequence of application of treatment. There was no significant difference in the inactivation kinetics of the two strains, but inactivation rates were affected by different treatments. For both strains, the inactivation rates, from fastest to slowest, resulted from: (i) lethal pH and then by lethal water activity, or lethal water activity and then by lethal pH; (ii) lethal pH and water activity applied simultaneously; (iii) lethal pH; and (iv) lethal water activity. The results demonstrated that the sequence of lethal stress application strongly influences L. monocytogenes inactivation, and that L. monocytogenes growth limits are not good predictors of survival in inimical environments.     

高渗胁迫下啤酒酵母的生理特性研究

研究了0．3mol／L、0．6mol／L、1．0mol/L的氯化钠对啤酒酵母活细胞数、细胞形态、糖代谢、pH值、菌体蛋白的影响。细胞动力学结果表明：随着NaCl浓度的增加,细胞的渗透胁迫加剧,细胞收缩。与对照组比较0．6mol／LNaCl、1．0mol／L NaCl延长细胞对数生长期,减缓葡萄糖消耗速率,且分别在2h-4h、6h-8h内出现糖代谢停滞,但并不影响细胞的增殖。在生理可接受的渗透范围内,与对照组比较0．3mol／LNaCl基本上不影响啤酒酵母的生物量、糖代谢速率和pH值。说明该菌株有较高的抗高渗能力。随着NaCl浓度的升高,渗透胁迫加剧,耐高渗有关的蛋白开始表达。     

COLLAGEN TYPES IN MECHANICALLY DEBONED CHICKEN MEAT

Mechanically deboned chicken meat (MDCM) from neck parts was digested with pepsin for 24 h at 4C. The solubilized collagens were subjected to salt fractionation both at acidic and neutral pH. The precipitates obtained after centrifugation, dialysis and lyophilization were quantitatively evaluated and collagen types were identified. The precipitate formed at 0.7 M NaCl pH 2.5, was dissolved in 1.0 M NaCl, 0.05 M-Tris-HCl, pH 7.5 buffer and further fractionated by sequential salt precipitation at 1.8, 2.5 and 4.5 M. Collagen types I, II, III and V were detected by electrophoresis techniques. Type I collagen was the major component. The presence of type II collagen indicated MDCM contained cartilaginous tissues.