共查询到20条相似文献,搜索用时 15 毫秒
1.
Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M. Lowder A. Unge N. Maraha J. K. Jansson J. Swiggett J. D. Oliver 《Applied microbiology》2000,66(8):3160-3165
The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost. 相似文献
2.
Sanjay K. S. Patel Rahul K. Gupta Sang-Yong Kim In-Won Kim Vipin C. Kalia Jung-Kul Lee 《Indian journal of microbiology》2021,61(1):45
In the present study, Rhus vernicifera laccase (RvLac) was immobilized through covalent methods on the magnetic nanoparticles. Fe2O3 and Fe3O4 nanoparticles activated by 3-aminopropyltriethoxysilane followed with glutaraldehyde showed maximum immobilization yields and relative activity up to 81.4 and 84.3% at optimum incubation and pH of 18 h and 5.8, respectively. The maximum RvLac loading of 156 mg/g of support was recorded on Fe2O3 nanoparticles. A higher optimum pH and temperature of 4.0 and 45 °C were noted for immobilized enzyme compared to values of 3.5 and 40 °C for free form, respectively. Immobilized RvLac exhibited better relative activity profiles at various pH and temperature ranges. The immobilized enzyme showed up to 16-fold improvement in the thermal stability, when incubated at 60 °C, and retained up to 82.9% of residual activity after ten cycles of reuses. Immobilized RvLac exhibited up to 1.9-fold higher bisphenol A degradation efficiency potential over free enzyme. Previous reports have demonstrated the immobilization of RvLac on non-magnetic supports. This study has demonstrated that immobilization of RvLac on magnetic nanoparticles is very efficient especially for achieving high loading, better pH and temperature profiles, and thermal- and solvents-stability, high reusability, and higher degradation of bisphenol A. 相似文献
3.
C. Trunet N. Mtimet A.-G. Mathot F. Postollec I. Leguerinel D. Sohier O. Couvert F. Carlin L. Coroller 《Applied and environmental microbiology》2015,81(2):562-568
The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory. 相似文献
4.
Abdollah Ghobakhlou Serge Laberge Hani Antoun David S. Wishart Jianguo Xia Ramanarayan Krishnamurthy Rupasri Mandal 《PloS one》2013,8(12)
Arctic Mesorhizobium sp. N33 isolated from nodules of Oxytropis arctobia in Canada’s eastern Arctic has a growth temperature range from 0°C to 30°C and is a well-known cold-adapted rhizobia. The key molecular mechanisms underlying cold adaptation in Arctic rhizobia remains totally unknown. Since the concentration and contents of metabolites are closely related to stress adaptation, we applied GC-MS and NMR to identify and quantify fatty acids and water soluble compounds possibly related to low temperature acclimation in strain N33. Bacterial cells were grown at three different growing temperatures (4°C, 10°C and 21°C). Cells from 21°C were also cold-exposed to 4°C for different times (2, 4, 8, 60 and 240 minutes). We identified that poly-unsaturated linoleic acids 18∶2 (9, 12) & 18∶2 (6, 9) were more abundant in cells growing at 4 or 10°C, than in cells cultivated at 21°C. The mono-unsaturated phospho/neutral fatty acids myristoleic acid 14∶1(11) were the most significantly overexpressed (45-fold) after 1hour of exposure to 4°C. As reported in the literature, these fatty acids play important roles in cold adaptability by supplying cell membrane fluidity, and by providing energy to cells. Analysis of water-soluble compounds revealed that isobutyrate, sarcosine, threonine and valine were more accumulated during exposure to 4°C. These metabolites might play a role in conferring cold acclimation to strain N33 at 4°C, probably by acting as cryoprotectants. Isobutyrate was highly upregulated (19.4-fold) during growth at 4°C, thus suggesting that this compound is a precursor for the cold-regulated fatty acids modification to low temperature adaptation. 相似文献
5.
Heat Stress Responses in Cultured Plant Cells : Heat Tolerance Induced by Heat Shock versus Elevated Growing Temperature 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Using cultured pear (Pyrus communis cv Bartlett) cells, heat tolerance induced by heat shock was compared to that developed during growth at high temperature. After growth at 22°C, cells exposed to 38°C for 20 minutes (heat shock) showed maximum increased tolerance within 6 hours. Cells grown at 30°C developed maximum heat tolerance after 5 to 6 days; this maximum was well below that induced by heat shock. Heat shock-induced tolerance was fully retained at 22°C for 2 days and was only partly lost after 4 days. However, pear cells acclimated at 30°C lost all acquired heat tolerance 1 to 2 days after transfer to 22°C. In addition, cells which had been heat-acclimated by growth at 30°C showed an additional increase in heat tolerance in response to 39°C heat shock. The most striking difference between heat shock and high growth temperature effects on heat tolerance was revealed when tolerance was determined using viability tests based on different cell functions. Growth at 30°C produced a general hardening, i.e. increased heat tolerance was observed with all three viability tests. In contrast, significantly increased tolerance of heat-shocked cells was observed only with the culture regrowth test. The two types of treatment evoke different mechanisms of heat acclimation. 相似文献
6.
Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L 总被引:4,自引:4,他引:0
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10−5 molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N2 after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10−2 molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10−4 molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT50 of −5°C to an LT50 of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days. 相似文献
7.
To explore chitin-binding domain (ChBD)-based cell immobilization, a tripartite gene fusion consisting of an in-frame fusion of ChBD to lpp and ompA was constructed and expressed in Escherichia coli. ChBD-displayed cells exhibited highly specific and stable binding to chitin within a wide range of pHs (5 to 8) and temperatures (15 to 37°C). These results illustrate the promising use of this approach for engineering applications. 相似文献
8.
Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant, Arthrobacter chlorophenolicus A6
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28°C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system (“viable cells”) whereas propidium iodide (PI) was used to stain cells with damaged membranes (“dead cells”). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28°C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28°C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5°C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates. 相似文献
9.
Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Amare Gessesse 《Applied microbiology》1998,64(9):3533-3535
Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers. 相似文献
10.
Three synthetically produced glycolipids, N-(β-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(β-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(β-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70°C. X-Ray diffraction revealed a direct Lβ' → H transition at 20°C, 60°C, and 45°C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of ≈20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H phase the binding of water is stronger than in the Lβ' phase. In the temperature range below 0°C, OSGA and OOGA show a “subzero transition” due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal → distorted hexagonal at 60°C. 相似文献
11.
A new strain of Prototheca zopfii Krüger was grown on acetate or on pure n-alkanes. A maximum acetate-supported exponential growth of 12 divisions day−1 occurred at pH 5 and 30°C. At 25°C, growth on n-alkanes was almost as fast, but no growth occurred at 30°C. After 4 days at 25°C, 34 to 45% of the n-alkanes had been removed, whereas at 21°C and slower growth, utilization was twofold greater after 15 days. Rates of growth and utilization increased markedly after a point of sudden emulsification. 相似文献
12.
The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT50) of about −3°C to an LT50 of about −6°C after 24 hours at 4°C; LT50 values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A+) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides. 相似文献
13.
Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication 总被引:4,自引:3,他引:1
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Yasuyoshi Ike Hajime Hashimoto Kazuo Motohashi Noriko Fujisawa Susumu Mitsuhashi 《Journal of bacteriology》1980,141(2):577-583
A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)+ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)+ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 106 and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 106. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid. 相似文献
14.
The metabolism of [2-14C]indole in the rat 总被引:3,自引:1,他引:2
1. [2-14C]Indole has been synthesized from [14C]formate and o-toluidine via N[14C]-formyltoluidine. 2. When fed to rats, the 14C of [14C]indole (dose 70–80mg./kg. body wt.) is fairly rapidly excreted, and in 2 days an average of 81% appears in the urine, 11% in the faeces and 2·4% as carbon dioxide in the expired air. 3. Radioactivity is excreted in the urine as indoxyl sulphate (50% of the dose), indoxyl glucuronide (11%), oxindole (1·4%), isatin (5·8%), 5-hydroxyoxindole conjugates (3·1%), N-formylanthranilic acid (0·5%) and unchanged indole (0·07%). The faeces contain indoxyl sulphate (0·4% of the dose) and indole (0·2%), but the major metabolites have not been identified. 4. Fed to rats with biliary cannulae an average of 5·6% of a dose of [14C]indole (20–60mg./kg. body wt.) is excreted in the bile in 2 days. Radioactivity is present as indoxyl sulphate (0·8% dose) and 5-hydroxyoxindole conjugates (0·6%). 5. Rats further metabolize indoxyl into N-formylanthranilic acid and anthranilic acid, and oxindole into 5-hydroxyoxindole. 6. With rat-liver microsomes plus supernatant under aerobic conditions, indole gives indoxyl, oxindole, possibly isatin, N-formylanthranilic acid and anthranilic acid, but under anaerobic conditions gives only oxindole. Similarly, under aerobic conditions, oxindole gives 5-hydroxyoxindole, anthranilic acid and o-aminophenylacetic acid. 7. Indole is metabolized by two pathways, one via indoxyl to isatin, N-formylanthranilic acid and anthranilic acid, and the other via oxindole to 5-hydroxyoxindole and possibly to o-aminophenylacetic and anthranilic acid. 8. The following new compounds are described: 4-hydroxy-2-nitrophenylacetic acid, 3-, 4- and 5-benzyloxy-2-nitrophenylacetic acid, 5- and 7-hydroxyoxindole and 5-aminoacridine indoxyl sulphate. 相似文献
15.
Renu Khanna-Chopra Vimal Kumar Semwal 《Physiology and Molecular Biology of Plants》2011,17(4):339-346
Thermal stability of antioxidant defense enzymes was investigated in leaf and inflorescence of heat adaptive weed Chenopodium album. Leaf samples were taken at early and late seedling stage in December (LD, 20 °C/4 °C) and March (LM, 31 °C/14 °C). Young inflorescence (INF) was sampled at flowering in April (40 °C/21 °C). LD, LM and INF crude protein extracts were subjected to elevated temperatures (5 to 100 °C) for 30′. Superoxide dismutase (SOD) was the most heat stable enzyme followed by Ascorbate peroxidase (APX). Two heat stable SOD isozymes were visible on native-PAGE at 100 °C in both leaf and INF. Some heat stable APX isozymes were more abundant in INF than leaf. Thermostability of catalase (CAT) increased with age and increasing ambient temperatures in leaves. CAT activity was observed up to 60 °C in leaves and INF while peroxidase (POX) retained activity up to 100 °C in INF due to one thermostable isozyme. Glutathione reductase (GR), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR) showed activity up to 70 °C in both leaves and INF. DHAR activity was stable up to 60 °C while GR and MDHAR declined sharply after 40 °C. Constitutive heat stable isozymes of SOD and APX in leaves and INF may contribute towards heat tolerance in C. album. 相似文献
16.
Simultaneous Direct Counting of Total and Specific Microbial Cells in Seawater, Using a Deep-Sea Microbe as Target 总被引:7,自引:3,他引:4
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
To rapidly and accurately enumerate total and specific microbes in aquatic samples, fluorescent in situ hybridization was combined with direct counting via direct immobilization of cells on a polymer-coated Nuclepore filter. The technique, named FISH-DC, achieved almost complete recovery of total cells and reproducibility of Psychrobacter pacificensis cells of deep-sea origin (error, ≤3%) in a mixed culture and in natural seawater. Target cells immobilized on the filter were also successfully enumerated after stringent 3-cycle hybridization and even after a 16-month preservation at −30°C. 相似文献
17.
Identification and Partial Characterization of the Denaturation Transition of the Light Harvesting Complex II of Spinach Chloroplast Membranes
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Differential scanning calorimetry was employed to investigate the structure of spinach (Spinacia oleracea) chloroplast membranes. In a low ionic strength Hepes-buffered medium, major calorimetric transitions were resolved at 42.5°C. (A), 60.6°C (B), 64.9°C (C1), 69.6°C (C2), 75.8°C (D), 84.3°C (E), and 88.9°C (F). A lipid melting transition was also commonly seen at 17°C in scans starting at lower temperatures. The D transition was demonstrated by four independent methods to derive from denaturation of the light harvesting complex associated with photosystem II (LHC-II). Evidence for this conclusion was as follows: (a) the endotherm of the isolated LHC-II (74.0°C) was very similar to that of D (75.8°C); (b) the denaturation temperature of the 27 kilodalton LHC-II polypeptide determined in intact chloroplast membranes by thermal gel analysis was identical to the temperature of the D transition at pH 7.6 and after destabilization by shifting the pH to 6.6 or by addition of Mg2+; (c) analysis of the stability of the LHC-II complex by electrophoresis in native gels demonstrated that the complex dissociates during the D transition, both at pH 7.6 and 6.6; and (d) the 77 Kelvin fluorescence maximum of LHC-II in chloroplasts was seen to shift to lower wavelengths (indicating gross denaturation of LHC-II), at the temperature of the D transition when examined at either of the above pHs. With this identification, five of the eight major endotherms of the chloroplast membrane have now been assigned. 相似文献
18.
Jun Chen Jing Shen Christian Solem Peter Ruhdal Jensen 《Applied and environmental microbiology》2013,79(19):6140-6147
Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen. 相似文献
19.
Michael B. Elowitz Michael G. Surette Pierre-Etienne Wolf Jeffry B. Stock Stanislas Leibler 《Journal of bacteriology》1999,181(1):197-203
The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient, Da, of GFP in E. coli DH5α was found to be 7.7 ± 2.5 μm2/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with Da of 2.5 ± 0.6 μm2/s. In addition, GFP mobility can depend strongly on at least two factors: first, Da is reduced to 3.6 ± 0.7 μm2/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces Da to 4.0 ± 2.0 μm2/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of Da reported here. These measurements have implications for the understanding of intracellular biochemical networks. 相似文献
20.
Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation. 相似文献