Site-directed mutagenesis of the temperature-sensing histidine protein kinase CorS from Pseudomonas syringae

Several plant pathogenic bacteria belonging to the species Pseudomonas syringae produce the phytotoxin coronatine to enhance their virulence. Pseudomonas syringae pv. glycinea PG4180 synthesizes coronatine at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, its optimal growth temperature. In contrast, temperature has virtually no effect on coronatine synthesis in P. syringae pv. tomato strain DC3000. A modified two-component system controlling coronatine synthesis and consisting of the histidine protein kinase (HPK), CorS, the response regulator, CorR, and a third essential component, CorP, had been identified previously in both strains. CorS had been identified previously as a potential thermo-sensor. Comparison of the amino acid sequences of the HPKs from the two organisms revealed distinct differences. Site-directed mutagenesis of CorS from PG4180 was used to identify amino acyl residues potentially important for temperature signal perception. Point mutations and combinations of these were introduced into corS of PG4180 to generate corS variants with increased similarities to the respective allele from strain DC3000. These mutations resulted in either loss of activity, increase of thermoresponsiveness, or had no effect on CorS activity. Although none of the introduced mutations resulted in a clear conversion of CorS activity from thermo-responsive to temperature-independent, amino acyl residues important for temperature-dependent CorS activity and coronatine biosynthesis were identified.     

Cloning and Expression of Genes Required for Coronamic Acid (2-Ethyl-1-Aminocyclopropane 1-Carboxylic Acid), an Intermediate in the Biosynthesis of the Phytotoxin Coronatine

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.     

Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium Pseudomonas syringae pv. glycinea

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.     

Cloning, Nucleotide Sequence, and Expression in Escherichia coli of Levansucrase Genes from the Plant Pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae, Erwinia amylovora, and other bacterial species. The lsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene in Escherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3.0-kb PstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to the E. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring the lsc gene. Expression of lsc was found to be dependent on the vector-based P_lac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.     

Interspecific variation in thermoregulation among three sympatric bats inhabiting a hot, semi-arid environment

Bats in hot roosts experience some of the most thermally challenging environments of any endotherms, but little is known about how heat tolerance and evaporative cooling capacity vary among species. We investigated thermoregulation in three sympatric species (Nycteris thebaica, Taphozous mauritianus and Sauromys petrophilus) in a hot, semi-arid environment by measuring body temperature (T _b), metabolic rate and evaporative water loss (EWL) at air temperatures (T _a) of 10?C42?°C. S. petrophilus was highly heterothermic with no clear thermoneutral zone, and exhibited rapid increases in EWL at high T _a to a maximum of 23.7?±?7.4?mg?g^?1?h^?1 at T _a????42?°C, with a concomitant maximum T _b of 43.7?±?1.0?°C. T. mauritianus remained largely normothermic at T _as below thermoneutrality and increased EWL to 14.7?±?1.3?mg?g^?1?h^?1 at T _a????42?°C, with a maximum T _b of 42.9?±?1.6?°C. In N. thebaica, EWL began increasing at lower T _a than in either of the other species and reached a maximum of 18.6?±?2.1?mg?g^?1?h^?1 at T _a?=?39.4?°C, with comparatively high maximum T _b values of 45.0?±?0.9?°C. Under the conditions of our study, N. thebaica was considerably less heat tolerant than the other two species. Among seven species of bats for which data on T _b as well as roost temperatures in comparison to outside T _a are available, we found limited evidence for a correlation between overall heat tolerance and the extent to which roosts are buffered from high T _a.     

Effect of culture conditions of Kluyveromyces marxianus on its autolysis, and process optimization

Cultivation of the lactose-metabolizing yeast Kluyveromyces marxianus var. marxianus (formerly K.?fragilis) on supplemented whey permeate resulted in cellular yield little affected by culture conditions in the ranges pH?=?2.3–5 and T?=?30–40?°C. When autolysis was induced only by energy source deficiency and thermal shock, cellular material solubilization depended slightly on autolysis temperature in the range T?=?45–60?°C. On the contrary, the process was under tight control of culture conditions; when autolysis was carried out at 50?°C with an initial dry cellular concentration of 50?g l^?1, a clear optimum was observed for cells cultivated at pH?=?4.5 and T?=?35?°C. So the critical step of the autolytic process consisted in biosynthesis of lytic enzymes (during cell growth) rather than enzymatic progress (during autolysis). These results were compatible with a model previously proposed for Saccharomyces cerevisiae [1].     

Bioremediation potential of Chondrus crispus (Basin Head) and Palmaria palmata: effect of temperature and high nitrate on nutrient removal

To evaluate the nutrient removal capabilities of two red macroalgae, apical blades were cultured in the lab for 4?weeks at either 6, 10, or 17°C and nitrate at either 30 or 300?μM, typical of the seasonal range of conditions at a land-based Atlantic halibut farm. Stocking density was 2.0?g?L^?1, irradiance 125?μmol?photons?m^?2?s^?1, photoperiod 16:8 (L:D), and nitrogen to phosphorus ratio 10:1. For both species, the highest growth rate was at 300?μM NO ₃ ^? with Palmaria palmata growing fastest at 6°C, 5.8%?day^?1, and Chondrus crispus growing best at 17°C, 5.5%?day^?1. Nitrogen and carbon removal by P. palmata was inversely related to temperature, the highest rate at 6°C and 300?μM NO ₃ ^? of 0.47?mg N and 6.3?mg C per gram dry weight per day. In contrast, C. crispus removal of N was independent of temperature, with mean removal of 0.49?mgN?gDW^?1?day^?1 at 300?μM NO ₃ ^? . The highest carbon removal by C. crispus was 4.4?mgC?gDW^?1?day^?1 at 10°C and 300?μM nitrate, though not significantly different from either 6 or 17°C and 300?μM nitrate. Tissue carbon:nitrogen ratios were >20 in both species at 30?μM nitrate, and all temperatures indicating nitrogen limitation in these treatments. Phycoerythrin content of P. palmata was independent of temperature, with means of 23.6?mg?gFW^?1 at 300?μM nitrate. In C. crispus, phycoerythrin was different only between 6°C and 17°C at 300?μM nitrate, with the highest phycoerythrin content of 12.6?mg?gFW^?1 at 17°C. Morphological changes were observed in P. palmata at high NO ₃ ^? concentration as curling of the fronds, whilst C. crispus exhibited the formation of bladelets as an effect of high temperature.     

Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production

Phaseolotoxin, a phytotoxin of Pseudomonas syringae pv. phaseolicola, is produced at 18°C but not at 28°C. Here we report that a fragment (24.4 kb) cloned from the wild-type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox^? mutant at both temperatures. A subclone harbouring a 465bp fragment contains motifs that are characteristic of DNA-binding sites. In mobility shift assays we have detected a protein(s) from the wild-type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA-binding motifs. We propose that the binding protein is a repressor which is ‘titrated’ by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.     

Light and temperature conditions affect bioflavonoid accumulation in callus cultures of Cyclopia subternata Vogel (honeybush)

Callus cultures of the endemic South-African legume Cyclopia subternata were cultivated under varying light and temperature conditions to determine their influence on biomass growth and bioflavonoids accumulation. Experimental modifications of light included complete darkness, light of different spectral quality (white, red, blue and yellow) and ultraviolet C (UVC) irradiation. The calli were also subjected to elevated temperature or cold stress. Among the tested light regimes, cultivation under blue light resulted in the highest levels of hesperidin (H)—118.00 mg 100 g^?1 dry weight (DW) on 28 days of experiment, as well as isoflavones: 7-O-β-glucosides of calycosin (CG), pseudobaptigenin (PG) and formononetin (FG)—28.74, 19.26 and 10.32 mg 100 g^?1 DW, respectively, in 14-days old calli. UVC irradiation applied on 20 days stimulated the accumulation of H (204.14 mg 100 g^?1 DW), CG (31.84 mg 100 g^?1 DW) and PG (18.09 mg 100 g^?1 DW) in 28 days culture by 140, 46 and 165 %, respectively, without negatively influencing callus growth. Low temperature (13 °C) increased CG content by over 1,500 % (235.29 mg 100 g^?1 DW) when applied during the whole 28-days growth cycle, at the same time causing 95 % decrease in culture growth in comparison to reference calli maintained at 24 °C. On the contrary, elevated temperature (29 °C) applied during the second half of the culture period resulted in over 300 and 500 % increase in CG and PG content (61.76 and 58.89 mg 100 g^?1, respectively) while maintaining relatively high biomass yield.     

Body temperature and respiratory dynamics in un-shaded beef cattle

In this study body temperature (BT, °C) and panting score (PS, 0–4.5; where 0?=?no panting/no stress and 4.5?=?catastrophic stress) data were obtained from 30 Angus steers housed outside over 120 days Steers were implanted with a BT transmitter on day ?31, BT was recorded at 30-min intervals to a data logger and downloaded each day to a database. The cattle were housed in ten outdoor un-shaded pens with an earthen floor, eight of which had a pen floor area of 144 m² (three transmitter steers plus five non-transmitter steers; 18 m²/steer) and two had an area of 168 m² (three transmitter steers and six non-transmitter steers; 18.7 m²/steer). Only data from the transmitter steers were used in this study. The PS of the steers was obtained daily (± 15 min) at 0600 hours (AM), 1200 hours (MD) and 1600 hours (PM). At the same times climate variables (ambient temperature, black globe temperature, solar radiation, relative humidity, wind speed and rainfall) were obtained from an on-site weather station. PS observations were made from outside the pens so as not to influence cattle responses. The two closest BT values to the time when PS was obtained were downloaded retrospectively from a logger and averaged. A total of 8,352 observations were used to generate second order polynomial response curves: (AM) y?=?39.08?+?0.009?x +?0.137x ² (R ²?=?0.94; P? y?=?39.09?+?0.914x ? 0.080x ² (R ²?=?0.89; P? y?=?39.52?+?0.790x ? 0.068x ² (R ²?=?0.83; P?x?PS. These data suggest that PS is a good indicator of body temperature. The BT at MD corresponded to slightly lower PS compared with PM, e.g., for PS 1; BT at MD?=?39.1?±?0.05 °C whereas BT at PM?=?39.5?±?0.05 °C. However during AM, BT was lower (P?相似文献   

Acute Toxicity of Arsenic under Different Temperatures and Salinity Conditions on the White Shrimp Litopenaeus vannamei

The aim of this study was to determine acute toxicity in the post larvae of the white shrimp Litopenaeus vannamei after 96 h of exposure to dissolved arsenic under three different temperatures and salinity conditions. Recent reports have shown an increase in the presence of this metalloid in coastal waters, estuaries, and lagoons along the Mexican coast. The white shrimp stands out for its adaptability to temperature and salinity changes and for being the main product for many commercial fisheries; it has the highest volume of oceanic capture and production in Mexican shrimp farms. Lethal concentrations (LC₅₀–96 h) were obtained at nine different combinations (3?×?3 combinations in total) of temperature (20, 25, and 30 °C) and salinity (17, 25, and 33) showing mean LC₅₀–96 h values (±standard error) of 9.13?±?0.76, 9.17?±?0.56, and 6.23?±?0.57 mgAs?L^?1(at 20 °C and 17, 25, and 33 salinity); 12.29?±?2.09, 8.70?±?0.82, and 8.03?±?0.59 mgAs?L^?1 (at 25 °C and 17, 25, and 33 salinity); and 7.84?±?1.30, 8.49?±?1.40, and 7.54?±?0.51 mgAs?L^?1 (at 30 °C and 17, 25, and 33 salinity), respectively. No significant differences were observed for the optimal temperature and isosmotic point of maintenance (25 °C–S 25) for the species, with respect to the other experimental conditions tested, except for at 20 °C–S 33, which was the most toxic. Toxicity under 20 °C–S 33 conditions was also higher than 25 °C–S 17 and 20 °C (S 17 or 25). The least toxic condition was 25 °C–S 17. All this suggests that the toxic effect of arsenic is not affected by temperature changes; it depends on the osmoregulatory pattern developed by the shrimp, either hyperosmotic at low salinity or hiposmotic at high salinity, as observed at least on the extreme salinity conditions here tested (17 and 33). However, further studies testing salinities near the isosmotic point (between 20 and 30 salinities) are needed to clarify these mechanisms.     

A study on intra-particle diffusion effects in enzymatic reactions: glucose-fructose isomerization

In this work different aspects of the glucose-fructose enzymatic isomerization, using immobilized glucose isomerase, are studied and quantified. Reaction temperatures range from 40?°C to 60?°C. Intra-particle effective diffusivities (D _e), determined after uptake experiments, are between 1.20?×?10^?6?cm²/s, at 40?°C, and 2.52?×?10^?6?cm²/s, at 60?°C. The estimated energy of activation for diffusion (E _aD) is 7.71?kcal/mol. No significant adsorption of the sugars on the support gel matrix is observed. Crushed particles (φ = 150–350?μ) are used during kinetic experiments. For this range of particle diameters, inherent kinetics is approached. A reversible Michaelis–Menten rate equation is fitted to the data, providing the following parameters at pH = 7.0: k ₀ = 2.15?×?10^?6?g/IU/s; E _a/R = 8998?K. Glucose (K _G) and fructose (K _F) affinity constants are essentially the same, ranging from 0.190?M, at 40?°C to 0.305?M, at 60?°C. The thermodynamic equilibrium constant is determined for the three temperatures, and the heat of reaction, estimated from a Van't Hoff plot, is ΔH = 1682?cal/mol. Independent experiments, where the reaction occurs in the presence of significant intra-particle mass transfer resistance, are used as validation tests.     

Effect of polyphenols extracted from tamarind (Tamarindus indica L.) seed coat on pathophysiological changes and red blood cell glutathione peroxidase activity in heat-stressed broilers

The purpose of this study was to determine the effect of polyphenols extracted from the tamarind seed coat (PETSC) on glutathione peroxidase (GPx) activity, red blood cell parameters and bilirubin in heat-stressed broilers. One hundred forty-seven broilers, 18-days old were divided into two groups. In group 1, broilers were maintained at an environmental temperature of 26?±?2 °C throughout the experimental period. In group 2, the broilers were maintained at 38?±?2 °C (cyclic temperature: 26?±?2 °C; ?38?±?2 °C; and ?26?±?2 °C, and broilers were maintained at 38?±?2 °C for 6 h/ day) and received PETSC at a concentration of 0, 100, 200, 300, 400 or 500 mg/kg in their diet ad libitum. Parameters were investigated on days 1, 7, 14 and 21 of the experimental period. Results showed that GPx activity of heat-stressed broilers that received 100 mg/kg of PETSC in their diet was lower (P?<?0.05) than that in broilers fed the other concentrations. The mean total red blood cell count and hemoglobin concentration of heat-stressed broilers that received 100 mg/kg PETSC was higher (P?<?0.05) than those in broilers in group 1 and those fed the other concentrations. The mean bilirubin level in the excreta of heat-stressed broilers that received 100 mg/kg of PETSC was lower (P?<?0.05) than that in broilers that received 0, 300, 400 and 500 mg/kg of PETSC. This showed that PETSC could reduce GPx activity and bilirubin in feces, and increase red blood cell parameters in heat-stressed broilers.     

Usefulness of an aluminized polyester film for reducing heat in polyethylene calf hutches

This study determined the efficacy of a radiant barrier material used in the construction industry to moderate summer temperatures in polyethylene calf hutches. The cover consisted of a single layer of two-sided reflective aluminized polyester film with a center polyester scrim reinforcement (reflectivity?=?95 %). At each of two dairies, six hutches containing a young calf were either uncovered (control) or had reflective covers across the top and sides of the hutch, leaving the front, back, and 1.2?×?1.8-m attached outdoor wire pen exposed. Duplicate loggers mounted 20 cm above the flooring in the center of each hutch recorded interior temperature at 30-min intervals over 22 days during late August to early September. The mean daily interior peak temperatures in each of the hutches over 21 days of observation were significantly less (P?P?=?0.77) between dairies. The mean daily interior peak temperatures in each of the hutches over the warmest 10 days of observation were significantly less (P?P?相似文献   

Temperature-dependent development and life table parameters of Thrips palmi (Thysanoptera: Thripidae) on eggplant

The life history of Thrips palmi Karny on eggplant (Solanum melongena L.) leaves was studied based on the age stage and two sex-life tables at 16, 19, 22, 25, and 31?°C. The intrinsic rate of increase (r) at these temperatures was 0.0427, 0.0566, 0.0979, 0.1738, and 0.2237?day^?1, respectively. The relationship among the gross reproductive rate (GRR), the net reproductive rate (R ₀), and the pre-adult survivorship (l _a) is consistent with R ₀?<?l _a?×?GRR?<?GRR for all results at different temperatures. The mean generation time was 47.52, 38.33, 29.52, 19.81, and 13.88?days, respectively. The developments of pre-adult and adult stages were faster in males than in females. The means of developmental periods for each developmental stage decreased with increases of temperature. The maximum life span of female adults was noted at 56.67?days, whereas that of males was 50.66?days at 16?°C. The maximum female fecundity (64.18?eggs/female) was recorded at 25?°C and the lowest (23.38 eggs/female) at 16?°C. Life table data could be used to project population growth, to design mass rearing programs, and to establish management tactics to control insect pests.     

High-level expression of a xylanase gene from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5?l fermentor culture, the total extracellular protein was 8.1?g?l^?1 with an activity of 52,940?U?ml^?1. The enzyme was purified to homogeneity with a recovery of 48?%. The recombinant xynA was optimally active at 75?°C, as measured over 10?min, and at pH 7. The enzyme was stable up to 80?°C for 30?min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.