Conditions for Maximum Enflagellation in Naegleria fowleri1

ABSTRACT. Ameba to flagellate transformation in Naegleria fowleri (Lovell strain) was affected by growth temperature, phase of growth, strain of ameba, culture agitation, enflagellation temperature, enflagellation diluent, and cell concentration. Amebae transformed best when they were grown without agitation and enflagellated with agitation. Regardless of growth temperature (23°, 30°, 37°, and 42°C were tested), amebae transformed best at 37°C. Enflagellation was greatest for cells harvested between 24 h (mid-exponential) and 84 h (late stationary) of growth.     

Combined effects of sulfites, temperature, and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae.

Analysis of variance was used to evaluate the simultaneous effects of strain, incubation temperature (15 to 25 degrees C), agitation time (0 to 24 h), and initial sulfite concentration (100 to 300 ppm) on glycerol production in grape juice by Saccharomyces cerevisiae. Fourteen strains were studied to determine their growth patterns in the presence of sulfites and ethanol. Baker's yeast strains were more sensitive to sulfite than wine strains, and little growth occurred at initial sulfite levels greater than 150 ppm. Sensitivity to sulfite increased with increasing levels of ethanol. Three strains exhibiting the best growth in the presence of sulfites and ethanol were selected for interaction studies. Fermentations were carried out until the solids content had decreased to less than 6 degrees Brix, which was the point that glycerol content became stable. For the three strains used, the greatest level of glycerol production was observed in the presence of 300 ppm of sulfite for most incubation temperatures and agitation times. There was significant interaction between the strain, incubation temperature, and agitation time parameters for glycerol synthesis, and a response surface method was used to predict the optimal conditions for glycerol production. Under static conditions, the highest level of glycerol production was observed at 20 degrees C, while incubation at 25 degrees C gave the best results when the cultures were agitated for 24 h. Response surface equations were used to predict that the optimum conditions for glycerol production by S. cerevisiae Y11 were a temperature of 22 degrees C, an initial sulfite concentration of 300 ppm, and no agitation, which yielded 0.68 g of glycerol per 100 ml.     

Biological factors affecting enflagellation of Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate that can be evoked to transform from amoebae to flagellates by subculture to nonnutrient buffer. More than half of the amoebae of strains KUL, nN68, and Lovell became enflagellated 300 min after subculture to amoeba-saline, whereas no amoebae of strains NF66, NF69, and HB4 did. N. fowleri nN68 enflagellated best when grown at 32 or 37 degrees C and subcultured to amoeba-saline at 37 or 42 degrees C. Amoebae from the stationary phase of growth enflagellated more readily than did actively growing amoebae. Incubation in expended culture medium from stationary-phase cultures enhanced the capability of growing amoebae to enflagellate after subculture to amoebasaline. Enflagellation was more extensive when the population density in amoebasaline did not exceed 2 x 10(5) amoebae per ml. Cycloheximide at 1 microgram/ml and actinomycin D at 25 micrograms/ml inhibited growth of N. fowleri nN68. Cycloheximide at 0.5 microgram/ml and actinomycin D at 25 micrograms/ml completely prevented enflagellation when added at time zero. Cycloheximide at 0.5 microgram/ml, added 120 to 300 min after initiation of enflagellation, prevented further differentiation and caused existing flagellates to revert to amoeboid cells. Similarly, actinomycin D at 25 micrograms/ml, added 90 to 300 min after initiation of enflagellation, retarded differentiation and caused flagellates to revert. Radiolabeled precursors were incorporated into macromolecules during differentiation in nonnutrient buffer. Enflagellation of N. fowleri is a suitable model for studying regulation of a eucaryotic protist.     

Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities

Bacillus proteolyticus CFR3001 isolated from fish processing wastes (both fresh water and marine) produced an alkaline protease. The optimum conditions for cell growth and protease production were 37 degrees C, 96 h, agitation speed of 100 rpm and medium pH 9. The partially purified protease obtained from had specific activity of 22.05 at 37 degrees C was active between 40 degrees C and 50 degrees C and lost >20% of its activity around 60 degrees C. Its molecular weight was approximately 29 kDa and it inhibited the growth of several pathogenic organisms such as Escherichia coli, Listeria monocytogenes, Bacillus cereus and Yersinia enterocolytica. The scanning electron microscopy (SEM) studies revealed that the protease produced by B. proteolyticus CFR3001 lysed the cells of these pathogenic bacteria.     

Assembly of the mitochondrial ribosomes in a temperature-conditional mutant of Saccharomyces cerevisiae defective in the synthesis of the var1 protein

An investigation of the role of the var1 protein in the assembly of the yeast mitochondrial ribosomes was carried out in a temperature conditional mutant, strain h56, which contains a mutation (tsv1) just upstream of the structural gene for the var1 protein. The mutation results in a marked decrease in the synthesis of the var1 protein at the permissive temperature of 28 degrees C and an apparently complete absence of var1 synthesis at the restrictive temperature of 36 degrees C. Long-term growth of strain h56 at the non-permissive temperature was found to result in the loss of the small (37 S) ribosomal subunit and the appearance of a novel 30 S ribonucleoparticle. Both the small (37 S) and the large (54 S) mitochondrial ribosomal subunits were found to be assembled in strain h56 for at least 3 h after transfer to the non-permissive temperature.     

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.     

Simian virus 40 A gene function: further characterization and growth of tsA transformed chinese hamster cells

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33 degrees C or 37 degrees C) and a "normal" phenotype at the nonpermissive temperature (40.5 degrees C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the "T" antigen (100 K) has an increase rate of synthesis and degradation at 40.5 degrees C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5 degrees C (13 hours vs. 100 hours). These results suggest that at 40.5 degrees C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5 degrees C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.     

Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion.

Bacillus subtilis strains carrying div-341 or sacU mutations, or both, have been characterized to reveal the roles of both genes in the initiation of sporulation, as well as in cell division and exoenzyme secretion. Both mutations were closely linked by transformation and caused the pleiotropic effects on sporulation and sporulation-associated events. Some sacU mutations (sacUh) resulted in hyperproduction of exoenzymes, reduced autolysis, and an ability to sporulate in the presence of excess nutrients. The div-341 mutation, on the other hand, resulted in filamentous growth at a higher temperature (45 degrees C) and showed spo0 properties at an intermediate permissive temperature (37 degrees C) in the usual sporulation medium. However, the div-341 strain sporulated better than wild-type strain at 37 degrees C in the presence of excess nutrients. Exoenzyme production and autolysis were reduced at 37 degrees C in the div-341 strain. A double mutant with sacUh32 and div-341 showed the complex phenotypes. It showed the sacUh32 property of autolysis and exoenyzme secretion. It showed the sacUh32 property of sporulation at 30 degrees C and the div-341 property at 37 degrees C. Slow growth and defective spore outgrowth of the div-341 strain at 37 degrees C were not observed in the double-mutant strain. Based on pleiotropic phenotypes and close linkages of both mutations, we discuss the relationship between the sacU and div-341 genes and their roles in sporulation, exoenzyme secretion, and cell division.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Production of hepatitis B core antigen in a stirred tank bioreactor: The influence of temperature and agitation

The influence of temperature and agitation on the growth ofEscherichia coli expressing hepatitis B core antigen (HBcAg) in stirred tank bioreactor were investigated. The highest specific growth rate forE. coli (0.844 h⁻¹) was achieved at a temperature of 37°C and an agitation speed of 250 rpm. The activation energy for the growth of theE. coli strain W3110IQ in the stirred tank bioreactor was estimated to be 11 kcal/mol. The highest protein yield was achieved at a temperature of 44°C and an agitation speed of 250 rpm. The relative protein concentration at 44°C is 30 and 6% higher compared to that at 30 and 37°C, respectively.     

乳糖诱导植物甜蛋白des-pGlu1-Brazzein在大肠杆菌中的表达

构建了植物甜蛋白des-pGlu1-Brazzein高效表达的工程菌株,对其乳糖诱导条件进行了优化。乳糖浓度、诱导时间和诱导温度对菌株生长和目的蛋白表达的试验结果显示,高浓度乳糖对菌株生长有抑制作用(P<0.01),对目的蛋白的表达无显著影响(P>0.05),时间延长对于菌株生长有利(P<0.01),对目的蛋白的表达因温度不同而不同。进一步的研究表明,以0.5%乳糖在28℃～30℃诱导4h对菌株生长和目的蛋白的表达有利,目的蛋白的表达量可达20%左右。     

Regulation of thyroidal inducibility of Na,K-ATPase and binding of epidermal growth factor in wild-type and cold-sensitive E1a mutant type 5 adenovirus-transformed CREF cells

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of hydrophobic and hydrophilic variants of Candida albicans

We have previously demonstrated that most isolates of C. albicans are hydrophobic when grown at room temperature (RT, ca. 22-24 degrees C) and hydrophilic when grown at 37 degrees C. Variants of our standard strain LGH1095 were isolated that are hydrophobic at 37 degrees C and hydrophilic at RT. After repeated phase partitioning with cyclohexane-water cell populations that were 6-16% hydrophobic at RT and 66-80% hydrophobic at 37 degrees C were obtained. Subsequent limiting dilution experiments provided clones which were more hydrophobic at RT or hydrophilic at 37 degrees C. These were then recloned until the resultant populations were consistently under 5% cell surface hydrophobicity (CSH) at RT or over 95% at 37 degrees C. Treatment with several detergents as well as sugars did not decrease the CSH of these cells. Lipase and several proteases also had no effect. When treated with trypsin at a concentration twice that used to lower CSH of normal cells to less than 5%, the hydrophobic variant only decreased in CSH by 50%. Both variants were capable of germinating, although at different levels depending on prior growth temperature. Sensitivity to the germination inhibitor morphogenic autoregulatory substance (MARS) was similar to that of the parent strain.     

Growth and fermentation characteristics of new selected strains of Saccharomyces at high temperatures and high cell densities

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38-40 degrees C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35 degrees C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35-38 degrees C.     

Transformation of Acidiphilium by electroporation and conjugation.

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.     

Evaluation of methods for the microbiological control of natural corks for sparkling wine bottles

The various parameters proposed in Norm 0.20/95 of Catalunya (Spain) for the microbiological analysis of natural corks for sparkling wines were evaluated. The best results were obtained through the use of 1/4 Ringer's solution or saline for rinsing with an agitation time of 30 min, and an agitation speed of 150-200 rpm. Tryptone soya agar (TSA) and Sabouraud dextrose agar (SDA) were used as a culture medium for the bacteria and fungi, respectively, and a cultivation time of 48 h and incubation temperatures of 37 +/- 2 degrees C for bacteria and 28 degrees C for yeast and filamentous fungi.     

Growth studies of plasmid bearing and plasmid cured Yersinia enterocolitica GER O:3 in the presence of cefsulodin, irgasan and novobiocin at 25 and 37 degrees C

AIM: In this study, the growth characteristics of Yersinia enterocolitica biotype 4, GER O:3 plasmid bearing (P+) and plasmid cured (P-) strain types were evaluated in brain heart infusion broth supplemented with cefsulodin, irgasan, and novobiocin alone or in combination. METHODS AND RESULTS: Growth curves were obtained for the two strain types in broth supplemented with selective agents at 25 or 37 degrees C for 32 h to obtain data on the lag phase durations and growth rates of the strains. Generally, the lag times and growth rates of the P+ and P- strains were similar for cultures incubated at 25 degrees C regardless of the selective agent added and where plasmid replication and expression were not under any significant burden. However, where the lag times and growth rates of the strains were examined at 37 degrees C, significant differences were observed in the lag phase durations of the plasmid bearing strain type compared the plasmid cured strain, an effect that was due to the burden of the plasmid and the influence of selective agents. Generally, when two or more agents were present, lag phase durations were longer for the plasmid bearing strain. Some exceptions noted where in the presence of irgasan or full selective agent (CIN) the opposite case was observed. When growth rates were compared, the plasmidless strain type was typically faster than the plasmid bearing strain in the presence of most selective agents at 37 degrees C and the growth rates of both strain types at 25 degrees C were similar where the temperature appeared to negate the effects of plasmid. CONCLUSIONS: The data obtained in these studies suggest that selective agents (in particular irgasan) and incubation temperature play a significant role in influencing the growth characteristics of plasmid bearing and plasmid cured strains of Y. enterocolitica. SIGNIFICANCE AND IMPACT OF THE STUDY: This data presented in this study has significant implications for enrichment methods used in the detection or recovery of plasmid bearing Y. enterocolitica strains from food, environmental or clinical samples.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.