Leishmania in the Chick Embryo. II. Effects of Inoculum Size,Strain Origin,and Stage Injected and Infectivity of Embryo-Derived Parasites for Hamsters*

SYNOPSIS. Amastigotes of Leishmani donovani strains 2S, 3S, 3K, Hm, Gm, and Et were inoculated intravenously into 14-day chick embryos. The course of infection was followed by examinations of liver impression smears. With strain 33 at 33 C incubation, there was a 29-fold increase at 6 days postinfection when the inoculum contained ～4 × 10⁶ amastigotes, but only a ～6.3-fold increase when ～64 × 10⁶ parasites were injected. Infection courses of several geographic strains were compared at 30, 33, and 35 C incubation. Although the results were variable, Sudan strains 2S and 3S appeared to be separate isolations of a single strain. The Burma (Et), Kenya (3K), and Mediterranean (Hm, Gm) strains appeared to be distinct, but confirming evidence of their distinctness should be sought using serologic, epidemiologic, clinical, and biochemical criteria. Strains 2S and 3S multiplied best at 33 C or below, but the embryos usually failed to survive at 28 or 30 C. Multiplication was inhibited partially at 35 C and completely at 37 C. Inoculation of strain 3S promastigotes into chick embryos resulted in a loss of parasites in 1 hr to 2 days postinfection. Only amastigotes were seen in embryos incubated at 28 and 33 C for 4 days. Hamsters infected with parasites passaged once in chick embryos died at median postinoculation times that were closely comparable to those noted among hosts infected with amastigotes from hamster spleen.     

Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium,in response to temperature upshocks

In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 41°C, were applied to the temperature-sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24-h fed-batch process. Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 33°C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production. Although increasing the culture temperature from 33 to 37, 38, 39 or 40°C resulted in final glutamate titers superior to 80 g/l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 40°C. Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 333–337 DOI: 10.1038/sj/jim/7000251 Received 26 September 2001/ Accepted in revised form 23 February 2002     

Glutamic Acid Production in Biotin-rich Media by Temperature-sensitive Mutants of Brevibacterium lactofermentum,a Novel Fermentation Process

Temperature-sensitive mutants were derived from Brevibacterium lactofermentum strain 2256 in a search for mutants which would produce a large amount of L-glutamic acid in biotin- rich media at the nonpermissive temperature. A total of 159 mutant strains was selected which showed adequate growth at 30°C but showed little or no growth at 37°C on minimal medium. Twenty of these were found to produce glutamic acid in a biotin-rich medium after a temperature shift from 30°C to 37°C, while the wild-type strain 2256 did not produce it under the same cultural condition.

One of the typical mutant strains, Ts-88, produced approximately 2g/dl of glutamic acid from beet molasses (the yield > 55%) in the presence of 33 µg/liter of biotin when tempera- , ture was shifted from 30°C to 40°C during the cultivation. It was concluded that, by controlling only temperature during fermentation, glutamic acid production could be realized in media containing biotin-rich natural carbon sources, without any chemical control such as the addition of expensive surface-active agents or antibiotics. Characteristics and merits of the novel fermentation process are discussed.     

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.     

Mechanisms involved in the increased sensitivity ofEscherichia coli to microcin 15m at 42°C

Escherichia coli cells show a markedly increased sensitivity to the antibiotic microcin 15m when briefly treated at 42°C as compared to the effect at 37°C. Furthermore, mutants resistant to the microcin at 37°C become sensitive at 42°C at microcin concentrations that are inactive at 37°C. This effect can be overcome byl-methionine. The mechanism involved seems to be based on an apparent inactivation of the homoserine-O-transsuccinylase activity. As previously established, this enzyme suffers a reversible partial inactivation when the cells are shifted to 42°C and the action of the microcin at this temperature seems to bring this process to a virtually irreversible stage. In mixed cultures of the microcin-producing strain and oneE. coli strain sensitive to the antibiotic, a much stronger growth inhibition of the latter strain has been observed at 42°C than at 37°C.     

Isolation and Characterization of Thermotolerant Gluconobacter Strains Catalyzing Oxidative Fermentation at Higher Temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10°C to 37°C and had average optimum growth temperature between 30-33°C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37°C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37°C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264^T was unable to do such fermentation at 37°C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30°C. Even oxidative fermentation of D-fructose done at 37°C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37°C was superior to that observed at 30°C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.     

Intraspecific variation in Trypanosoma cruzi: effect of temperature on the intracellular differentiation in tissue culture.

Inhibition of T. cruzi amastigote-trypomastigote differentiation in tissue culture at 37 C is a strain-dependent event. When eight T. cruzi strains were submitted to two environmental temperatures (33 and 37 C), the following patterns of differentiation were obtained: in three strains, transformation was inhibited at 37 C but readily occurred at 33 C; in three other strains differentiation took place at both temperatures; finally, in the two remaining strains, a partial inhibition was detected at 37 C. The authors discuss the meaning of this intraspecific variation and the possible relationship with the occurrence of temperature-sensitive mutants among protozoa.     

Cell-associated hemolysis activity in the clinical strain of <Emphasis Type="Italic">Pseudomonas fluorescens MFN1032</Emphasis>

Background  

MFN1032 is a clinical Pseudomonas fluorescens strain able to grow at 37°C. MFN1032 cells induce necrosis and apoptosis in rat glial cells at this temperature. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides. Under laboratory conditions, this activity is not expressed at 37°C. This activity is tightly regulated and is subject to phase variation.     

Effect of temperature on competition amongst strains of Rhizobium trifolii for nodulation of two white clover varieties

The effect of temperature and soil type on the relative success in nodulating cultivars of white clover (Trifolium repens) by mixtures of antibiotic-resistant mutants of Rhizobium trifolii was studied. Under aseptic test-tube culture, 75 str nodulated the plants 5 days earlier than 33 spc at the lowest temperature, but the temperature ×Rhizobium strain interaction was not significant. 33 spc was more effective than 75 str at 25°C and, although no significant difference was found between the two mutant strains at the lower temperatures, die temperature ×Rhizobium strain interaction was highly significant. In soil, when inoculated with mixed inoculum, cv. S100 was more uniformly nodulated by 75 str (81%) than S184 (49%). Success in nodulation could be altered by temperature and the temperature × bacterial strain interaction was significant. In the mixed inoculum treatments, 75 str was most compatible with S100 at 12°C, whereas 33 spc was most compatible with SI84 at 25°C; the bacterial strain × variety × temperature interaction was highly significant. The results are discussed from the point of view of improving symbiotic nitrogen fixation by selecting effective strains of Rhizobium which are compatible with the particular host cultivar, which are competitive with the indigenous population and whose optimum temperatures for nodulation and competitiveness are similar to the soil temperature at the times of inoculation.     

Thermal conditioning improves quality and speed of keratinocyte sheet production for burn wound treatment

Background aimsCultured patient-specific keratinocyte sheets have been used clinically since the 1970s for the treatment of large severe burns. However, despite significant developments in recent years, successful and sustainable treatment is still a challenge. Reliable, high-quality grafts with faster availability and a flexible time window for transplantation are required to improve clinical outcomes.MethodsKeratinocytes are usually grown in vitro at 37°C. Given the large temperature differences in native skin tissue, the aim of the authors’ study was to investigate thermal conditioning of keratinocyte sheet production. Therefore, the influence of 31°C, 33°C and 37°C on cell expansion and differentiation in terms of proliferation and sheet formation efficacy was investigated. In addition, the thermal effect on the biological status and thus the quality of the graft was assessed on the basis of the release of wound healing-related biofactors in various stages of graft development.ResultsThe authors demonstrated that temperature is a decisive factor in the production of human keratinocyte sheets. By using specific temperature ranges, the authors have succeeded in optimizing the individual manufacturing steps. During the cell expansion phase, cultivation at 37°C was most effective. After 6 days of culture at 37°C, three times and six times higher numbers of viable cells were obtained compared with 33°C and 31°C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33°C. Keratinocytes showed increased differentiation potential and formed better epidermal structures, which led to faster biomechanical sheet stability at day 18. In addition, a cultivation temperature of 33°C resulted in a longer lasting and higher secretion of the investigated immunomodulatory, anti-inflammatory, angiogenic and pro-inflammatory biofactors.ConclusionsThese results show that by using specific temperature ranges, it is possible to accelerate the large-scale production of cultivated keratinocyte sheets while at the same time improving quality. Cultivated keratinocyte sheets are available as early as 18 days post-biopsy and at any time for 7 days thereafter, which increases the flexibility of the process for surgeons and patients alike. These findings will help to provide better clinical outcomes, with an increased take rate in severe burn patients.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

Aims: The potential effect of in‐premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free‐living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Methods and Results: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington‐2 increased in growth at 30, 32 and 37°C while strains Philadelphia‐1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington‐2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia‐1 in the presence of N. fowleri. Yet, strain Bloomington‐2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human‐pathogenic legionellae and point to future research required to better understand Legionella ecology.     

Growth and macromolecular synthesis phenotypes of a heat-sensitive mutant strain, rip-1, of Neurospora crassa

Summary A heat-sensitive mutant of Neurospora crassa, strain 4M(t), was isolated using ultraviolet-light mutagenesis followed by the inositol-less death enrichment technique. The heat-sensitivity is the result of a single gene mutation which maps to the distal end of the right arm of linkage group II. The mutation defines the rip-1 gene locus. Both conidial germination and mycelial extension are inhibited in the mutant at 35°C and above (the nonpermissive temperature) but prolonged incubation at that temperature is not lethal to either cell type. Analysis of the lateral mycelial growth rates of wild type and of the rip-1 mutant at a variety of temperatures between 10 and 40°C indicated that the maximal growth rate occurs at 35°C in the wild type, and at 25°C in the rip-1 strain. The rip-1 mutant grows 239-times slower at 35°C than at 25°C, whereas the wild type grows 1.4-times faster. Temperature shift-up experiments showed that even 3 h at 20°C is not sufficient to allow germination at 37°C, thereby showing that the mutant cannot accumulate enough heat-sensitive product at the permissive temperature to contribute to germination at 37°C. The reciprocal temperature shift-down experiments showed that the molecular events at 37°C may be qualitatively useful for germination after shifting to 20°C. Studies of macromolecular synthesis showed that the biochemical defect in the heat-sensitive strain appears to affect RNA synthesis before protein synthesis, although there were differences in the relative effects depending on the age of the germinating conidia and the inhibition of the two processes was never complete. Messenger RNA synthesis is normal in the mutant at 37°C. Previous work has shown that the rip-1 mutant strain has a conditional defect in the accumulation of 25S rRNA and, hence, in 60S ribosomal subunit production (Loo et al. 1981). There are also indications from those studies that the 60S ribosomal subunit may be functionally impaired at the higher temperature. Thus, the growth and macromolecular synthesis phenotypes may result as a consequence of a conditional, ribosome function defect and leads to the hypothesis that the mutation in the rip-1 strain may be in a gene for a 60S ribosomal subunit component, perhaps a ribosomal protein.     

Temperature management strategy for efficient gene expression in a thermally inducible <Emphasis Type="Italic">Escherichia coli</Emphasis>/bacteriophage system

In a two-phase operation, E. coli containing λSNNU1 (Q ⁻ S ⁻) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.     

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.     

Conjugative transfer of plasmid pTd33 in agrobacteria

Supramembrane structures that connect conjugating agrobacterial cells were visualized for the first time by transmission electron microscopy. The primary contact of cells during conjugation was shown to occur through the formation of long pili containing no VirB1 protein. Pretreatment of agrobacterial cells with acetosyringone resulted in a six-to tenfold increase in the transfer frequency of plasmid pTd33 at 19–25°C and had almost no effect at 30°C. The transfer of plasmid pTd33 fromA. tumefaciens strain GV3101 to plasmid-freeA. tumefaciens strain UBAPF-2 was 16 times decreased after the centrifugation of cells. The transfer efficiency of plasmid pTd33 fromA. tumefaciens strain LBA2525 (virB2::lacZ) to plasmid-freeA. tumefaciens strain UBAPF-2 was one order of magnitude lower than the transfer from the wild-typeA. tumefaciens strain GV3101. Treatment of donor cells with 0.01% SDS before mating decreased the transfer efficiency by a factor of 26. The role of pili in the establishment of contact between conjugating cells of agrobacteria is discussed.     

Temperature Effects on Legionella pneumophila Killing by and Multiplication in Phagocytes of Guinea Pigs

We examined the effects of temperature on the interaction between Legionella pneumophila and phagocytes of guinea pigs. The body temperatures of guinea pigs infected with a sublethal dose (1.2 × 10⁴ CFU) or a lethal dose (1.0 × 10⁵ CFU) of L. pneumophila elevated from 38.4±0.15 C to 40.2±0.42 C or 40.3 ± 0.62 C, respectively. The intracellular bacterial killing by and bacterial proliferation in the phagocytes were examined at 33, 37, 40, and 42 C, using in vitro culture systems of peritoneal macrophages or polymorphonuclear leukocytes (PMN) of guinea pigs. In all the macrophages incubated at different temperatures, significant intracellular bacterial killings were observed at 4 hr after in vitro phagocytosis. After 24 hr of incubation, there was about a 100-fold increase of CFU and the number reached a maximum after 48 hr of incubation in the macrophages incubated at 42 C as well as 37 and 40 C, suggesting that macrophages support the intracellular bacterial growth in hyperthermia. In the PMN, L. pneumophila CFU 4 hr or 12 hr after the infection were significantly lower at 42 C than those at 37 C (P<0.05), indicating that the bactericidal capacity of PMN was enhanced at 42 C compared to 37 C. However, in all the PMN incubated at different temperatures, there were about 10-fold increases of CFU 24 hr after the infection, suggesting that PMN as well as macrophages support intracellular bacterial growth in hyperthermia. The extracellular bacterial growth was examined at 33, 37, 40, and 42 C in buffered yeast extract (BYE) broth or RPMI 1640 medium containing 50% guinea pig serum as a permissive or non-permissive liquid medium for the bacterial growth, respectively. Inhibition of bacterial growth in BYE broth at 42 C, and a decrease of CFU in RPMI 1640 medium containing 50% guinea pig serum at 42 C were observed. In conclusion, hyperthermia may be beneficial by restricting extracellular bacterial survival, but it exerts no beneficial effect on the restriction of intracellular bacterial growth in phagocytes, though PMN showed enhanced initial killing at 42 C. These results suggest that fever, or hyperthermia itself, may not largely contribute as a nonspecific host defense early in the course of legionellosis.     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.     

Temperature sensitive mutant of Trichoderma reesei defective in secretion of cellulase

Summary Temperature sensitive mutants of Trichoderma reesei derived from hypersecretory strain RL-P37 were isolated and characterized. Compared to the parent strain, one mutant (LU-ts 1) grew well in the mycelial phase at both permissive (25°C) and non-permissive (37°C) temperatures. However, the secretion of overall protein and active cellulases was significantly reduced in the mutant at the higher temperature. No accumulation of active cellulases or intracellular proteins was observed in the mycelia of LU-ts 1 at 37°C. The inhibitory effects of temperature on cellulase secretion in LU-ts 1 were reversible. Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses confirmed that the secretion of the major cellulases was greatly reduced in LU-ts 1 at 37°C. Molecular characterization of the various temperature sensitive secretion mutants of T. reesei should help elucidate the crucial aspects of the secretory pathway of this cellulolytic fungus.