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.     

L-serine production by a methionine-auxotrophic mutant of methylotrophic Pseudomonas

A methionine-auxotropic mutant deficient in homocysteine transmethylation activity was induced from a methylotrophic L-serine-producing derivatives of Pseudomonas MS31. This mutant grown with limited L-methionine had more than 1.7-fold higher serine hydroxymethyltransferase (SHMT) activity than its parent strain. The elevated SHMT activity significantly contributed to the improvement of L-serine accumulation from glycine and methanol. Under the optimum conditions, this mutant accumulated up to 23.9 mg/ml of L-serine. The yield coefficient L-serine from consumed glycine was 89% (mol/mol). The maximum conversion rate of added glycine (19 mg/ml) to L-serine was 77% (mol/mol).     

甲基营养型L-丝氨酸产生菌的选育

以甲基营养型假单胞菌J-12为出发菌株,经硫酸二乙酯（DES）和亚硝基胍（NTG）诱变处理,D-丝氨酸结构类似物平板和高浓度甘氨酸平板定向筛选,获得1株三-丝氨酸高产菌N-13,其发酵液中L-丝氨酸产量较出发菌株提高97．9％。在含有20g/L甘氨酸和7mL／L甲醇的培养基中,L-丝氨酸积累可达4．81g／L。     

A New Synthesis of (E,E)-8, 10-Dodecadien-1-ol,Sex Pheromone of Codling Moth

A methanol-utilizing bacterium, which produced red to pink pigments and assimilated methanol via icl^- serine pathway, was isolated from soil and tentatively designated as Pseudomonas MS 31. This bacterium produced L-serine when glycine was added to the growth medium at the late exponential phase of growth. The cells showed high L-serine degradation activity. Chelating agents and some metal ions, which inhibited L-serine degradation, stimulated the L-serine accumulation. In the presence of 0.1 ? 1 mM EDTA, o-phenanthroline, 8-hydroxyquinoline, α,α'-dipyridyl, cobalt sulfate or nickel sulfate, this bacterium produced 0.7?2.1mg L-serine from 4mg glycine per ml culture.     

Isolation and Characterization of a Temperature-sensitive Sporulation Mutant of Bacillus subtilis

Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 of sporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.     

Production of L-serine by Sarcina albida.

Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.     

Production of L-serine by Sarcina albida.

Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.     

Pseudomonas corrugata (NRRL B-30409) Mutants Increased Phosphate Solubilization, Organic Acid Production, and Plant Growth at Lower Temperatures

A study for screening and selection of mutants of Pseudomonas corrugata (NRRL B-30409) based on their phosphate solubilization ability, production of organic acids, and subsequent effect on plant growth at lower temperatures under in vitro and in situ conditions was conducted. Of a total 115 mutants tested, two (PCM-56 and PCM-82) were selected based on their greater phosphate solubilization ability at 21°C in Pikovskaya’s broth. The two mutants were found more efficient than wild-type strain for phosphate solubilization activity across a range of temperature from psychotropic (4°C) to mesophilic (28°C) in aerated GPS medium containing insoluble rock phosphate. High-performance liquid chromatography analysis showed that phosphate solubilization potential of wild-type and mutant strains were mediated by production of organic acids in the culture medium. The two efficient mutants and the wild strain oxidized glucose to gluconic acid and sequentially to 2-ketogluconic acid. Under in vitro conditions at 10°C, the mutants exhibited increased plant growth as compared to wild type, indicating their functionality at lower temperatures. In greenhouse trials using sterilized soil amended with either soluble or rock phosphate, inoculation with mutants showed greater positive effect on all of the growth parameters and soil enzymatic activities. To the best of our knowledge, this is the first report on the development of phosphate solubilizing mutants of psychotropic wild strain of P. corrugata, native to the Indian Himalayan region.     

Xanthomonas campestris, a novel stress tolerant, phosphate-solubilizing bacterial strain from saline–alkali soils

A total of 198 bacterial strains were isolated from various niches of saline–alkali soils, out of which 85 strains were able to solubilize phosphate on plates at 30 °C. The strain RMLU-26, identified as Xanthomonas campestris, was the most efficient with its ability to solubilize P, subjected to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) for development of mutants. The P solubilizing ability of X. campestris is reported for the first time. The wild type and mutant strains of X. campestris revealed a differential response to various stress factors (high pH, temperature, and salt concentration). The mutant strain revealed maximum P solubilization (67.1%) at 30 °C and pH 8.0 while the wild type strain showed maximum solubilization (41.9%) at 35 °C and pH 7.0. Percent P₂O₅ solubilization by both strains revealed a steep decline in tricalcium phosphate solubilization with an increase in NaCl concentration from 0.5 to 10% along with a concomitant drop in pH of the medium from 8.0 to 4.5 in wild type and 4.0 in mutant strain. However, a 1.5- to 2-fold increase in ‘P’ solubilization was observed in the mutant strain when compared to the wild type strain in the presence of NaCl. The overall improved tolerance of the strains to alkalinity and salinity could be due to accumulation and/or secretion of specific solute (xanthan).     

利用原生质体紫外诱变技术选育耐高温香菇菌株

【目的】运用原生质体紫外诱变技术选育香菇耐高温新菌株。【方法】以香菇菌株18为出发菌株,紫外诱变处理其原生质体,通过47°C热激3 h后菌丝恢复生长的情况来筛选获得耐高温诱变株,测定18及其所有诱变株在木屑培养基中的恒温长速、高温长速以及恢复长速,并进行高温出菇试验。【结果】筛选得到57株耐高温诱变株,其中诱变株N6、N44和N24的综合性状较好。恒温长速、高温长速以及恢复长速与出菇性状具有相关性,恢复长速与出菇产量、单菇性状、耐高温能力呈正相关,可初步作为预测耐高温菌株综合性状的指标。【结论】利用原生质体紫外诱变技术,可初步选育出耐高温香菇新菌株。     

Global Analysis of the Genes Involved in the Thermotolerance Mechanism of Thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

Involvement of digalactosyldiacylglycerol in cellular thermotolerance in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The effects of digalactosyldiacylglycerol (DGDG) deficiency on photosynthesis at high temperatures were examined using a dgdA mutant of Synechocystis sp. PCC 6803 incapable of DGDG biosynthesis. The dgdA mutant cells showed significant growth retardation when the temperature was increased from 30 to 38°C, although wild-type cells grew normally. The degree of growth retardation was enhanced by increasing light intensity. In addition, dgdA mutant cells showed increased sensitivity to the photoinhibition of photosynthesis when illuminated at 38°C. Analysis of photosynthesis in intact cells suggested that the inhibition of repair processes and accelerated photodamage resulted in growth retardation in dgdA mutant cells at high temperatures.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Production of L-serine by the methanol utilizing bacterium,Pseudomonas 3ab

Summary A bacterium capable of growth on methanol and some organic acids as sole source of carbon and energy has been isolated and designated Pseudomonas 3ab. This facultative methylotrophic organism apparently utilizes the serine pathway of formaldehyde fixation.When methanol was used as the sole carbon source for growth, L-serine production by Pseudomonas 3ab occurred upon the addition of glycine and methanol at the end of the exponential growth phase. The maximum yield of L-serine (4.7 g/l) was obtained when 20 g/l glycine and 8 g/l methanol were added and the pH of the culture medium was changed to 8.5.Although Pseudomonas 3ab is unable to grow on L-serine or glycine, it is very active in decomposing these amino acids. The degradation of L-serine and glycine has been shown to be pH-dependent with a minimum at pH 8.5–9.0.     

Ion-beam and gamma-ray irradiations induce thermotolerant mutants in the entomopathogenic fungus Metarhizium anisopliae s.l.

Entomopathogenic fungi, such as Metarhizium anisopliae (Metch.) Sorokin, are important agents for the biological control of insect pests. However, these fungi are not compatible with high temperatures. In this study, mutagenesis using ion beams or gamma rays was used to generate five potentially thermotolerant mutants from two wild-type isolates of M. anisopliae (two using ion beams and three using gamma rays). The mutant isolates had a higher upper thermal limit for vegetative growth compared to the wild types (by 2–3°C) and enhanced tolerance to wet–heat stress of 45°C for conidial germination. At 25°C and 30°C, most mutants were as virulent to maize weevil adults as the wild type, however, one mutant produced using ion beams almost lost virulence entirely. These results indicate that ion beams and gamma rays are useful tools for improving biological characteristics, such as thermotolerance, in entomopathogenic fungi, but that mutants must be carefully evaluated for unpredictable negative side effects.     

Flavonoids in the Flowers of Gnaphalium affine D. Don

Thermonsenstivie division mutants were derived from Bacillus subtilis Marburg 168 thy trp₂ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48°C was investigated.

In the absence of uracil, the mutant cells grew normally at 37°C and stopped dividing after temperature shift to 48°C resulting in filaments of two to four times length of normal rods. The total cell number after temperature shift from 37 to 48°C, increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48°C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48°C or when uracil was introduced to the culture at 48°C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48°C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature.

No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.     

Temperature-sensitive mutants of a thermotolerant yeast,Hansenula polymorpha

Temperature-sensitive mutants (TS-1 and TS-7) of a thermotolerant yeast, Hansemula polymorpha CK-1, were isolated. The mutants were unable to grow at 50°C, the maximum growth temperature of the wild type. Mutants TS-1 and TS-7 grown at 20°C showed 33 and 50% viabilities after 6 h of incubation of 50°C, respectively. Mutant TS-1 showed little variation of the degree of fatty acid unsaturation (1.26–1.28/mol) and mutant TS-7 had an almost constant sterol/phospholipid molar ratio (0.31–0.34) at 20, 30 and 40°C, although the wild type had a decrease of the degree of fatty acid unsaturation from 1.56 at 20°C to 1.30 at 40°C and an increase of the sterol/phospholipid molar ratio from 0.26 at 20°C to 0.54 at 40°C.     

Genetic and physiological alterations occurring in a yeast population continuously propagated at increasing temperatures with cell recycling

This work investigated the effects of increasing temperature from 30°C to 47°C on the physiological and genetic characteristics of Saccharomyces cerevisiae strain 63M after continuous fermentation with cell recycling in a system of five reactors in series. Steady state was attained at 30°C, and then the temperature of the system was raised so it ranged from 35°C in the last reactor to 43°C in the first reactor or feeding reactor with a 2°C difference between reactors. After 15 days at steady state, the temperature was raised from 37°C to 45°C for 25 days at steady state, then from 39°C to 47°C for 20 days at steady state. Starter strain 63M was a hybrid strain constructed to have a MAT a/α, LYS/lys, URA/ura genotype. This hybrid yeast showed vigorous growth on plates at 40°C, weak growth at 41°C, positive assimilation of melibiose, positive fermentation of galactose, raffinose and sucrose. Of 156 isolates obtained from this system at the end of the fermentation process, only 17.3% showed the same characteristics as starter strain 63M. Alterations in mating type reaction and in utilization of raffinose, melibiose, and sucrose were identified. Only 1.9% of the isolates lost the ability to grow at 40°C. Isolates showing requirements for lysine and uracil were also obtained. In addition, cell survival was observed at 39–47°C, but no isolates showing growth above 41°C were obtained.     

Temperature-sensitive mutants of Corynebacterium ammoniagenes ATCC 6872 with a defective large subunit of the manganese-containing ribonucleotide reductase

Chemical mutagenesis of the nucleotide-producing strain Corynebacterium ammoniagenes ATCC 6872 with N-methyl-N-nitro-N-nitrosoguanidine followed by an enrichment protocol yielded 46 temperature-sensitive (ts) clones. A rapid assay for the allosterically regulated Mn-ribonucleotide reductase (RRase) was developed with nucleotide-permeable cells of C. ammoniagenes in order to screen for possible defects in DNA precursor biosynthesis at elevated temperature. Three mutants (CH 31, CH 32, and CH 33) grew well at 30° C but did not proliferate at 40° C because they did not reduce ribonucleotides to 2′-deoxyribonucleotides. They were designated nrd ^ts (nucleotide reduction defective). When the cultures were shifted from 30 to 40° C, the nrd ^ts mutants immediately ceased to incorporate radiolabeled nucleic acid precursors into the DNA fraction, while DNA chain elongation was barely affected. Thus, exhaustion of the deoxyribonucleotide pool ultimately inhibited cell division, leading to a filamentous growth morphology. In contrast to the wild-type, all three nrd ^ts mutants displayed a distinctly enhanced sensitivity of ribonucleotide reduction towards hydroxyurea (in permeabilized cells and in vitro) at 30° C. The results from assays for biochemical complementation of heat-inactivated (2 min, 37° C) mutant enzyme with either the small or the large subunit of wild-type Mn-RRase located the mutational defect on the large subunit. Received: 28 December 1995 / Revision received: 22 January 1997 / Accepted: 29 January 1997     

Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937-derived phagocytes

In the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat-shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37°C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937-derived phagocytes resulted in long-lasting DnaK induction in the wild-type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30°C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild-type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37°C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature-independent contribution of DnaK to intracellular multiplication of the pathogen B. suis.