Production of l-Threonine by Auxotrophic Mutants of Escherichia coli

Two auxotrophic mutants of Escherichia coli have been shown to accumulate significant amounts of l-threonine in the culture medium. One mutant, 13071, is deficient in α,ε-diaminopimelic acid (DAP), and the other, 13070, is deficient in both DAP and methionine.     

Production of O-Succinyl-l-homoserine by Auxotrophic Mutants of Aerobacter aerogenes

Ten of Nineteen methionine-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 by treatment with N-methyl-N′-nitro-N-nitrosoguanidine were found to accumulate in a culture broth a large amount of O-succinyl-l-homoserine (OSH) which was an intermediate in the biosynthesis of methionine in Escherichia coli and Salmonella typhimurium. OSH was isolated from the culture broth and identified by the behavior in paper chromatography, elementary analysis, melting point, optical density and infrared spectrum. Among these mutants, A. aerogenes KY 7056 which responds to methionine, homocysteine or systathionine was used to investigate culture conditions for OSH production. The amount of OSH accumulation reached a level of 8.36 mg/ml with the medium containing 10% fructose and 1% ammonium sulfate. Addition of l-homoserine (10 mg/ml) increased the amount of OSH accumulation to a level of 15.8 mg/ml. Methionine or cystathionine suppressed the accumulation of OSH. Addition of δ-hydroxylysine to the fermentation medium almost abolished the OSH accumulation.     

Histidine Production by Auxotrophic Histidine Analog-resistant Mutants of Corynebacterinm glutamicum

Corynebacterium glutamicum mutants carrying both auxotrophy and histidine analog-resistance were derived by a mutagenic treatment, and their histidine productivity was compared with that of a triazolealanine (TRA)-resistant histidine producer, C. glutamicum KY-10260. As a result, a leucine auxotrophic TRA-resistant mutant, Rα-88 was selected out of 164 auxotrophic derivatives of KY-10260. It produced histidine at a distinctly higher concentration than the parent strain under every condition tested. The concentration reached 11 mg/ml or 5.8% (w/w) of the initial sugar. Addition of an excessive amount of leucine to the medium inhibited the histidine production together with the by-production of valine by this mutant. Thiazolealanine-resistant mutants derived from a tyrosine auxotroph, a phenylalanine auxotroph and a tryptophan auxotroph gave the same or lower production in comparison with KY-10260.     

Inhabitancy and Colonization on Healthy Rice Plants by Glomerella cingulata

An omnivorous phytopathogenic fungus, Glomerella cingulata, was isolated from rice plants in Japan. We evaluated the effect of the fungus as an inoculum source for anthracnose to other plants. Leaf sheaths of rice plants were monitored in mid‐June and mid‐July for 2 years to evaluate the quantitative inhabitancy of G. cingulata in the fungal community. The pathogenicity of G. cingulata to 20 plant leaves was elucidated. A hygromycin B‐resistant green fluorescent protein (GFP) mutant of the fungus was used to observe fungal infection and development processes on rice plants. Glomerella cingulata was detected on rice sheaths in mid‐June at a low frequency, but was not detected in mid‐July. The fungus has a broad pathogenic spectrum, whereas development of the GFP mutant in tissues of rice plants was limited. Thus, the effect of G. cingulata inhabiting rice plants as a source of inoculum for other crops is likely to be low, although the fungal potential to infect various plants was ascertained.     

Sex-Linked Auxotrophic and Putative Auxotrophic Mutants of DROSOPHILA MELANOGASTER

Thirty-two mutants with improved growth response on a yeast-sucrose compared with a defined medium have been characterized with respect to ribonucleoside supplementability. Twenty mutants respond to either pyrimidine ribonucleoside. Four mutants respond to one or both purine ribonucleosides. Eight mutants ("putative" auxotrophs) do not respond to dietary RNA supplementation. Mapping and complementation studies suggest that eleven loci are represented: one, rudimentary, probably accounts for all pyrimidine requirers; there are three purine loci and seven at which the putative auxotrophs are found.     

Production of N-Succinyl-l-diaminopimelic Acid by Auxotrophic Mutants of Aerobacter aerogenes and Serratia marcescens

α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.

The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP^?) and S. marcescens KY 8921 (DAP^?/Lys^?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml.     

Fermentative Production of l-Leucine with Auxotrophic Mutants of Corynebaeterium glutamicum

An auxotrophic mutant of Corynebaeterium glutamicum was found to accumulate a large amount of l-leucine in the culture medium. The nutritional requirement of the mutant is rather complex but it’s growth was most remarkably stimulated by l-phenylalanine. Acetate (1.5~3.0%) or pyruvate (3%) stimulated the l-leucine production. By a further mutagenic treatment, 329 mutants earring some defect in addition to phenylalanine auxotrophy were derived from the mutant No. 190. Among them, a histidine auxotrophic derivative produced twice as much l-leucine as the parent strain, i.e., the level of l-leucine production by this derivative reached 16 mg/ml in a medium containing 12% glucose, 1 % (NH₄)₂SO₄ and 2.5% CH₃COONH₄ as carbon and nitrogen sources. Some other auxotrophic markers such as isoleucine- (or threonine-), threonine-, purine(s)-, homoserine-, or methionine- auxotrophy also improved the L-leucine production by No, 190.     

Asymmetric Reduction of 2-Methyl-2-aryloxyacetic Acids by Glomerella cingulata

The mycotoxin citrinin had antifungal activity under acidic conditions. At the minimum inhibitory concentration, it completely inhibited cellular respiration and partially inhibited the incorporation of radioactive precursors into macromolecules in Saccharomyces cerevisiae. It had no effect on cell permeability. In mitochondrial preparations, it significantly inhibited succinate oxidase and NADH oxidase. Rhizopus chinensis was more sensitive than S. cerevisiae; its growth and mycelial respiration at acidic pH were completely inhibited by lower concentrations of citrinin. The pH-dependent antifungal activity of citrinin seems to be associated with its uptake by fungi. Approximately half of the citrinin taken up was found in mitochondria. The main site of the antifungal action of citrinin, therefore, appears to be the mitochondrial electron transport system.     

The Characterization of a Cerebroside Sulfate from the Mycelia of Glomerella cingulata

A sulfur-containing lipid with chromatographic properties in several systems equal to the commercial sulfolipid (extracted from bovine spinal cord, Applied Science) has been isolated in 0.6 % yield (dry weight basis) from the mycelia of Glomerella cingulata. About 35 % of exogenous Na³⁵SO₄ incubated with the mycelial medium for 72 hours was recovered in the cerebroside sulfate fraction, with only traces of ³⁵S activities in other lipid fractions.     

Mutants Showing Heterothallism from a Homothallic Strain of SACCHAROMYCES CEREVISIAE

Reverse and forward mutation, induced by photoaddition of 8-methoxypsoralen (8-MOP) and 3-carbethoxypsoralen (3-CPs) or ultraviolet light (UV), are reduced in three pso mutants of Saccharomyces cerevisiae. The pso1–1 strain exhibits a lower frequency of spontaneous reversion (antimutator) and is almost entirely unaffected by the three agents in both the haploid and diploid states. The pso2–1 strain demonstrates very reduced frequencies of 8-MOP and 3-CPs plus 365 nm radiation-induced mutations in happloid and diploid cells. UV-induced mutations are slightly reduced, whereas survival is almost normal. The pso3–1 strain is mutable by 8-MOP and 3-CPs photoaddition only in the low-dose range. After UV treatment, survival of pso3–1 is nearly normal, whereas the frequencies of induced mutants are diminished as compared to the normal PSO⁺. An analogue of adenine, 6-N-hydroxyaminopurine, is capable of inducing reversions in wild type, as well as in pso and rad6–1 mutant strains, indicating that this drug may act as a direct mutagen in yeast. The comparison of photoaddition of the bifunctional agent (8-MOP) to that of the monofunctional one (3-CPs) confirms that cross-links, as well as monoadditions, are mutagenic in S. cerevisiae. Repair, of the recombinational type, taking place in diploid cells or in haploid cells in G2 phase leads to higher survival, but appears to be error-free.     

Identification of Glomerella cingulata f. sp phaseoli recombinants by RAPD markers

We examined the capacity of strains of Glomerella cingulata f. sp phaseoli fungus (Colletotrichum lindemuthianum sexual stage) to form recombinants, using random amplified polymorphic DNA (RAPD). Crosses of all possible combinations between strains 40, 42, 20, 21, 22, 23, 24, 25, and 26 were made on Petri dishes using M3 culture medium. The 42 x 21 cross produced the largest number of perithecia and five asci; the respective ascospores were isolated. RAPD analysis was performed on the parents and descendants. The 62 polymorphic RAPD bands obtained were used to assess the genetic similarity using the method of Sorence and Dice and clustering analysis in the form of a dendrogram by the UPGMA method. The RAPD markers allowed identification of recombinants from the cross between strains 42 and 21 of G. cingulata f. sp phaseoli and 40 ascospores presented 63 and 49% genetic similarity with parents 2 (strain 42) and 1 (strain 21), respectively.     

Accumulation of Xanthosine by Auxotrophic Mutants of Bacillus subtilis

Several guanineless mutants derived from Bacillus subtilis IAM 1145 were found to accumulate xanthosine in the culture broth. Further mutation of the guanineless mutants to adenine dependence led to remarkable increase in the accumulation of xanthosine. One of the guanine-adenine doubleless mutants, strain Gu-Ad-3-35, accumulated 8.9g of xanthosine per liter. Xanthosine was isolated in a crystalline form from the culture broth by a procedure involving charcoal treatment and ion-exchange chromatography.     

Clinical and experimental keratitis caused by the Colletotrichum state of Glomerella cingulata and Acrophialophora fusispora

Two cases of mycotic keratitis caused by the Colletotrichum state of Glomerella cingulata and Acrophialophora fusispora are reported for the first time. Both the isolates produced experimental corneal lesions in rabbit eyes but A. fusispora was more pathogenic. The experimental infection was more severe, with both the fungi, in rabbits pretreated with cortisone as compared with untreated animals. In vitro A. fusispora was most sensitive to miconazole and tolciclate followed by clotrimazole, amphotericin B and lactones while clotrimazole exerted maximum inhibitory effect on Colletotrichum followed by miconazole, lactones, amphotericin B and arnebins. Arnebins and tolciclate were inactive respectively against A. fusispora and Colletotrichum. Of the 3 drugs tested in vivo, against A. fusispora keratitis in rabbit, amphotericin B showed better results than tolciclate and miconazole.     

围小丛壳Glomerella cingulata子囊孢子交配繁殖、生物学特性及致病性

炭疽叶枯病（Glomerella leaf spot）是我国苹果上新发现的一种病害。为了解围小丛壳Glomerella cingulata子囊孢子的交配方式、生物学特性和致病性,从安徽砀山、山东牟平等地采集病害样品,经分离培养和纯化获得单孢菌株。在适宜条件下单孢菌株可产生子囊和子囊孢子,经过毛细管破子囊壁后单孢分离,获得12个子囊,每个子囊有8个子囊孢子。其中10个子囊中有4个“正”孢子（+）和4个“负”孢子（-）,2个子囊中只有“负”孢子。子囊孢子单孢菌株培养72h,“正”菌株菌落白色,以营养生长为主;“负”菌株菌落灰白色,直径略小于正菌株,菌丝稀疏,边缘菌丝白色,中部有大量橙色的分生孢子堆。“正”、“负”菌株异宗配合后,可产生大量可育子囊壳;单独的“正”菌株有性生殖产生稀疏丛簇状的可育子囊壳;单个的“负”菌株只能产生分散且不育的子囊壳。“正”、“负”菌株菌落的生长速度没有差异,对温度、营养、光照和pH值的敏感性也没有差异,但“正”、“负”菌株的致病性存在差异。正菌株的有性生殖没有导致rDNA-ITS、β-tubulin基因碱基序列变异。     

Isolation of Auxotrophic Mutants of Methylophilus methylotrophus by Modified-Marker Exchange

A method for stabilizing a transposon (Tn5) has been developed which allows the isolation of stable auxotrophic mutants of Methylophilus methylotrophus ASI. Insertion of Tn5 into a cloned M. methylotrophus ASI DNA fragment encoding anthranilate synthase followed by transfer of the vector with the modified trpE gene to M. methylotrophus ASI resulted in unstable auxotrophs among the recombinants. Deletion of IS50R, which encodes transposase production from Tn5, stabilized the transposon after mobilization to M. methylotrophus ASI. When trpE genes with the modified Tn5 inserts were mobilized into M. methylotrophus ASI, stable, kanamycin-resistant tryptophan auxotrophs were obtained by double-crossover homologous recombination with the chromosome.