Induction of Mutants from the Tartrate Producing Bacterium,Gluconobacter suboxydans and their Properties

The purpose of the present investigation is to obtain the superior mutants from the tartrate producing strain, Gluconobacter suboxydans 2026Y2 previously isolated from nature. Some mutant strains obtained by treatment with N-methyl-N′-nitro-N-nitrosoguanidine were found to accumulate L(+) tartaric acid in culture broth with much higher yield than in the case of the wild strain.

The high tartrate productivity of the mutants was followed by the low accumulation of 2-ketogluconic acid. The mutants having high assimilability of 5-ketogluconate showed high tartrate productivity.

The culture conditions for tartaric acid production by a mutant, Gl. suboxydans N-3874, were investigated. As a result, the amount of tartaric acid accumulated in culture broth reached to a level of 14.6g/liter in the medium containing 5% glucose and 0.3% corn steep liquor.     

Studies on Histidine Fermentation

Mutants resistant to 1,2,4-triazolealanine (TRA) or 2-thiazolealanine (TA) were derived from Corynebacterium glutamicum ATCC-13761 by mutagenic treatment with N-methyl-N′-nitro-N-nitrosoguanidine. More than eighty percent of these mutants were found to accumulate a large amount of l-histidine in culture broth. Among these histidine producers, KY-10260 which was selected on TRA-containing agar, was used to investigate the cultural conditions for histidine production. The amount of histidine accumulation reached to a level of 6~8 mg/ml with a medium containing 15% molasses (as glucose) and 4.5% ammonium sulfate.

According to the similar procedure, some histidine producers were derived from other bacteria, Arthrobacter citreus, Brevibacterium flavum, Bacillus megaterium, Bacillus subtilis and Nocardia globerula.     

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.     

Biosynthesis of Streptomycin

A method for the accumulation of the streptomycin precursor (L) in the culture broth of Streptomyces griseus was developed and the precursor was successfully isolated from the broth.

When the microorganism was cultured under shaking in the glucose-meat extract-peptone medium (0.5% glucose, 0.2% yeast extract, 0.2% meat extract, 0.4% peptone, 0.5% sodium chloride, 0.025% magnesium sulfate, pH 7.0), the accumulation of the precursor in the broth was induced by the addition of supplementary glucose (e.g., 2 g glucose per 100 ml broth) 24 hr after inoculation followed by further cultivation for 48 hr. Increased accumulation of L component was obtained merely by increasing glucose content in the culture medium (e.g., 5% glucose-containing medium in the above-indicated one) instead of glucose supplement on the way of fermentation. For the accumulation of a large amount of L component in a culture broth, it looked to be necessary for pH value of the broth to be maintained between 6 and 7 during fermentation.

L component was isolated from the culture broth by adsorption on Amberlite IRC-50 and elution with 2% NaCl solution. The L component was separated on this column from contaminated streptomycin which requires 5% NaCl solution to be eluted. The L component in the 2% NaCl eluate was adsorbed on active carbon at neutral or slightly alkaline pH and eluted with 95% methanol at acidic pH, Partially purified L component precipitated as hydrochloride by addition of acetone to the methanol extract which had been concentrated in vacuo.     

Steroid 1-Dehyclrogenation by a Crude Enzyme Preparation from Arthrobacter simplex

Nonexacting purineless mutants were isolated from an inosine-forming adenine auxotroph of Bacillus pumilus. Some of them accumulated Bratton-Marshall reaction-positive material in their culture fluid. The product was isolated in crystalline form and identified with 5(4)-amino-4(5)-imidazolecarboxamide riboside (AICA-Riboside).

AICA-Riboside accumulated by the nonexacting purineless mutants was less than inosine accumulated by their parent adenine auxotroph.

A number of mutants that require adenine specifically were isolated from AICA-Riboside-forming purineless mutants. More than half of them accumulated a large amount of AICA-Riboside as compared with their parents, nonexacting purine auxotrophs. The rest of adenine-requiring mutants from purineless mutants lost the ability to accumulate AICA-Riboside.

The effect of hypoxanthine on the accumulation of ACIA-Riboside by these auxotrophs was also examined.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

During the course of the investigation on the production of nucleotide by fermentative processes, it was found that a large amount of ATP and ADP or GTP and GDP, in addition to a smaller amount of AMP or GMP, accumulated in the culture broth when Brevibacterium ammoniagenes ATCC 6872 was incubated in a medium containing adenine or guanine.

After treatment of the culture filtrate with charcoal, the nucleotides were isolated by ion-exchange chromatography on Dowex-1 × 2 (Cl^?-form). They were identified by paper-chromatography, ultraviolet absorption spectra and analyses of base, ribose and phosphate. The ATP preparation from the broth had the same activity with that of authentic sample in the β-aspartokinase system from Corynebacterium glutamicum.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

An adenine-requiring mutant (KY7208) of Brevibacterium ammoniagenes ATTC 6872 was found to accumulate an appreciable quantity of IMP and hypoxanthine in the culture liquid.

Crystalline IMP was isolated from culture broth of KY7208 by the use of ion-exchange columns. The preparation obtained was definitely identified as 5′-IMP, based on the results on paperchromatography, UV and IR absorption spectra, and analyses of its hydrolysates.

Growth responses of this mutant were demonstrated to adenine and adenosine, but not to 5′-AMP, 3′-AMP and 5′-AMP.

Over 5 mg of IMP per ml of broth were produced by the organism in natural medium consisting of glucose, yeast extract, urea, high concentrations of phosphate and magnesim salts, and others. The chemical changes showed that hypoxanthine first accumulated in the earlier stage of fermentation, and IMP synthesis then took place with the disappearance of hypoxanthine in the later stage of fermentation.     

Carbohydrate Metabolism-Mutants of a Bacillus Species

During the course of studies on the effects of mutation in carbohydrate metabolism on the synthesis of purine derivatives, it was found that three mutants of a Bacillus species, which lacked transketolase or d-ribulose 5-phosphate 3-epimerase, accumulated a large amount of d-ribose in the culture medium. The amount of d-ribose was about 35 mg per ml of the broth incubated for 6 days. d-Ribose in the broth was purified in crystalline form and was identified from its chemical and physical properties.     

Stabilization of Adenosine-producing Mutants Derived from Bacillus sp. No. 1043

In the case of fermentative production of adenosine by the mutants derived from a Bacillus strain, abnormal fermentations due to the instability of mutants were frequently observed, and therefore studies were performed on the stabilization of mutants.

Among the genetic characteristics of adenosine-producing mutants, the xanthine-requirement was the most important factor and the adenosine productivity was found to decrease significantly as the number of revertants on this genetic marker increased. By using the media supplemented with excess amount of guanine sources, the increase of xanthine-non-requiring revertants both during the transfers on slants and in the preservation periods was perfectly suppressed. Secondly, an attempt was made to derive mutants in which no revertants would appear. Such mutants (‘NB-strains’) were selected by using a medium on which revertants appeared in a high frequency. One strain of the above mutants was found defective in XMP aminase.     

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.     

Xylitol Production by an Enterobacter Species

A xylose-utilizing bacterial strain was isolated from soil.

The strain, No. 553, was identified as Enterobacter liquefaciens from the result of the taxonomical studies. This bacterium grew well on D-xylose as a sole carbon source and accumulated pentitol extracellularly in shaking culture.

Pentitol produced was isolated from the culture broth and identified as xylitol.

The xylitol production reached the maximum after the cessation of the cell growth with a yield of 33.3 mg per ml in a medium containing 10% D-xylose as a sole carbon source and no significant decline of the amount of xylitol was observed through the period of the cultivation.     

Isolation and Characterization of Staphylococcus aureus Mutants Which Form Altered Cell Clusters

Staphylococcus aureus FDA 209P produces two extracellular bacteriolytic enzymes, 51-kDa endo-β-N-acetylglucosaminidase (GL) and 62-kDa N-acetylmuramyl-l -alanine amidase (AM), both of which can disperse cell clusters. To characterize the physiological roles of these enzymes in vivo, mutants with altered autolysin activity were isolated, and their degree of cluster formation in broth culture was assessed. Bacteriolytic activities of GL and AM, produced and secreted from these mutants into the culture fluid and detected with activity gels, coincided well with the degree of cluster formation of the mutants. The mutants with little or no enzyme activity grew in clusters, whereas those with high activity grew as well-separated cocci, suggesting that these enzymes are involved in cell separation of S. aureus in vivo.     

Formation of Desthiobiotin from Oleic Acid by Brevibacterium sp.

Microbial formation of biotin-vitamers from oleic acid was investigated. Many strains of bacteria which were able to utilize oleic acid as a sole carbon source were isolated from soils and other natural materials. Among these bacteria, some strains formed a biotin-vitamer from oleic acid in the culture broth during the cultivation. The vitamer was purified from the culture broth of strain No. 23, and identified as desthiobiotin by chromatographical and biological methods.

From the results of investigation on the taxonomical characteristics, the bacterial strain No. 23 was assumed to be Brevibacterium sp.     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of investigation of alicyclic hydrocarbon-utilizing microorganisms, five strains of ethylcyclohexane-utilizing bacteria were isolated from soil samples.

Among those bacteria, the strain S6B1 that was identified as Alcaligenes faecalis, showed the best growth in shaking culture.

The strain S6B1 was found to produce 4-ethylcyclohexanol from ethylcyclohexane.

This substance separated from culture broth was purified and identified to be trans-4-ethylcyclohexanol by the use of NMR.     

Ricerche Sulla Cultura Sommersa di Cellule Singole di Piante Superiori

Abstract

Research on submerged culture of single cells of higher plants. — The author describes a method which allows to obtain submerged cultures of single cells of Phaseolus vulgaris and Nicotiana tabacum. The medium composition in macroelements in the culture on agar appears to effect to a great extent the ability of tissues to dissociate into single cells in the subsequent liquid culture. In this respect Heller's solution results to be more suitable than Gautheret's and Hildebrandt and Ri-ker's.

Cells are grown at 24 [ddot]C in 300 ml flasks containing 60 ml of broth on a rotary shaker at 220 rpm.

To prevent contaminations some antibacterial agents were added to cultures of Phaseolus vulgaris. Among these Penicillin and Neomycin were not tossic at 20 and 5 ppm concentrations respectively.

The presence of septa, which are observed also in largely vacuolate cells, seems to confirm the ability of single cells to divide.

The optimum 2,4-D concentration for growth decreases from 6 × 10^-8 to 6 × 10^-8 during successive liquid cultures, each of them being inoculated with on amount of the previous one. This fact, showing the adaptation of liquid cultures to decreasing concentrations of the growth hormone, is in agreement with previous observations in solid cultures by several authors.     

Effect of a Microbial Acid Protease Inhibitor (S-PI) on the Production of Fungal Acid Protease

Cladosporium sp. No. 45–2, an acid protease-producing microorganism, was cultured in medium containing a microbial acid protease inhibitor (S–PI). By the addition of S–PI, the amount of acid protease in the culture broth showed an increase of 50~80% over those of normal culture (S–PI-free). Acid protease was purified from the S–PI-added culture filtrate, and its enzymatic and physicochemical properties were compared with those of acid protease obtained from normal culture. It was determined that the acid protease obtained from S–PI-added culture was the same as that of normal culture, but that the productivity was increased by the addition of S–PI.

The increase in acid protease productivity is assumed to be due to a change in metabolism by the addition of S–PI.     

Cultivation conditions for extracellular production of penicillinase by Escherichia coli carrying pEAP31 on a semi-large scale

Summary Cultivation conditions for extracellular production of penicillinase on a semi-large scale were established by using Escherichia coli K-12 HB 101 carrying the plasmid pEAP31 with the penicillinase gene from alkalophilic Bacillus sp. no. 170. Extracellular production of the enzyme was affected by several parameters such as concentration of carbohydrates and Nacl, pH value of culture broth, culture temperature, culture volume and shaking speed of the cultivation flask. The organism produced a large amount of the enzyme in culture broth under the optimal conditions established. For example, 180 units/ml of the extracellular enzyme was produced when the organism was inoculated in 300 ml broth in a 500-ml volume cultivation flask and shaken at 30°C on a reciprocal shaker at 172 oscillations/min with 3.2-cm strokes.     

Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone

The finding that most strains of microbes produce a growth stimulating substance for microorganisms was demonstrated and confirmed with the culture broth of Escherichia coli grown on a glucose-mineral medium. Addition of culture broth of E. coli to the culture media of the others markedly reduced the lag phase in microbial growth but not growth rate in the subsequent exponential phase nor the total cell yield in the stationary phase. The growth stimulation causing reduction of the lag phase was dependent on the amount of culture broth added. Occurrence of cell growth was essential for the excretion of the growth stimulating substance by E. coli. Under identical inoculum size, even with a heavy inoculum, a further reduction of the lag phase was observed by the addition of culture broth of E. coli. The substance was only effective at the initial growth phase but inert when the substance was added to a growing culture at the exponential phase. Finally, the substance was identified as pyrroloquinoline quinone, a newly established coenzyme, through chromatographic, spectroscopic and enzymatic criteria.     

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures

Aerobacter aerogenes No. 505 isolated from soil by Uyeda produced l-valine extracellularly by an aerobic shaking culture. Under anaerobic conditions the production of this amino acid was inhibited while lactic acid as well as a small amount of alanine were produced. The changes in ORP during the incubation under both conditions were investigated. When l-valine was the main product under aerobic conditions the ORP showed a constant value (rH 8.0) from 16 to 40 hr after inoculation. But when lactic acid was the main product and alanine was produced as the only amino acid under anaerobic conditions, the ORP drifted to rH 0 (zero). The phenomenon of the conversion of fermentation was shown clearly by the ORP of the culture broth.

The endpotentiai of lactic acid fermentation by Rhizopus G-36 was rH 13 to 14 when measured in the presence of trace amounts of redox dye mixtures. Without dyes, the rH was 18 to 22 and this fungal culture was slower in reaching endpotentials than bacterial cultures. It was postulated that the amount of redox substances exhibiting electromotive activity was not sufficient in this culture.

rH value 13 to 14 was not obtained under such conditions that lactic acid was not produced; that is in a medium with higher concentration of the nitrogen source in the presence of Fe²⁺ and Zn²⁺, or in a medium containing acetate in place of glucose as the carbon source.

Mycelia of Rhizopus G-36 after 36 hr-culture produced lactic acid even in the absence of oxygen. But unexpectedly, the ORP under anaerobic secondary culture was exactly the same as that in the aerobic shaking culture (rH 13.2).

A method for homogenization of the culture without secondary oxidation was improved. The ORP of anaerobically homogenized cultures was rH 11, and was thought to be due to the activities of all redox systems in the mycelium.

The respiration system of this strain was switched from cytochrome system to flavin system at the point of change in KGN-sensitivity. The ORP of this strain may be influenced by respiration through the flavin system.