As methods for selecting auxotrophic mutants of filamentous fungi, two methods were re-examined and two methods were newly divised. The screening effects of these methods were evaluated by a reconstruction test as well as by a practical test. As a result, the permanganate method and the heating method devised by the author were proved to be as useful as the sulfite method and the filtration method. Details of the procedures are described.
1. Gentisic acid oxidase was obtained as cell-free extract from the cells of Pseudomonas ovalis S-5 which had been induced by m-hydroxybenzoic acid. Its purification and investigation of its properties were carried out, and the facts that this enzyme was a new ring splitting enzyme and required ferrous ion as a sole co-factor, were demonstrated. 2. Fumarylpyruvic acid which is the product of the ring cleavage, was obtained as crystalline form, and identified. Maleylpyruvic acid which is initial intermediate was recognized spectrophotometrically, and the metabolic pathway of gentisic acid oxidation was established. 3. Protocatechuic acid oxidase which was contaminated in crude extract, is constitutive enzyme in the cells of Ps. Ovalis, and was purified. As the results from investigation of its properties, this enzyme also requires ferrous ion as a co-factor, and produces β-carboxymuconic acid. 4. In secondary induction procedure, the inductive formation of m- hydroxybenzoic and gentisic acids oxidizing enzymes by intact cells prepared from the stationary phase were the strongest. Oxygen was necessary to this enzyme formation, however, the addition of glucose as energy source was ineffective. And other factors which influence to secondary induction procedure, were determined.
1. m-Hydroxybenzoic acid hydroxylase A which catalizes m-hydroxybenzoic acid to potocatechuic acid was obtained as cell-free enzyme from the cells of Ps. dacunhae A-7-2 and R-10-2 and m-hydroxybenzoic acid hydroxylase B which catalyzes m-Hydroxybenzoic acid to gentisic acid was obtained from the cells of Ps. sp. 15-1A and strain 23-1A, 42-1A. Each enzyme requires TPN, probably TPNH, as a cofactor. 2. The constitutive enzymatic pathway via protocatechuic acid in the cells of Ps. ovalis S-5, was demonstrated, and its activity is strongest at the stationary phase during the growth of the cells. Shikimic acid is converted to protocatechuic acid by the incubation with the cells and this pathway is not inhibited by the addition of streptomycin 10-3M. 3. From the incubation mixture of Ps. ovalis S-5 with shikimic acid, 5-dehydroshikimic acid was crystallized which is produced from shikimic acid and converted to protocatechuic acid. Thus, 5-dehydroshikimic acid was recognized as a precursor to protocatechuic acid. 4. The pathway from m-hydroxybenzoic acid was examined among the one hundred and thirtythree strains isolated from the soil and sewages. It was concluded that one hundred and nine strains had protocatechuic acid path. And a new pathway from salicylic acid to gentisic acid was recognized in the cells of Ps. sp. 15-1A, 15-4A and 40-2A.
The tumor-inhibitory antibiotic, raromycin is produced by a soil strepmyces which differs from any previously described members of the genus. We therefore regard it as representing a new species for which the name S. albochromogenes is assigned. Morphological, physiological and serological characteristics of the cultures of this organism is described in detail. It is closely related to S. griseochromogenes.
The enzymatic studies and some experiments with intact cells have been made on the biosynthetic pathway of L-ornithine in Micrococcus glutamicus. The pathway thus revealed is as follows: N-Acetylglutamic acid, which is probably formed via glutamic acid, is phosphorylated and reduced to form N-acetylglutamic-γ-semialdehyde. N-Acetylornithine is produced as the result of transamination between N-acetylglutamic-γ-semialdehyde and glutamic acid, and it is then deacetylated by the transfer of its acetyl group to glutamic acid, forming ornithine. The last-mentioned reaction seemed to be catalized by a new transacetylase. The conceivable relationship between these results and the efficient production of ornithine in the mutant of M. glutamicus has been discussed.
The change of the fermentation product, the production of glutamic acid instead of ornithine, occurred by the addition to the medium of a large amount of L-arginine which is the growth factor of the amino acid producing organism, a mutant of Micrococcus glutamicus. The mechanism responsible for this change has been studied, referring to the biosynthetic pathway of ornithine of the organism. Evidence has been presented that arginine inhibits the phosphorylation of N-acetylglutamic acid, thus preventing the conversion of glutamic acid to ornithine. This inhibition is probably specific in action and according to the concept of the regulatory mechanism of biosynthesis is of a feedback-type in nature.
Leifson's findings, that two different groups exist in currently adopted genus Acetobacter, the one including peritrichously flagellated or non-motile acetate-oxidizing species and the other including polarly flagellated or non- motile species which are unable to oxidize acetate, were fully confirmed. In our present experiments, however, the latter group was considered to be assignable to Gluconobacter gen. nov. Asai, fomerly proposed by one of the authors. It is therefore demonstrated, that the generic name of Gluconobacter should be revived instead of Acetomonas gen. nov. Leif son given for the latter group. The reasons supporting this view were discussed, and the following characters are to be mentioned especially: Genus Acetomonas Leifson lacks in producing a remarkable amonnt of acetic acid from ethanol as compared with redefined Acetobacter and it is rather proper to accumulate glu- conic acid to a high extent. Accordingly, from general considerations the generic name of Acetomonas was considered to be inappropriate and Gluconobacter more reasonable. Several new findings on the biochemical characters of Gluconobacter genus were also discussed.