Production of Nucleic Acid-Related Substances by Fermentative Processes. XXVIII. Accumulation of 5′ Inosinic Acid by a Manganese-Insensitive Mutant of Brevibacterium ammoniagenes

Abstract

A manganese-insensitive mutant, KY 13105, of Brevibacterium ammwniagenes which accumulates considerable amounts of 5' inosinic acid (IMP) in the presence of 100 to 1,000 Mg of Mn2+ per liter was obtained from an IMP-producing mutant of a manganese-sensitive strain, KY 13102. The effects of Mn2 at 0 to 30 pug/liter on IMP accumulation by KY 13105 were similar to those by KY 13102. However, the accumulation of IMP by KY 13105 was not affected by 100 to 1,000 ,Mg of MnS per liter, showing a clear difference from KY 13102. The accumulation MATERIALS AND METHODS Strain. Two IMP-accumulating microorganisms were used: KY 13102 (manganese-sensitive) derived from B. ammoniagenes ATCC 6872 and obtained by ultraviolet light irradiation (2), and KY 13105 (manganese-insensitive) derived from KY 13102 by the same procedure. Media and cultivation. Cultivation of microorganisms was carried out by the procedures reported previously (2). Fermentation medium contained the following constituents per liter: glucose, 100 g; KH2PO4, 10 g; K2HPOi, 10 g; MgSO4.7H20, 10 g; CaCl,62H20, 0.1 g; FeSO4.7H20, 10 mg; ZnSO.-7H20, 1 mg; cysteine, 20 mg; thiamine, 5 mg; calcium pantothenate, 10 mg; nicotinic acid, 5 mg; biotin, 30 Aug; urea, 6 g (sterilized separately) in deionized water and adjusted to pH 8.3 with 5 N NaOH. Zero to 1,000 jug of Mn2+ per liter and 0 to 100 mg of adenine per liter were added as needed. Complete medium used for mutation contained (w/v) 1% peptone, 1% meat extract, 0.5% yeast extract, and 0.3% NaCl (pH 7.3).

Production of Nucleic Acid-Related Substances by Fermentation Processes

“…2, the cells accumulating IMP showed marked morphological changes. In IMP accumulation, it is known that hypoxanthine is first accumulated and then is followed by IMP accumulation which is accompanied by morphological changes (10). It thus appears that, under limitation of Mn2 , strain KY 13714 first accumulates inosine and thereafter excretes IMP into the medium without degrading it to inosine.…”

“…the parent strain (Table 3). We had also previously found that cellular morphology of the parent strain is severely affected by Mn2+ (8,10). Therefore, the morphological change resulting from various Mn2+ levels were examined in inosine accumulation (Fig.…”

“…Purine nucleoside-degrading activities were measured with the same enzyme preparation used in the above experiment. When 1 ml of enzyme preparation trom the IMP-producing strain was used, 10 ,umoles of a purine nucleoside was completely de- a Organisms were cultivated at 30 C for 2 days. bAbbreviations: 5' AMP, 5'-adenosine monophosphate; 5' IMP, 5'-inosine monophosphate; 5' GMP, 5'-guanosine monophosphate; 5' XMP, xanthosine monophosphate.…”

“…MATERIALS AND METHODS Strains. B. ammoniagenes KY 13102 was used as the IMP-producing strain (8). The strain is manganesesensitive; i.e., the accumulation of IMP is strongly inhibited by 100 to 1,000 jug of manganese ion (Mn2+) per liter (9).…”

“…Inosine, hypoxanthine, and IMP were separated by paper chromatography in isobutyric acid-concentrated acetic acid-I N ammonia (10:1:5; BuAA solvent). The spots were extracted with 5 ml of hot water (100 C, 30 min) and were measured spectrophotometrically (8). Cell growth was determined by the same procedure as reported previously (8) and was expressed as dry cell weight.…”