Bacterial Synthesis of Nucleotides

The synthesis of inosinic acid from inosine and p-nitrophenylphosphate by the partially purified enzyme, nucleoside phosphotransferase, prepared from Escherichia coli (B-25) is described.

The results presented in this paper represent that the nucleotide, inosinic acid, synthesized by the nucleoside phosphotransferase of E. coli, used as an example of bacterial enzymes, is not always 5′-isomer and that most of inosinic acid synthesized are 3′(& 2′)-isomer, together with a small amount of 5′-isomer. It was pointed out that cupric ion accelerated both the synthesis of inosinic acid and the liberation of p-nitrophenol, and that the nucleoside phosphotransferase and the phosphatase may be different from each other.     

Stereospecific Metabolism of O-Ethyl O-2-Nitro-5-methylphenyl N-Isopropyl Phosphoramidothioate (S-2571) by Liver Microsomal Mixed Function Oxidase

The distribution of lipoprotein lipase activity in microorganisms was examined, Rhizopus japonicus KY 521 showed the highest activity of lipoprotein lipase in the culture fluid among microorganisms tested, Lipase was also excreted in addition to lipoprotein lipase by this organism. The effect of cultural conditions on the extracellular production of the two lipases by this organism was investigated. The addition of phospholipid such as lecithin brought about a remarkable increase in the extracellular production of both lipases. It was found that lecithin did not increase significantly the net synthesis of the enzyme, but accelerated the secretion of the enzyme formed in the mycelium into the culture medium.     

Production of Thermostable Clostridium thermohydrosulfuricum Xylose Isomerase in Bacillus brevis

The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned into Bacillus brevis. Under control of the strong cell wall protein promoter, the gene was efficiently expressed during the early stationary phase of growth, when cell densities were high. The expressed gene product was a soluble cytoplasmic protein and made up more than 20% of the total cellular protein. A simple heat treatment at 85°C for 10 min gave a virtually pure enzyme. Final isomerase yields were about 0.5 g isomerase per liter culture. The purified isomerase has an optimum temperature at 85°C, and an optimum pH around 7. The isomerase is stable at 85°C for several hours, opening possibilities for new uses.     

Cloning and Expression of the Anti tumor Glycoprotein Gene of Streptococcus pyogenes Su in Escherichia coli

The structural gene for the streptococcal acid glycoprotein (SAGP), an antitumor glycoprotein produced by Streptococcus pyogenes Su, was cloned in Escherichia coli, and the nucleotide sequence was determined. The deduced amino acid sequence of SAGP was composed of 410 amino acids, and was highly homologous with colicin E1, a bacterial antimicrobial protein. The SAGP gene was subcloned into an expression plasmid and was expressed in E. coli. The gene product was demonstrated to have almost the same molecular weight and an immuno-chemical property with those of the native SAGP.     

Production of Polysaccharide from Starch and Identification of Constituent Sugars of the Preparation

A bacterium which belongs to Achromobacter sp. was isolated and named as Achromobacter mucosum nov. sp. Starch and dextrin were essential carbon sources to produce a polysaccharide effectively by the bacterium. Maltotriose was as effective as starch for the production of the polysaccharide, and glucose, maltose, isomaltose and panose were little effective. This indicates that the bacterium requires a definite configuration of carbon source to produce the polysaccharide effectively. The dry powder of the polysaccharide was prepared from 10 liters of broth in the yield of 34.9 g. Glucose, galactose, mannose and uronic acid were confirmed as the constituent sugars of the polysaccharide and it was most probable that the uronic acid was d-glucuronic acid.     

Accumulation of Diphosphorylated Butirosin A Derivatives by Phosphatase-negative Mutants of Bacillus vitellinus

It has been reported that Bacillus vitellinus, a butirosin A-producing bacterium, has two enzymes: butirosin A-3′-phosphotransferase, catalyzing phosphorylation of butirosin A, and phosphatase, catalyzing dephosphorylation of butirosin A-3′-phosphate.

A phosphatase-negative mutant, P-15, was derived from a potent butirosin A producer, BA-44, by NTG treatment. The mutant, P-15, was found to produce a butirosin A derivative when it was grown in a medium containing a relatively high concentration of inorganic phosphate. This compound was isolated and confirmed to be butirosin A-6′-N-diphosphate by physico-chemical analysis and ¹³C-NMR spectrometry.

Furthermore, a mutant strain, AP-165, was derived from the phosphatase-negative mutant, P-15. This strain, AP-165, was isolated as a nonproducer on an agar piece of bouillon medium, but was found to accumulate a major product, 6′-deamino-6′-hydroxybutirosin A-6′-O-diphosphate, and a minor one, 6′-deamino-6′-hydroxybutirosin A-6′-O-monophosphate.

It seems likely that these phosphorylated compounds are possible intermediates of butirosin A biosynthesis in B. vitellinus.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was formed from the various carbohydrates including lactose, maltose, sucrose, d-glucose, d-fructose, d-galactose, d-mannose, d-mannitol, l-rhamnose, d-xylose, l-arabinose and glycerin. The influence of pH of the reaction mixture were tested, and inorganic phosphate was observed to be indispensable for α-ketoglutarate-fermentation. A cell of E. coli grown statically on glucose was found to reveal an ability of producing α-ketoglutarate under aerobic conditions. Optically dextro lactic acid was potent in the formation of a-ketoglutaric acid. The following reagents revealed the inhibiting effect on α-ketoglutarate-fermentation; CuSO₄, AgNO₃, iodoacetate, 2, 4-dinitrophenol, NaN₃, 3-sulfanilamido-6-methoxypyridazine and arsenite, while, kanamycin and 8-azaguanine has no inhibiting effect. When E. coli was grown in a glucose-medium, a small supply of air increased the yield of acetate against decreasing α-ketoglutarate.