Two peptide antibiotics katanosins A and B were isolated from the culture broth of a strain related to the genus Cytophaga.These antibiotics are basic peptides soluble in aqueous alcohols. The molecular formulae C57H95N15Oi7for A and C58H97N15Oi7for B were indicated. The constituent amino acids of katanosin A are suggested to be Thr (1), Ser (1), Val (1), Leu (3), Arg (1) and three unusual amino acids. In katanosin B, the Val residue is replaced by He. Katanosins A and B are active against Gram-positive bacteria in vitro and in vivo.In the course of our screening program for new antibiotics from bacterial strains, a strain numbered PBJ-5356 related to the genus Cytophaga was found to produce an antibiotic principle, which proved to be a cell wall synthesis-inhibitor by inhibition of incorporation of radioisotopic diaminopimelic acid into the cell wall peptidoglycan of a Bacillus strain1}. It was isolated as a complex of components A and B, which was then separated by HPLC. These are peptide antibiotics, whose structures (Fig. 1) will be discussed in an accompanying paper2).In this paper, the taxonomy of the producing strain, the production and isolation of the antibiotics as well as the physico-chemical and biological properties are presented. TaxonomyThe producing organism designated PBJ-5356 was isolated from a soil sample collected in Katanocity, Osaka Prefecture, Japan.

In recent years, it has become to be recognized that bacteria can produce antibiotics of wide structural diversity, some of which are known also as the products of Actinomycetales1~5) Here we report the isolation of fosfomycin (phosphonomycin)6~8) from a bacterial culture, which was noticed in our screening work by causing spheroplast formation of Escherichia coli LS-1 (a supersensitive mutant to 0-lactam antibiotics) in a hypertonic medium.The producing organism, PB-5,123, was isolated from pond water in Kyoto city, and identified as Pseudomonas syringae9~10) by the following characteristics. The organism was aerobic, Gram-negative, non-sporulating rods (1.0 x 1.8 -2.0 Elm) with rounded ends. Motility was observed with polar multi-trichous flagellation. Colonies on a nutrient agar were circular, convex, smooth and shining with orange color. Glucose was metabolized oxidatively. The following tests gave positive results; catalase, gelatin liquefaction, citrate utilization, arginine dihydrolase, and formation of fluorescent pigments. But, the following were negative; oxidase, starch hydrolysis, nitrate reduction, and accumulation of poly-8-hydroxybutyrate.Growth was observed at 28°C and 37°C, but not at 5°C or 42°C.The producing organism was cultivated by jar fermentation using a medium consisting of glycerol 3.0%, glucose 0.1%, Polypeptone 0.5%, beef extract 0.5%, NaCl 0.5%, under aerobic conditions for 24 hours. The antibiotic in the culture filtrate (ca. 160 liters) was adsorbed on a Dowex-1X2 (Cl-) column and eluted with 5 yo NaCl. The active eluate was freeze-dried to give a powder (120 g). Extraction with methanol from the powder was repeated several times. The extract was concentrated and passed through a carbon column with water. The effluent was concentrated and applied to a Biogel P-2 column with water. The active eluate from the column was freeze-dried to give a glassy residue (3 g). It was chromatographed on a DEAE-Sephadex column in linear gradient manner with water and 0.4 M NH4HCO3. The active eluate was once freeze-dried and then passed through an Amberlite CG-50 (Na+) column with water. Freeze-dry of the effluent gave a crude powder (230 mg) of the antibiotic as a sodium salt. It was then purified by chromatographies on an Avicel cellulose column with 75 % propanol followed by 75% acetonitrile. Concentration and freeze-dry of the active eluate gave a colorless powder (21 mg) of the antibiotic as a sodium salt.The antibiotic obtained as above is a watersoluble acidic substance. No UV absorption was observed with an aqueous solution of the sodium salt. The IR spectrum (KBr) and the 1H NMR spectrum (D 2O) were substantially identical with those of a commercially available fosfomycin sodium salt (Meiji Seika Kaisha, Ltd.). The identity with fosfomycin was also shown by color reaction and TLC experiments. The same color tones were observed by reaction with molybdate reagent. Identical mobilities on TLC were shown as follows: Silica gel plate (Merck); CHCl3 -MeOH -28% ammoniacal water (1:3:2), Rf 0.52; ...

Newantibiotics, plusbacins At~A4and Bt~B4, were isolated from the culture broth ofa strain of Pseudomonas sp. These antibiotics were isolated as a complexby columnchromatographies on Diaion HP-20 and silica gel, and then separated by HPLC. They are amphoteric in nature. The hydrochlorides are obtained as colorless powders, soluble in methanol and alkaline water. From their physico-chemical properties, these antibiotics are presumed to be acyloctapeptides containing a lactone linkage, and their differences occur in amino acid and fatty acid residues. The antibiotics are active against Gram-positive bacteria in vitro and in vivo. 817 In the course of our screening work for newantibiotics from bacterial strains, a strain numbered PB-6250 related to the genus Pseudomonas was found to produce an antibiotic principle, which showed inhibitory activity against methicillin-resistant Staphylococcus aureus and proved to be a cell wall synthesis-inhibitor. It was isolated as a complex of peptide antibiotics, which was then separated by HPLC into eight components. They were shown to be new antibiotics and named plusbacins A1~A4 and Bj~B4. Their structures will be presented in the succeeding paper1}.In this paper, the taxonomy of the producing strain, the production and isolation of the antibiotics as well as the physico-chemical and biological properties are presented. TaxonomyThe producing organism numbered PB-6250 was isolated from a soil sample collected in Okinawa-honto, Okinawa Prefecture, Japan. The organism is an aerobic Gram-negative, non-sporulating rod (0.5~0.7 x 2.0~5.0 /mi) with rounded ends. It is weakly motile by one or several polar flagella. It exhibits good growth at 28°C and on nutrient agar it forms circular, convex, opaque, entire, glistening and wet colonies with brownish cream color. Soluble, brown pigment is diffused around colonies. Poly-/?-hydroxybutyrate is not accumulated as an intracellular carbon reserve. Other physiological characteristics are shownin Table 1.Acid formation was observed from D-glucose, D-fructose, maltose and trehalose, but not from D,L-arabinose, D-xylose, D-mannitol, lactose and sucrose. No gas formation was observed from the above carbohydrates.From comparison of these characteristics with those of bacteria registered in the Volume1 of Bergey's Manual of Systematic Bacteriology2), the organism should be ascribed to the genus Pseudomonas. According to further comparison with the registered species of the genus, the organism is most related to Pseudomonas paucimobilis, especially in the feeble motility, but differed in the inability of starch hydrolysis and mole% G+C ofDNA.

2H and 13C NMR studies on katanosin A confirmed the presence of eight usual amino acid residues which were previously deduced by amino acid analysis and suggested the presence of /3-hydroxyaspartic acid, /3-hydroxyleucine and /3-phenylserine residues. These amino acids were isolated and confirmed, including their stereochemistries, by comparison with the respective authentic specimens. Stereochemistries of the usual aminoacids weredetermined by comparing the L-leucylated amino acids with reference compoundsby HPLC.Lithium borohydride reduction and chromic acid oxidation of katanosin A and alkali-treated katanosin A elucidated a lactone linkage between the C-terminal Ser and phenylserine residues. Edman degradation on alkali-treated katanosin A clarified the total amino acid sequence. The difference in katanosins A and B was determined to be replacement of Val in A by He in B. Thus, the structures of katanosins A and B were elucidated.

The constituent amino acids of plusbacins AX~A4 were determined to be two moles of L-?ra«s-3-hydroxyproline and one mole each of D-^/zreo/Miydroxyaspartic acid, L-threo-phydroxyaspartic acid, D-a//o-threonine, D-serine, D-alanine and L-arginine. In plusbacins Bl~B4, one mole of L-/r