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Several taxonomically unrelated true fungi produce penicillins,1 and one species of Streptomyces has been reported to yield penicillin N.2 Cephalosporin C (1) has been isolated from only one species of Cephalosporiumf We report here the identification of penicillin N, and isolation and structure elucidation of three new /3-lactam antibiotics of the cephalosporin C type from two species of Streptomyces. Metabolite 2 was produced by a strain of Streptomyces lipmanii NRRL 3584. A new streptomycete species,4 Streptomyces clavuligerus NRRL 3585, afforded antibiotics 3 and 4.The antibiotics present in the broth filtrate were concentrated by carbon and anion exchange resin column chromatography. Final purification was achieved by chromatography on cellulose and silica gel to yield the purified antibiotics. The three antibiotics exhibited some common properties. All had a band at about 1770 cm-1 in the ir spectra, suggesting the presence of a /3-lactam carbonyl group.56 The uv spectra showed absorption maxima at ca. 260 nm, characteristic of the 3-cephem chromophore3•7•8 (Table I). Potentiometric titration revealed the presence of three ionizable groups, and amino acid determination on acid hydrolysates by the Spackman-Stein-Moore method9 yielded about 2 µ /ing of a-aminoadipic acid. An acetyl determination with 2 gave a value (1) A

A new species of Streptomyces which produces two new cephalosporin antibiotics is named and described. Sporophores are produced on an extensive aerial mycelium and consist of networks of short, sympodially branched hyphae. One to four spores are borne usually on short, club-shaped side branches. Sporophores eventually segment to form chains of spores. The spores are oblong to slightly cylindrical, smooth-walled, and gray to grayish green en masse. The species name proposed, Streptomyces clavuligerus, refers to the production of club-like side branches.A previously undescribed streptomycete isolated from a South American soil sample by a selective isolation procedure was found to produce two new cephalosporin antibiotics, Henrici, strain NRRL 3584, was isolated from a different soil sample from the same locality and likewise was found to produce a new cephalosporin antibiotic, 7-(5-amino-5-carboxyvaleramid 0)-7 -methoxycephalosporanic acid (6). Both organisms also produce penicillin N, an antibiotic previously reported from a streptomycete by Miller et al. (Bacteriol. Proc., p. 32, 1962). The former organism is believed to be a new species in the genus Streptomyces Waksman and Henrici, and in this report the new species is named, described, and assigned a type strain. MATERIALS AND METHODSIsolation of bacterial strain. The organism was selected from a series of soil isolations that were plated at dilutions of 1:103 to 1:105 on a modified Pridham and Gottlieb basal medium (7), supplemented with 0.5% glucose. Unsterilized nystatin (E. R. Squibb and Sons, New York, N.Y.) at 30 mg/liter was added to the melted agar before dilution plates were poured. Inoculated plates were incubated at 30 C for 10 days.Method. The methods and media recommended by the International Streptomyces Project (ISP; 10) were used primarily, along with several supplementary tests.Stock slant cultures were maintained on ISP no. 2 (yeast-malt) agar. These slants, as well as subsequent cultures used in this study, were incubated at 30C. Inocula were prepared by transferring a loopful of spores from an agar slant into a 250-ml flask containing 70 ml of tryptone-yeast extract broth (7). Flasks were incubated on a rotary shaker [2-inch (5 cm) stroke, 250 rev/min] for 72 hr. Broth cultures were macerated for 30 sec in an homogenizer, centrifuged, and then suspended in 0.85% sterile saline. Inocula for carbon-utilization tests were washed twice and suspended in saline.Microscopic observations were made on cultures that were grown from 4 to 21 days on ISP no. 4 (inorganic saltsstarch), Bennett's, and ISP no. 5 (glycerol-asparagine) media. Sporophore morphology was observed on undisturbed plate cultures, and slide mounts were stained with 0.1% Acid Fuchsin in lactic acid-glycerol (1 :

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