Biologically pure culture of Streptoverticillium stramineum sp. nov.

Abstract

This invention relates to two new antibacterial and anti-tumor agents designated LL-BO1208.alpha. and LL-BO1208.beta. produced during microbiological fermentation, under controlled conditions, using the novel microorganism Streptoverticillium stramineum and mutants thereof.

Description

BACKGROUND OF THE INVENTION

1. Field of Art

This invention relates to two new antibacterial and antitumor agents designated LL-BO1208.alpha. and LL-BO1208.beta. and to their production by fermentation of the novel microorganism Streptoverticillium stramineum or mutants thereof.

2. Background of the Invention

LL-BO1208.alpha. and LL-BO1208.beta. are novel antibiotics and anti-tumor agents of the bleomycin/phleomycin family. See, e.g., S. M. Hecht (Ed.), Bleomycin: Chemical, Biochemical and Biological Aspects, Springer-Verlag, New York, 1979. LL-BO1208.alpha. and LL-BO1208.beta. are differentiated from all other members of this family by their structure, chemical and physical properties, and effects on specific microorganisms. The most closely related known compound is Tallysomycin A [M. Koniski et al., J. Antibiotics 30: 789 (1977)] which, like LL-BO1208.beta., contains .beta.-lysine. These two compounds are the only members of the bleomycin/phleomycin family which contain this particular amino acid. However, Tallysomycin A differs significantly from LL-BO1208.beta. in that Tallysomycin A contains spermidine and an additional sugar moiety, and in addition LL-BO1208.beta. contains a reduced bithiazole ring system.

BRIEF SUMMARY OF THE INVENTION

This invention relates to the novel microorganism Streptoverticillium stramineum and mutants thereof derived spontaneously or by conventional mutagenic or recombinant techniques.

This invention also relates to two new antibacterial and anti-tumor agents designated LL-BO1208.alpha. and LL-BO1208.beta., to their production by fermentation, to methods for their recovery and concentration from crude solutions and to processes for their purification. The present invention includes within its scope, these agents in dilute forms, as crude concentrates and in pure crystalline forms. Hereinafter, the term LL-BO1208 refers to a mixture of LL-BO1208.alpha. and LL-BO1208.beta. in any proportions.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention have the following postulated structure: ##STR1##

The above postulated structures are based on Plasma Desorption Mass Spectra and Fast Atom Bombardment Mass Spectra, which give apparent calculated molecular ions of M.sup.+ =m/e 1383 for LL-BO1208.alpha. and M.sup.+ =m/e 1470 for LL-BO1208.beta.; Infrared (IR) Spectrum, Ultraviolet (UV) Spectrum, Proton Nuclear Magnetic Resonance ('H-NMR) Spectrum, C-Thirteen Carbon Nuclear Magnetic Resonance (.sup.13 C--NMR) Spectrum, and Electron Impact Mass Spectrum (ElMS) of each compound as shown in the accompanying figures:

FIG. I: IR Spectrum of LL-BO1208.beta. in KBr.

FIG. II: 'H-NMR Spectrum of LL-BO1208.beta.. 79.5 MHz in D.sub.2 O, internal TMS reference equivalent.

FIG. III: .sup.13 C-NMR Spectrum of LL-BO1208.beta.. 20 MHz in D.sub.2 O, internal TMS reference equivalent.

FIG. IV: UV Spectrum of LL-BO1208.beta.. 10 ug/ml in H.sub.2 O.

FIG. V: .sup.13 C-NMR Spectrum of LL-BO1208.alpha.. 20 MHz in D.sub.2 O, internal TMS reference equivalent.

FIG. VI: 'H-NMR Spectrum of LL-BO1208.alpha.. 79.5 MHz in D.sub.2 O, internal TMS reference equivalent.

FIG. VII: EIMS of LL-BO1208.beta. silylated methyl glycoside. Gulose derivative. 4.6 eV, ion source temperature 250.degree. C.

FIG. VIII: EIMS of LL-BO1208.beta. silylated methyl glycoside. Mannose derivative. 46 eV, ion source temperature 250.degree. C.

FIG. IX: EIMS of N-trifluoroacetyl-n-butyl ester of threonine. 46 eV, ion source temperature 250.degree. C.

FIG. X: EIMS of N-trifluoroacetyl-n-butyl ester of .beta.-alanine. 46 eV, ion source temperature 250.degree. C.

FIG. XI: EIMS of N-trifluoroacetyl-n-butyl ester of 4-amino-3-hydoxy-2-methylvaleric acid. 46 eV, ion source temperature 250.degree. C.

FIG. XII: EIMS of N-trifluoroacetyl-n-butyl ester of 2,3-diaminopropionic acid. 46 eV, ion source temperature 250.degree. C.

FIG. XIII: EIMS of N-trifluoroacetyl-n-butyl ester of .beta.-lysine. 46 eV, ion source temperature 250.degree. C.

The chemical shifts of significant peaks in the .sup.13 C-NMR spectra of LL-BO1208.alpha. and LL-BO1208.beta. acetate salts are given in Table I:

TABLE I______________________________________Chemical Shifts of Significant Peaks in the .sup.13 C--NRR Spectra.sup.a of LL-BO1208.alpha. and LL-BO1208.beta. Acetate SaltsLL-BO1208.alpha. LL-BO1208.beta.______________________________________182.3 182.5178.8 178.8178.6 178.6177.5 177.5173.3 173.5173.1 173.3172.7 173.1170.4 170.3170.2 --169.0 169.0166.6 166.6165.9 165.9164.7 164.6159.2 159.2153.7 153.7149.4 149.3138.2 138.2135.8 135.9126.7 127.1119.1 119.1113.4 113.499.4 99.498.6 98.676.8 76.775.6 (.times.2) 75.6 (.times.2)74.8 74.774.1 74.271.5 71.570.4 70.469.6 69.669.2 69.268.5 68.568.3 68.366.0 66.062.2 62.261.5 61.560.9 60.960.2 60.258.3 58.353.8 53.9-- 49.648.7 48.6 (.times.2)48.3 ---- 45.843.9 43.8-- 42.041.4 41.4-- 40.339.8 39.838.7 38.737.9 37.934.2 34.234.0 ---- 30.324.4 24.5-- 24.120.1 20.116.0 16.013.1 13.112.0 12.0______________________________________ .sup.a 50.3 MHz spectra obtained in D.sub. 2 O, chemical shifts in parts per million relative to tetramethylsilane.

The novel agents of the present invention are organic bases and thus are capable of forming acid-addition salts with a variety of organic and inorganic salt-forming reagents. Thus, acid-addition salts are formed by admixture of the antibacterial free base in a neutral solvent with an acid such as, for example, sulfuric, phosphoric, hydrochloric, hydrobromic, sulfamic, citric, maleic, fumeric, tartaric, acetic, benzoic, gluconic, ascorbic, and the like. The acid-addition salts of the agents of this invention are, in general, crystalline solids, soluble in water, methanol and ethanol but insoluble in non-polar organic solvents such as diethyl ether, benzene, toluene and the like. For the purposes of this invention, the antibacterial free bases are equivalent to their pharmaceutically acceptable non-toxic, acid-addition salts.

The new antibacterial agents LL-BO1208.alpha. and LL-1208.beta. are formed during the cultivation, under controlled conditions, of a new strain of Streptoverticillium stramineum sp. nov.

A representative strain of this new antibiotic-producing microorganism was isolated from a grassland soil sample collected in South Dakota and is maintained in the culture collection of the Lederle Laboratories Division, American Cyanamid Company, Pearl River, N.Y. 10965, as culture number LL-BO1208. A viable culture of this new microorganism has been deposited with the Culture Collection Laboratory, Northern Utilization Research and Development Division, U.S. Department of Agriculture, Peoria, Ill. 61604, and has been added to its permanent collection under the accession number NRRL 12292.

The following is a general description of the representative strain NRRL 12292 of the microorganism Streptoverticillium stramineum, based on diagnostic characteristics observed. Observations were made of the cultural, physiological and morphological features of the organism in accordance with the methods detailed by Shirling and Gottlieb, Internat. J. of Syst. Bacteriol. 16: 313-340 (1966). Media used in the study were selected from those recommended for the cultivation of actinomycetes by Pridham, et al., Antibiotics Annual pp. 947-953 (1956). The culture was also streaked on media especially employed for the study of Streptoverticillia, Locci, et al., Giorn. Microbiol. 17: 1-60 (1969). Details are recorded in Tables II-IV, wherein the underscored descriptive colors are taken from Jacobson, et al., Color Harmony Manual, 3rd Edition [Container Corp. of America, Chicago, Ill. (1948)].

Micromorphology and Chemotaxonomy

Streptoverticillium stramineum NRRL 12292 bears umbels of smooth-surfaced, ovate to rectangular (0.4 .mu.m-0.6 .mu.m.times.0.75 .mu.m-1.0 .mu.m) spores borne on verticillately-arranged straight sporophores. The cell walls of this strain contain the L-isomer of diaminopimelic acid.

Amount of Growth

Good on most media; moderate to good on Czapek's Solution Agar, Benedict's Agar, Czapek's Agar plus casamino acids, and Emerson's Agar.

Aerial Mycelium and/or en masse Spore Color

Aerial mycelium yellowish; spore masses in yellowish shades, ranging from Light Ivory 2ca to Light Wheat 2ea. Sporulation moderate to heavy depending upon medium.

Soluble Pigments

Light yellowish to yellowish to tannish when present; absent on several media.

Reverse Color

In yellowish to light brownish shades.

Miscellaneous Physiological Reactions

Nitrates not reduced to nitrites; complete liquefaction of gelatin in 14 days; melanoid pigments produced on peptone-iron agar. Carbon source utilization, according to the method of Pridham and Gottlieb, J. Bacteriol. 56: 107-114 (1948) as follows: good utilization of adonitol, d-fructose, glycerol, i-inositol, d-mannitol, d-mannose and dextrose; fair utilization of d-trehalose; and no utilization of l-arabinose, d-galactose, lactose, d-melezitose, d-melibiose, d-raffinose, l-rhamnose, salicin, sucrose and d-xylose. Tolerates 7% sodium chloride but not 10% sodium chloride.

TABLE II__________________________________________________________________________Cultural Characteristics of Streptoverticillium stramineum NRRL 12292Incubation: 14 days AMOUNT OF AERIAL MYCELIUM Temperature: 28.degree. C.MEDIUM GROWTH AND/OR SPORES SOLUBLE PIGMENT REVERSE COLOR REMARKS__________________________________________________________________________Kuster's Oatflake Good Aerial mycelium yel- Light Yellowish MaizeAgar lowish becoming Light 2 hb Ivory 2 ca in sporu- lating areas. Sporulation heavy.Yeast-Malt Agar Good Aerial mycelium yel- Yellowish Honey Gold lowish becoming Light 2 ic Ivory 2 ca in sporu- lating areas. Sporulation heavy.Bennett's Agar Good Aerial mycelium yel- Light Yellowish Maize lowish becoming Light 2 hb Ivory 2 ca in sporu- lating areas. Sporulation heavy.Tomato-Paste Good Aerial mycelium yel- Tannish Yellow MapleOatmeal Agar lowish becoming Light to Wheat 2 ea in sporu- Light Brown lating areas. 3 ng-4 ng Sporulation heavy.Hickey & Tresner's Good Aerial mycelium Light Yellowish Yellow MapleAgar pinkish-yellowish be- 3 ng coming Light Wheat 2 ea in sporulating areas. Sporulation heavy.Asparagine- Good Aerial mycelium yel- None MaizeDextrose Agar lowish becoming Light 2 hb Wheat 2 ea in sporu- lating areas. Sporulation heavy.Czapek's Solution Modarate Aerial mycelium yel- None Cream Good medium onAgar to lowish becoming Light 11/2 ca which to ob- Good Wheat 2 ea in sporu- serve verti- lating areas. cils Sporulation moderate.Pablum Agar Good Aerial mycelium yel- Tannish Light Brown lowish becoming Light 4 ng Wheat 2 ea in sporu- lating areas. Sporulation heavy.Potato Dextrose Good Aerial mycelium yel- None Light WheatAgar lowish becoming Light 2 ea Wheat 2 ea in sporu- lating areas. Sporulation heavy.Benedict's Agar Moderate Aerial mycelium whit- Light Yellowish Light Maple to ish becoming Light 3 ng Good Wheat 2 ea in sporu- lating areas. Sporulation moderate.Rice Agar Good Aerial mycelium yel- Light Yellowish Bamboo lowish becoming Light to 2 gc Wheat 2 ea in sporu- None lating areas. Sporulation heavy.Weinstein's Moderate Aerial mycelium whit- Yellowish Yellow MapleAgar ish. No sporulation. 3 ngInorganic Salts- Good Aerial mycelium yel- None Cream Good medium onStarch Agar lowish becoming Light 11/2 ca which to ob- Wheat 2 ea in sporu- serve vertic- lating areas. ils. Sporulation heavy.ADDITIONAL MEDIA FOR OBSERVING SPECIES OF STREPTOVERTICILLIABennett's Agar Good Aerial mycelium yel- Light Yellowish Maize(different lowish becoming Light 2 hbformulation) Ivory 2 ca in sporu- lating areas. Sporulation heavy.Casamino Acids Moderate Aerial mycelium whit- None PearlCzapek's Agar to ish to yellowish be- 3 ba Good coming Light Ivory 2 ca in sporulating areas. Sporulation light to moderate.Emerson's Agar Moderate Aerial mycelium yel- Light Yellowish Honey Gold to lowish becoming Light 2 ic Good Ivory 2 ca in sporu- lating areas. Sporulation moderate.Glucose-Asparagine Good Aerial mycelium yel- None Light WheatAgar (ISP #5) lowish becoming Light 2 ea Wheat 2 ea in sporu- lating areas. Sporulation heavy.Glycerol Good Aerial mycelium yel- None Light WheatAsparagine Agar lowish becoming Light 2 ea(ISP #5) Wheat 2 ea in sporu- lating areas. Sporulation heavy.Inorganic Salts- Good Aerial mycelium yel- None Light TanStarch (ISP #4) lowish becoming Light 3 gc Wheat 2 ea in sporu- lating areas. Sporulation heavy.__________________________________________________________________________ TABLE III__________________________________________________________________________Miscellaneous Physiological Reaction of Streptoverticillium stramineumNRRL 12292Temperature: 28.degree. C.MEDIUM INCUBATION PERIOD AMOUNT OF GROWTH PHYSIOLOGICAL REACTION__________________________________________________________________________Peptone-Iron 48 hours Good Moderate amount of melanin pigmentAgar produced.Organic Nitrate 7 days Good Nitrates not reduced to nitritesBroth 14 days Good Nitrates not reduced to nitritesPurple Milk 7 days Good Fair to good peptonization 14 days Good Good peptonizationGelatin 7 days Good Partial liquefaction 14 days Good Complete liquefactionYeast Extract 10 days .gtoreq.7% <10%Agar Plus(4,7,10,13%)NaCl__________________________________________________________________________ TABLE IV______________________________________Carbon Source Utilization Pattern ofStreptoverticillium stramineum NRRL 12292Incubation: 10 days Temperature: 28.degree. C.CARBON SOURCE UTILIZATION SPORULATION______________________________________Adonitol Good Good**l-Arabinose None --d-Galactose None --d-Fructose Good lightGlycerol Good very lighti-Inositol Good noneLactose None --d-Mannitol Good noned-Mannose Good good**d-Melibiose None --d-Raffinose None --d-Rhamnose None --Salicin None --Sucrose None --d-Trehalose Fair noned-Xylose None --Dextrose Good Very lightd-Melezitose None --Negative Control None --______________________________________ **carbon sources especially bringing out color of spores

It is to be understood that the term Streptoverticillium stramineum is not limited to strain Streptoverticillium stramineum NRRL 12292 or to strains fully answering the above growth and microscopic characteristics, which are given for illustrative purposes only. Streptoverticillium stramineum described herein includes all strains of Streptoverticillium stramineum which produce the antibiotics LL-BO1208.alpha. or LL-BO1208.beta. or combinations thereof and which cannot be definitely differentiated from Streptoverticillium stramineum NRRL 12292 and its subcultures, including mutants and variants thereof. The term "mutants" includes the natural (spontaneous) mutants of this organism as well as induced mutants produced from this organism by various mutagenic means known to those skilled in the art, such as exposure to x-ray radiation, ultraviolet radiation, nitrogen mustard, actinophages, nitrosamines, and the like. It is also desired and intended to include inter- and intraspecific genetic recombinants produced by genetic techniques known to those skilled in the art, such as for example conjugation, transduction, and genetic engineering techniques.

Fermentation Process

Cultivation of Streptoverticillium stramineum may be carried out in a wide variety of liquid media. Media which are useful for the production of these novel antibacterial agents include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc. are supplied as impurities of other constituents of the media. Aeration in tanks, bottles and flasks is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An anti-foaming agent such as lard oil or silicone defoamer may be added as needed.

Inoculum Preparation

Shaker flask inoculum of Streptoverticillium stramineum is prepared by inoculating 100 ml. portions of sterile liquid medium in 500 ml. flasks with scrapings or washing of spores from an agar slant of the culture. The following is an example of a suitable seed medium.

______________________________________Corn starch 24 g.Dextrose 5 g.Yeast extract 5 g.tryptone.sup.1 5 g.Beef extract 3 g.Calcium carbonate 4 g.Water to 1000 ml.______________________________________ [.sup.1 A peptone, registered trademark of Difco Laboratories, Detroit, Michigan

These flasks are incubated at 23.degree.-35.degree. C., preferably at 28.degree. C., with agitation for 22 to 26 hours, and are then used to inoculate 12 liters of the same sterile medium in a bottle which, after incubation with aeration at 28.degree. C. for 24 hours, is used to inoculate 300 liters of the same medium in a seed tank. This inoculum is incubated at 28.degree. C. for 24 hours with impeller agitation at 175 r.p.m. and sterile air flow of 150 liters per minute and then used to inoculate tank fermentors.

Tank Fermentation

The following is a suitable medium for the production of LL-BO1208.alpha. and -.beta. in fermentation tanks.

______________________________________Lard Oil 30 kg.Pharmamedia .RTM..sup.2 15 kg.Corn steep liquor 15 kg.Ammonium sulfate 4.5 kg.Zinc sulfate heptahydrate 45 kg.Calcium carbonate 6 kg.Water to 1500 liters______________________________________ [.sup.2 defatted cotton seed meal, registered trademark of Trader's Protein Div. of Trader's Oil Mill, Ft. Worth, Texas

Each tank is inoculated with 3 to 10% of inoculum prepared as described above. Aeration is supplied at 750 liters of air per minute and the mixture is agitated by an impeller driven at 100 r.p.m. The temperature is maintained at 24.degree.-35.degree. C. The fermentation is continued for 80-120 hours, at which time the mash is harvested.

Isolation of LL-BO1208

During fermentation under the controlled conditions described above, Streptoverticilloium stramineum sp. nov. produces antibiotic agent LL-BO1208 (defined as a mixture of LL-BO1208.alpha. AND LL-BO1208.beta. in any proportions). This LL-BO1208 may be isolated as follows (all procedures described below are carried out at 5.degree.-30.degree. C., preferably at 20.degree.-25.degree. C., unless otherwise specified):

A fermentation is carried out as described above and the harvest mash is filtered to remove cellular debris. The antibiotic LL-BO1208 is then collected and purified by conventional techniques well-known to those skilled in the art, such as for example adsorption chromatography, column chromatography, cation exchange chromatography, thin-layer chromatography, or selective precipitation.

The preferred process for collecting and purifying LL-BO1208 comprises adjusting the pH of the harvest mash filtrate to a pH of about 7.0-7.5 and adsorbing said filtrate batchwise on a cation-exchange material. Suitable cation-exchange materials are those having a weakly acidic function and an open structure such as for example CM-Sephadex.RTM.C-25(Na.sup.+), carboxymethyl cellulose, or Amberlite IRC-50.RTM.. CM-Sephadex.RTM.25(Na+) is preferred. The adsorbed LL-BO1208 is eluted with a high concentration of a high-ionic strength salt such as for instance about 7-14% sodium chloride (preferably about 8%) or about 7-14% potassium chloride (preferably about 8%). 8% sodium chloride is preferred. The salt is then removed by conventional means for desalting water-soluble materials, such as for example adsorption chromatography, high-pressure liquid chromatography, or mixed-bed resin chromatography. The preferred method of desalting is adsorption chromatography. The antibiotic salt is adsorbed on a suitable adsorbant column such as for example Amberlite.RTM.XAD-2, granular carbon, Amberlite.RTM.XAD-4, or Amberlite.RTM.XAD-7. The preferred adsorbant is Amberlite.RTM.XAD-2. The interstitial salt is washed out with water and the LL-BO1208 is then eluted with a 10:90 to 30:70 (v/v) solution of demineralized water:neutral polar water-miscible solvent, such as for instance ethanol, methanol, acetone, etc. Preferably, a 30:70 water:methanol (v/v) solution is used.

The resulting salt-free antibiotic LL-BO1208 eluate may be desolventized in vacuo and freeze-dried for storage.

If desired, the LL-BO1208 may be separated into its constituents LL-BO1208.alpha. and LL-BO1208.beta. by conventional means well-known to those skilled in the art, such as for example column chromatography, adsorption chromatography, cation-exchange chromatography, thin-layer chromatography, or selective precipitation. The preferred methods of separating LL-BO1208.alpha. and LL-BO1208.beta. from the mixture LL-BO1208 obtained as above are as follows:

Isolation of LL-BO1208.alpha. and LL-BO1208.beta.

Salt-free eluent of LL-BO1208 as described above, or a freeze-dried portion of said eluent dissolved in water, is adsorbed on a cation-exchange material. Examples of suitable cation-exchange materials are those having a weakly acidic function and an open structure, such as for example CM-Sephadex.RTM.25(Na.sup.+) (preferred), carboxymethyl cellulose, Amberlite IRC-50(Na.sup.+).RTM., or Amberlite IRC-72.RTM.. The antibiotic LL-BO1208 is then separated into its components LL-BO1208.alpha. and LL-BO1208.beta. by chromatographic development with an appropriate high-ionic-strength linear salt gradient. Examples of appropriate gradients are, for example, 0.1M to 2M ammonium acetate (0.1M to 1M preferred), 0.1M to 1M ammonium formate, or 1% to 7% sodium chloride. Of these, 0.1M to 1M ammonium acetate is most preferred. The eluate fractions are monitored for the presence of antibiotic by conventional techniques well-known to those skilled in the art, such as for example by determining light absorption at 290 nm or by assaying for biological activity. The LL-BO1208.alpha. is eluted first at the lower salt concentration; followed by the LL-BO1208.beta. at the higher salt concentration. The active fractions for each antibiotic are pooled.

In the most preferred process, the components LL-BO1208.alpha. and LL-BO1208.beta. are isolated by chromatographic development in separate gradients. In this process, LL-BO1208.alpha. is separated from LL-BO1208.beta. by development on CM-Sephadex.RTM.25(Na.sup.+) with a 0.1M-0.4M ammonium acetate gradient and only LL-BO1208.alpha. is collected. The LL-BO1208.beta. is isolated by separation from LL-BO1208 on CM-Sephadex.RTM.25(Na.sup.+) with a 0.1-1.0M ammonium acetate gradient, and only LL-BO1208.beta. is collected.

Purification of LL-BO1208.alpha. and LL-BO1208.beta.

Once separated, the antibiotics LL-BO1208 and LL-BO1208 may be purified by conventional means, such as for example adsorption on and elution from Amberlite XAD-2.RTM. as described above.

The preferred process for purification of either antibiotic LL-BO1208.alpha. or LL-BO1208.beta. is as follows: the appropriate fractions from the salt gradient eluate are pooled and a sufficient amount of complexing agent is added to completely complex the antibiotic. Examples of appropriate complexing agents are those having the properties of forming insoluble sulfides and the ability to complex with the antibiotic at stable pH ranges, such as for example cupric acetate, cobalt acetate, or zinc acetate. The cupric ion is the preferred agent. The amount of complexing agent needed can be readily determined by those skilled in the art. The complexed antibiotic is desalted as described above, and the desalted antibiotic-metal complex is concentrated in vacuo and freeze-dried, giving a blue solid. This solid is dissolved in a neutral polar water-miscible solvent such as for example ethanol, methanol, or acetone. To this is added an equal volume of the same solvent saturated with a compound suitable for decomplexing the antibiotic, such as for example hydrogen sulfide. A small amount of clarifying agent, e.g. activated charcoal, diatomaceous earth, clays or silica gel, is added and the mixture is stirred for about 5 to 30 minutes, preferably for about 15 minutes, and then filtered. The filtrate is diluted with 1/4 to 2 volumes (preferably about 1/3 volume) of water and concentrated in vacuo at 25.degree.-50.degree. C. (preferably at about 35.degree. C.) to an aqueous phase, which is then filtered through an ultra-fine filter, e.g. a Nalgene.RTM. plain membrane filter with pores of 0.20 microns, Pall Ultipore.RTM. membrane filters, or Pyrex.RTM. brand funnels with ultrafine or fine fritted discs (preferred), and freeze-dried.

These antibacterial agents were compared in vitro using a variety of gram positive and gram negative bacteria by the standard agar dilution procedure. The results are reported as minimal inhibitory concentrations in mcg./ml. in Table V. The antibacterial agents were not active against strains of Pseudomonas, Enterococcus, and anaerobic bacteria at concentrations of 128 mcg./ml. or less.

TABLE V______________________________________ MINIMAL INHIBITORY CONCENTRATION (mcg./ml.) LL- LL-ORGANISM BO1208.alpha. BO1208.beta.______________________________________Salmonella sp. Arizona QHC 77-3 2 0.5Salmonella sp. Indiana 1Salmonella infantis 2Salmonella sp. Heidelberg 1Salmonella typhimurium K 1Acinetobacter sp. Stamford 7743 32 2Acinetobacter sp. K 77-1 64 32Acinetobacter sp. K 77-5 16Acinetobacter sp. UR 75-20 32Acinetobacter sp. MAYO 75-10 16Staphylococcus aureus SSC 18 4Staphylococcus aureus SSC 80 4Staphylococcus aureus SSC 22 4Staphylococcus aureus SSC 73 8Staphylococcus aureus FU 19-4 8Staphylococcus aureus FU 19-2 8 2Staphylococcus aureus SSC 79-18 8 1Klebsiella sp. Stamford 6404 8 1Klebsiella sp. SSC 78-1 8 1Enterobacter aerogenes Stamford 6315 16 2Enterobacter aerogenes K 79-16 8 4Enterobacter aerogenes K 79-17 4Enterobacter aerogenes K 79-18 4Enterobacter cloacae Schaefler 60 32Enterobacter cloacae K 79-16 8 4Enterobacter agglomeraus SM 77-1 8Serratia sp. TUL 78-15 128 8Serratia sp. QHC 77-2 64 8Serratia sp. TUL 78-17 64Serratia sp. UCLA 79-1 64Serratia sp. UCLA 79-3 64Proteus morganii Schaefler 71 16Proteus morganii K 79-25 64 8Proteus rettgeri Schaefler 72 2Proteus rettgeri K 77-5 256Proteus rettgeri N 76-1 32Proteus vulgaris SM 77-1 32Proteus vulgaris K 77-3 32Escherichia coli Stamford 7037 0.5 0.25Escherichia coli 311 2 0.25Escherichia coli Schaefler 59 0.5Escherichia coli Stamford 6341 1Escherichia coli K 79-1 2Escherichia coli K 79-4 0.5Escherichia coli SM 77-16 0.5Klebsiella pneumoniae Schaefler 58 4Klebsiella pneumoniae Schaefler 67 4Klebsiella pneumoniae Stamford 6404 4Klebsiella pneumoniae SSC 78-1 4Klebsiella pneumoniae SSC 78-10 8Klebsiella pneumoniae K 79-8 2______________________________________

The antibacterial agents LL-BO1208.alpha. and LL-BO-1208.beta. are also active in vivo against a variety of organisms. These new antibacterials are thereby potentially useful as therapeutic agents in treating bacterial infections in mammals.

The usefulness of these new antibacterial agents is demonstrated by their ability to control systemic lethal infections in mice. These new substances show high in vivo antibacterial activity in mice against Klebsiella pneumoniae AD and Escherichia coli 311 when administered subcutaneously in a single dose to groups of Charles River CD-1 female mice weighing 20.+-.2 g. each, infected intraperitoneally with a lethal dose of these bacteria in a trypticase soy broth (TSB) dilution of a 5 hour TSB culture. Table VI gives the in vivo activity of LL-BO1208.alpha. and -.beta. against these bacteria.

TABLE VI__________________________________________________________________________ ALIVE/TOTAL MICE TESTED 7 DAYS AFTER INFECTION KLEBSIELLASINGLE SUBCUTANEOUS PNEUMONIAE AD ESCHERICHIA COLI 311DOSE (mg./kg.) LL BO1208.alpha. LL BO1208.beta. LL BO1209.alpha. LL BO1208.beta.__________________________________________________________________________16 5/5 5/58 4/5 0/54 0/5 5/5 0/5 5/52 4/5 3/51 2/5 0/50.5 3/50.25 0/5__________________________________________________________________________

In addition to their antibacterial activity, these agents are effective in suppressing the growth of tumors in warm-blooded animals as substantiated by the results of the following tests.

Activity Against B16 Melanoma in Mice

Male mice (BDF.sub.1) are inoculated subcutaneously with 0.5 ml. of a 10% homogenate of B16 melanoma on day zero. The test compounds are administered intraperitoneally, at the indicated dosages, on days one through nine following tumor inoculation. Tumor size is measured on day 13 with a positive response being indicated by a 58% or greater inhibition of tumor size as compared to controls. The results of this test appear in Table VII.

TABLE VII__________________________________________________________________________Effect on Tumor Growth in Mice with Subcutaneous B16 Melanoma NO. SURVIVORS AVG. TUMOR % TUMORCOMPOUND DOSE (mg./kg.) NO. TREATED WEIGHT (g.) INHIBITION__________________________________________________________________________Control (saline) 10/10 605LL-BO1208.alpha. 16 8/10 212 65.0 8 10/10 217 64.1 4 10/10 240 60.3LL-BO1208.beta. 8 9/10 127 79.0 4 8/10 200 66.9__________________________________________________________________________

In addition to inhibiting tumor growth, these agents also increase the life span of warm-blooded animals bearing B16 melanoma. Table VIII gives the results of a test conducted in accordance with the above described procedure, with an increase in life span of 25% or more being considered significant.

TABLE VIII__________________________________________________________________________Effect on Survival Time of Mice BearingSubcutaneous B16 Melanoma MEDIAN SURVIVAL % INCREASE INCOMPOUND DOSE (mg./kg.) (DAYS) LIFE SPAN__________________________________________________________________________Control (saline) 29.0LL-BO1208.alpha. 16 38.0 31 8 41.5 43 4 46.5 60LL-BO1208.beta. 4 42.0 45 2 43.0 48 1 38.5 33__________________________________________________________________________

Activity Against Lewis Lung Carcinoma in Mice

Mice (BDF.sub.1) receive 0.5 ml. of a 10% tumor homogenate subcutaneously. The test compounds are administered intraperitoneally at the indicated doses on days one through nine following tumor inoculation. Tumor size is measured on day 13 with a positive response being indicated by a 58% or greater inhibition of tumor size as compared to controls. The results of this test appear in Table IX.

TABLE IX__________________________________________________________________________Effect on Tumor Growth in Mice with Subcutaneous Lewis Lung Carcinoma NO. SURVIVORS AVG. TUMOR % TUMORCOMPOUND DOSE (mg./kg.) NO. TREATED WEIGHT (g.) INHIBITION__________________________________________________________________________Control (Saline) 9/10 3836LL-BO1208.beta. 12 2/10 0 100 8 9/10 76 98 4 9/10 389 90 2 10/10 928 76 1 10/10 1103 71__________________________________________________________________________

In addition to inhibiting tumor growth, these agents also increase the life span of warm-blooded animals bearing Lewis lung carcinoma. Table X gives the results of a test conducted in accordance with the above described procedure, with an increase in life span of 25% or more being considered significant.

TABLE X__________________________________________________________________________Effect on Survival Time of Mice Bearing Lewis Lung Carcinoma MEDIAN SURVIVAL % INCREASE INCOMPOUND DOSE (mg./kg.) (DAYS) LIFE SPAN__________________________________________________________________________Control (saline) 26LL-BO1208.beta. 8 32 25 4 33 27 2 33 27__________________________________________________________________________

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1

Inoculum Preparation

An inoculum medium was prepared according to the following formulation:

______________________________________Corn starch 24 g.Dextrose 5 g.Yeast extract 5 g.tryptoneTM. 5 g.Beef extract 3 g.Calcium carbonate 4 g.Water to 1000 ml.______________________________________

Washed or scraped spores from an agar slant of Streptoverticillium stramineum NRRL 12292 were used to inoculate a 500 ml. flask containing 100 ml. of the above sterile medium. The flask was placed on a rotary shaker and agitated vigorously at 28.degree. C. for 24 hours. The resulting flask inoculum was transferred to a 12-liter bottle containing the same sterile medium and incubated at 28.degree. C. for 24 hours with aeration. The resulting bottle medium was used to inoculate a tank containing 300 liters of the same sterile medium which was then incubated at 28.degree. C. for 24 hours with agitation by an impeller driven at 175 r.p.m. and aeration by a sterile air flow of 150 liters per minute, producing the tank inoculum.

EXAMPLE 2

Fermentation

A fermentation medium was prepared according to the following formula:

______________________________________Lard oil 30 kg.Pharmamedia .RTM. 15 kg.Corn steep liquor 15 kg.Ammonium sulfate 4.5 kg.Zinc sulfate heptahydrate 45 g.Calcium carbonate 6 kg.Water to 1500 liters______________________________________

This fermentation medium was sterilized at 120.degree. C. for 60 minutes and then inoculated with 150 liters of the tank inoculum prepared in Example 1. The fermentation was carried out at 28.degree. C. using Hodag.RTM.FD82 [a silicone antifoam, registered trademark of Hodag Chemical Corp., Skokie, Ill.] as a defoaming agent. Aeration was supplied at 750 liters of sterile air per minute. The mash was agitated by impellers driven at 100 r.p.m. At the end of approximately 87 hours of fermentation time, the mash was harvested.

EXAMPLE 3

Preliminary Isolation of LL-BO1208.alpha. and .beta.

A fermentation was carried out as described in Example 2. The 1500 liters of harvest mash was combined with 3% (w/v) Celite.RTM.512 [a diatomaceous earth filter aid, registered trademark of Johns-Manville Sales Co., Englewood Cliff, N.J.] and filtered through a plate and frame press. The press cake was washed with 150 liters of water and the water wash and filtrate were combined. The pH was 7.1 and no adjustment to the desired 7.0-7.5 range was required. The antibiotics were adsorbed batchwise on CM-Sephadex.RTM.C-25 (Na.sup.+) [a carboxylated dextran ion exchange material, registerred trademark of Pharmacia Fine Chemicals Div. of Pharmacia, Inc., Piscataway, N.J.]. The resin was stirred for 11/2 hours, then allowed to settle for 2 hours and the supernatant was syphoned off and discarded. The resin was washed batchwise 3-5 times with water and then slurried in a volume of 8% sodium chloride solution equal to 1/2 the settled resin volume. the mixture was stirred 10 minutes, then transferred to a large (18.times.22 inch) stainless steel column. The "batch salt" effluent was collected by gravity flow from the column. When the liquid head above the resin in the column reached the top of the resin bed, fresh 8% sodium chloride solution was fed into the column. Twenty liter fractions were collected. The active fractions were determined by bioassays. These active fractions from the sodium chloride elution were percolated through a 5 liter bed volume of Amberlite.RTM.XAD-2 [a synthetic polymeric non-ionic adsorbant, registered trademark of Rohm and Haas, Philadelphia, Pa.] resin in a 10.2.times.152.4 cm. glass column. This resin was washed with demineralized water and fractions were collected in one to four liters which were pooled. The resin was then eluted with a 70% methanol:30% demineralized water mixture, and fractions were collected in four liter units which were pooled. Each of these pooled fractions was separately concentrated and freeze-dried.

EXAMPLE 4

Isolation of LL-BO1208.beta.

A 9.0 g. portion of the freeze-dried material from the pooled water elution fractions of the charged Amberlite.RTM.XAD-2 resin column of Example 3 was chromatographed on a CM-Sephadex.RTM.C-25 (Na+) column. The solid was dissolved in water and allowed to seep into the column. The charge was then washed into the column with water and then developed with a gradient between 0.1M and 1.0M ammonium acetate. A series of column fractions of 75 ml. were collected and monitored both by U.V. absorbance of a 1:20 dilution at 202 nm and by bioautography. Fractions 74-83 were combined, 200 mg. of copper acetate were added and the combined fractions were desalted using 300 ml. of Amberlite.RTM.XAD-2 resin, giving 1548 mg. of a blue solid. A 600 mg. portion of this solid was dissolved in 75 ml. of methanol. A 75 ml. portion of methanol saturated with hydrogen sulfide was added, together with a small portion of activated charcoal and the mixture was stirred for 15 minutes. This suspension was filtered. The filtrate was diluted with 50 ml. of water and concentrated in vacuo to an aqueous phase, which was then filtered through an ultra-fine filter. This filtrate was freeze-dried, giving 398 mg. of LL-BO1208.beta., having the following characteristics:

Microanalysis: C, 44.82; H, 6.50; N, 15.41; S, 3.94.

Specific Rotation [alpha].sub.D.sup.25 =+17.+-.2.

LL-BO1208.beta. has an Infrared Absorption Spectrum as shown in FIG. I; a Proton Magnetic Resonance Spectrum as shown in FIG. II; a C.sup.13 -Nuclear Magnetic Resonance Spectrum as shown in FIG. III; and an Ultraviolet Absorption Spectrum as shown in FIG. IV, all of the attached drawings.

EXAMPLE 5

Isolation of LL-BOL208.alpha.

A series of freze-dried preparations from the pooled water and pooled methanol elution fractions from charged Amberlite.RTM.XAD-2 resin columns, prepared as described in Example 3, and from fermentations conducted essentially as described in Example 2, totalling 5.552 g. were combined and dissolved in 150 ml. of water. The solution was allowed to percolate into a 4.5.times.70 cm. column of CM-Sephadex.RTM.-25(Na+). The column was then developed with a gradient between 0.1M and 0.4M ammonium acetate. Fractions of about 40 ml. were collected and monitored for the presence of LL-BO1208.alpha. by taking optical density readings at 202 nm., after a 21 fold dilution with water. Fractions 62-71 were combined and desalted by adsorption and elution from an Amberlite.RTM.XAD-2 column as described in Example 4. The eluates were freeze-dried giving 387 mg. of solid. This solid was dissolved in 100 ml. of methanol saturated with hydrogen sulfide. The solution was stirred with a small portion of activated charcoal for 20 minutes, filtered, desolventized and freeze-dried, giving 303 mg. of LL-BO1208.alpha. having the following characteristics: Microanalysis: C, 43.18; H, 5.74; N, 15.47; S, 3.39.

Specific Rotation [alpha].sub.D.sup.25 =+19.

LL-BO1208.alpha. has a C.sup.13 -Nuclear Magnetic Resonance Spectrum as shown in FIG. V and Proton Magnetic Resonance Spectrum as shown in FIG. VI, both of the attached drawings.

EXAMPLE 6

Gas Chromatographic/Mass Spectrum of LL-BO1208.alpha. and LL-BO1208.beta. Silylated Methyl Glycoside

A one mg. portion of LL-BO1208.beta. in one ml. of 1.5M methanolic hydrogen chloride was heated in a sealed glass ampoule for 4 hours at 110.degree. C. The methanoylsate was transferred to a one ml. tapered vial fitted with a Teflon.RTM. (Dupont Co., Wilmington, Del.) coated rubber seal and the acid methanol was removed in a stream of nitrogen with some warming and then freeze-dried for 16 hours. A 100.mu. portion of Tri-Sil "Z" [trimethylsilylimidazole, trimethylsilylating agent, registered trademark of Pierce Chemical Co., Rockford, Ill.] was added to the freeze-dried residue and this mixture is allowed to stand at room temperature for 30 minutes prior to injection on a gas chromatographic unit. The silylated methyl glycoside of LL-BO1208.alpha. was prepared in a similar manner. The gas chromatographic/mass spectrum analysis for each was performed as follows:

Column: 3% OV-17 [an essentially 50:50 methyl:phenyl silicone, registered trademark of Ohio Valley Chemicals, Marietta, Ohio] on 100/120 GCQ (GAS CROM Q.RTM. [a silanized activated diatomaceous earth solid support, registered trademark of Applied Science Laboratories, Inc., State College, PA.]) (6 feet.times.2 mm.).

Program: 140.degree.-280.degree. C., 6.degree. C. per minute.

Helium (carrier gas): 30 ml. per minute; hydrogen: 30 ml. per minute; air: 30 ml. per minute.

Detector temperature: 275.degree. C.

Injector temperature: 218.degree. C.

Chart speed: 1/4 inch per minute.

Instrument: Varian CH-7 Mass Spectrometer.

Samples of bleomycin sulfate, zorbamycin, mannose and glucose were treated in exactly the same manner.

By mass spectral data the silyated methyl glycoside of LL-BO1208.alpha. and LL-BO1208.beta. contain a derivative identical to the L-gulose derivative obtained from the silyated methyl glycoside of bleomycin: ##STR2##

Retention time 4.6 minutes; G.C. intensity 1.2 volts; calculated molecular weight 482; G.C./M.S. as shown in FIG. VII of the attached drawings for LL-BO1208.beta..

By the same procedure, LL-BO1208.alpha. and LL-BO1208.beta. are shown to contain a mannose derivative identical to the silylated methyl glycoside of D-mannose: ##STR3##

Retention time 5.4 minutes; G.C. intensity 3.3 volts; calculated molecular weight 482; G.C./M.S. as shown in FIG. VIII of the attached drawings for LL-BO1208.beta..

EXAMPLE 7

Gas Chromatographic/Mass Spectrum of N-trifluoroacetyl-n-butyl Esters of Amino Acids Obtained by Acid Hydrolysis of LL-BO1208.alpha. and LL-B1208.beta.

A 3 mg. portion of LL-BO1208.beta. in 3 ml. of 6N hydrochloric acid was heated for 24 hours at 110.degree. C. The acid residue was washed several times with water on a rotary evaporator to remove excess hydrochloric acid and then placed under high vacuum for 16 hours.

The process for preparing the N-trifluoroacetyl-n-butyl ester was essentially that of Pandey, et al., J.A.C.S. 99(26): 8469-8483 (Dec. 21, 1977). A 0.5 ml. portion of acidic butanol (2.4 ml. of acetyl chloride in 10 ml. of n-butanol) was added to the acid hydrolysis product and the mixture was heated for 30 minutes at 100.+-.2.degree. C. in a sealed glass ampoule. This partial derivative was placed under high vacuum for 16 hours. A one ml. portion of methylenechloride and 0.5 ml. of trifluoroacetic anhydride were added to the freeze-dried partial derivative and the mixture was heated for 5 minutes at 150.+-.2.degree. C. The derivative was cooled to 0.degree. C. under nitrogen and any residual solvent was removed. Fresh methylene chloride was added to give a final concentration of 1%. The N-trifluoroacetyl-n-butyl ester of LL-BO1208.alpha. was prepared in the same manner.

The gas chromatographic/mass spectrum analysis of LL-BO1208.alpha. and LL-BO1208.beta. was performed as follows:

Column: 3% OV-17.RTM. in GCQ (100-120 mesh) (6 feet.times.2 mm.)

Program: 80.degree.-280.degree. C., 8.degree. C. per minute.

Helium flow: 33 ml. per minute.

Detector temperature: 277.degree. C.

Injector temperature: 215.degree. C.

Chart speed: 1/4 inch per minute.

Instrument: Varian CH-7 Mass Spectrometer.

Samples of .beta.-alanine, DL-threonine, streptothricin and bleomycin sulfate were treated in exactly the same manner.

By mass spectral studies the amino acids obtained from acid hydrolysis of LL-BO1208.alpha. and -.beta. and detected as their N-trifluoroacetyl-n-butyl esters include the following:

______________________________________ ##STR4## Retention time 4.4 minutes; G.C. intensity 8.0 volts; calculated mole- cular weight 367; G.C./M.S. as shown in FIG. IX of the at- tached drawings for LL-BO1208.beta.. ##STR5## Retention time 6.3 minutes; G.C. intensity 6.2 volts; calculated mole- cular weight 241; G.C./M.S. as shown in FIG. X of the at- tached drawings for LL-BO1208.beta.. ##STR6## Retention time 7.6 minutes; G.C. intensity 3.9 volts; calculated mole- cular weight 395; G.C./M.S. as shown in FIG. XI of the at- tached drawings for LL-BO1208.beta.. ##STR7## Retention time 9.4 minutes; G.C. intensity one volts; calculated mole- cular weight 352; G.C./M.S. as shown in FIG. XII of the attached drawings for LL-BO1208.beta...beta.-amino-.beta.-(4-amino-6-car- Retention timeboxy-5-methylpyrimidin-2- 10 minutes;yl)propionic acid deriva- GC intensity 6.6 volts;tive: calaulated molecular weight 448. ##STR8##______________________________________

In addition, LL-BO1208.beta. contains a .beta.-lysine derivative:

__________________________________________________________________________ ##STR9## Retention time 15.6 minutes; G. C. intensity 8.1 volts; calculated molecular weight 394; G.C./M.S. as shown in FIG. XIII of the attached drawings.__________________________________________________________________________

Claims

1. A biologically pure culture of the microorganism Streptoverticillium stramineum sp. nov., having the identifying characteristics of NRRL 12292, which culture upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, is capable of producing recoverable quantities of:

(a) the antibiotic LL-BO1208.alpha. wherein the substantially pure form:

(i) exhibits the following significant carbon-13 nuclear magnetic resonance chemical shifts in parts per million relative to the absorption of tetramethylsilane:

______________________________________182.3 169.0 126.7 71.5 60.9 38.7178.8 166.6 119.1 70.4 60.2 37.9178.6 165.9 113.4 69.6 58.3 34.2177.5 164.7 99.4 69.2 53.8 34.0173.3 159.2 98.6 68.5 48.7 24.4173.1 153.7 76.8 68.3 48.3 20.1172.7 149.4 75.6 66.0 43.9 16.0170.4 138.2 74.8 62.2 41.4 13.1170.2 135.8 74.1 61.5 39.8 12.0______________________________________

(ii) has an optical rotation [.alpha.].sub.D.sup.25 of +19

(iii) has an elemental analysis (percent) of about: C, 43.18; H, 5.74; N, 15.47; S, 3.39

(iv) has a characteristic carbon-13 nuclear magnetic resonance spectrum substantially as shown in FIG. V

(v) yields, upon hydrolysis, amino acids which include threonine, .beta.-alanine, 4-amino-3-hydroxy-2-methyl valeric acid, 2,3-diaminopropionic acid, and .beta.-amino-.beta.-(4-amino-6-carboxy-5-methylpyrimidin-2-yl)propionic acid

(vi) yields, upon methanolysis, L-gulose and D-mannose

(vii) has a characteristic Proton Magnetic Resonance Spectrum substantially as shown in FIG. VI;

(b) the pharmaceutically acceptable salts of antibiotic LL-BO1208.alpha.;

(c) the antibiotic LL-BO1208.beta. wherein the substantially pure form:

(i) exhibits the following significant carbon-13 nuclear magnetic resonance chemical shifts in parts per million relative to the absorption of tetramethyl silane:

______________________________________182.5 159.2 74.7 60.2 37.9178.8 153.7 74.2 58.3 34.2178.6 149.3 71.5 53.9 30.3177.5 138.2 70.4 49.6 24.5173.5 135.9 69.6 48.6 24.1173.3 127.1 69.2 45.8 20.1173.1 119.1 68.5 43.8 16.0170.3 113.4 68.3 42.0 13.1169.0 99.4 66.0 41.4 12.0166.6 98.6 62.2 40.3165.9 76.7 61.5 39.8164.6 75.6 60.9 38.7______________________________________

(ii) has an optical rotation [.alpha.].sub.D.sup.25 of +17+2

(iii) has an elemental analysis (percent) of about: C, 44.82; H, 6.50; N, 15.41; S, 3.94

(iv) has a characteristic carbon-13 nuclear magnetic resonance spectrum substantially as shown in FIG. III

(v) yields, upon hydrolysis, amino acids which include threonine, .beta.-alanine, 4-amino-3-hydroxy-2-methyl valeric acid, 2,3-diaminopropionic acid, .beta.-lysine, and .beta.-amino-.beta.-(4-amino-6-carboxy-5-methylpyrimidin-2-yl)propionic acid

(vi) yields, upon methanolysis, L-gulose and D-mannose

(vii) has a characteristic Infrared Absorption Spectrum substantially as shown in FIG. I, a characteristic Untraviolet Absorption Spectrum substantially shown in FIG. IV, and a characteristic Proton Magnetic Resonance Spectrum substantially as shown in FIG. II;

(d) the pharmaceutically acceptable non-toxic salts of antibiotic LL-BO1208.beta.; or

(e) mixtures thereof.

2. A biologically pure culture of the microorganism Streptoverticillium stramineum sp. nov. according to claim 1, wherein said microorganism has spontaneously mutated such that the organism is genetically altered but still retains the ability to synthesize antibiotic LL-BO1208.alpha., antibiotic LL-BO1208.beta. or mixtures thereof.

3. A biologically pure culture of the microorganism Stroptoverticillium stramineum sp. nov. according to claim 1, wherein said microorganism has been subjected to mutagenic means such that the microorganism is genetically altered but still retains the ability to synthesize antibiotic LL-BO1208.alpha., antibiotic LL-BO1208.beta. or mixtures thereof.