Abstract:
Novel antibiotic 354 (U-54,703) producible in a fermentation under controlled conditions using the new microorganism Streptomyces puniceus subsp. doliceus, NRRL 11160. This antibiotic is active against Gram-negative bacteria, for example, Pseudomonas and Proteus species. Thus, antibiotic 354 can be used in various environments to eradicate or control such bacteria.

Description:
BACKGROUND OF THE INVENTION 
     The disclosed fermentation which produces antibiotic 354 also produces the known antibiotic gougerotin. This known antibiotic is described in the publication, JACS 94: 3272 (1972). Gougerotin was also known as aspiculamycin and is disclosed and claimed in U.S. Pat. No. 3,849,398. 
     BRIEF SUMMARY OF THE INVENTION 
     Antibiotic 354 is producible in a fermentation under controlled conditions using a biologically pure culture of the new microorganism Streptomyces puniceus subsp. doliceus, NRRL 11160. Concomitantly produced with antibiotic 354 is the known antibiotic gougerotin. Antibiotic 354 is readily separated from gougerotin during the recovery procedure by subjecting a preparation containing the two antiobiotics to absorption on a cellulose column followed by elution with methanol followed by water. Antibiotic 354 elutes with the methanol and gougerotin elutes with the water. 
     Antibiotic 354 is active against Gram-negative bacteria, and is especially very active against species of Pseudomonas and Proteus. For example, antibiotic 354 is active against Pseudomonas aeruginosa GN-315 (UC 6149) which is resistant to gentamycin, kanamycin and nalidixic acid. Thus, antibiotic 354 can be used to treat topical Pseudomonas infections resistant to gentamycin, kanamycin or nalidixic acid. It can also be used an an oil preservative, for example, as a bacteriostatic agent for inhibiting the growth of Proteus vulgaris which is known to cause spoilage in cutting oils. Also, it is useful in wash solutions for sanitation purposes, as in the washing of hands and the cleaning of equipment, floors, or furnishings of contaminated rooms or laboratories; it is also useful as an industrial preservative, for example, as a bacteriostatic rinse for laundered clothes and for impregnating paper and fabrics; and it is useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media. It can also be used as a feed supplement to promote the growth of animals. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Chemical and Physical Properties of Antibiotic 354: 
     Molecular Weight: 172 (field desorption mass. spec.) 
     Elemental Analysis: (C 7  H 9  ClN 2  O) 2 .H 2  SO 4  (M.W. 474) 
     Found: C, 37.08; H, 4.79; N, 12.38; Cl, 15.52; S, 7.48; O, 22.75. 
     Ultraviolet Absorption Spectrum: 
     The ultraviolet absorption maxima of antibiotic 354, as reproduced in FIG. 2 of the drawings, are: 
     In 0.01 N HCl, λ, a, (ε): 213 nm, 38.54, (6,650) and 251 nm, 9.02, (1,550). 
     Infrared Absorption Spectrum: 
     Antibiotic 354, as the sulfate salt, has a characteristic infrared absorption spectrum in a mineral oil mull as shown in FIG. 1 of the drawings. Peaks are observed at the following wave lengths expressed in reciprocal centimeters. 
     Key: S = Strong M = Medium W = Weak sh = shoulder 
     
         ______________________________________Band Frequency(Wave Numbers)     Intensity______________________________________3170               S (sh)3070               S2950               S (oil)2920               S (oil)2850               S (oil)2750               S (sh)1687               S1572               M1462               S (oil)1377               M (oil)1342               M1300               W1285               W1252               W1217               M1187               M1100               S1062               S (sh) 992               M 975               M 940               M 925               M 890               W 862               M 800               M 730               M 705               M 660               M 608               S______________________________________ 
    
     Solubilities: 
     Antibiotic 354 is soluble in water, and poorly soluble in methanol, dimethylsulfoxide and dimethylformamide. 
     Nuclear Magnetic Resonance (NMR) Spectrum: 
     The &#39;H-NMR spectrum of antibiotic 354 (as the sulfate) at 60 megacycles is shown in FIG. 3 of the drawings. The NMR spectrum was observed on a Varian XL-100 Spectrometer on a solution (ca. 0.5 ml., ca. 15% concentration) of the sample of antibiotic 354 in deuterium oxide (D 2  O). The spectrum was calibrated against external tetramethylsilane and the precision of Δγ was &gt;±1 c.p.s. Frequencies were recorded in c.p.s. downfield from tetramethylsilane. 
     Antibacterial Spectrum of Antibiotic 354: 
     Antibiotic 354 shows the following zones of inhibition in millimeters (mm) on a standard disc plate assay (12.7 mm assay discs) at a concentration of 1 mg/ml. 
     
         ______________________________________                   Zone OfMicroorganism           Inhibition______________________________________Bacillus subtilis       25 mmPseudomonas mildenbergii                   30 mm______________________________________ 
    
     On testing antibiotic 354 by a microplate broth dilution assay using nutrient broth the following spectrum was observed. 
     
         ______________________________________               Minimum InhibitoryMicroorganism       Concentration (mcg/ml)______________________________________Staphylococcus aureus UC 76               31.2Streptococcus fecalis UC 694               125Escherichia coli UC 45               15.6Klebsiella pneumoniae UC 57               31.2Klebsiella pneumoniae UC 58               7.8Salmonella schottmuelleri UC 126               7.8Proteus vulgaris UC 93               15.6Proteus mirabilis   31.2Pseudomonas aeruginosa UC 95               15.6Serratia marcescens UC 131               3.9Shigella flexneri UC 143               15.6Salmonella typhi    15.6______________________________________ 
    
     &#34;UC®&#34; is a registered trademark of The Upjohn Company Culture Collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Michigan, upon request. 
     Antibiotic 354 has been shown to be active against Pseudomonas aeruginosa strains obtained from Bronson Hospital, Kalamazoo, Michigan. These strains were relatively resistant to the well-known antibiotics kanamycin, gentamycin, nalidixic acid, and Polymyxin B. The results of this comparative test, which was performed on a standard agar disc plate assay using 6.35 mm paper discs with 0.03 ml of antibiotic (1 mg/ml) per disc, follows. 
     
         ______________________________________  Zones of Inhibition (mm)                                   Anti-P. aeruginosa    Kana-   Genta-  Nalidixic                           Polymyxin                                   bioticStrain No.    mycin   mycin   Acid   B       354______________________________________6429     11      14      13     trace   226430     11      14      13     trace   336431      0      trace   12     trace   226433     trace   11      13     trace   216434     13      22      13     trace   206435      0      13      17     trace   246436      0       9      13     9       22______________________________________ 
    
     Antibiotic 354 was also tested on a nutrient broth dilution test against the same Pseudomonas strains listed above. The test tubes were incubated at 32° C. for 18 hours. The results are as follows: 
     
         ______________________________________P. aeruginosa  Minimum InhibitoryStrain No.     Concentration (mcq/ml)______________________________________6429           256430           256431           256433           256434             12.56435             12.56436           25______________________________________ 
    
     THE MICROORGANISM 
     The microorganism used for the production of antibiotic 354 and gougerotin is Streptomyces puniceus subsp. doliceus, NRRL 11160. 
     A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL 11160. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action. 
     The microorganism of this invention was studied and characterized by Alma Dietz and Grace P. Li of The Upjohn Research Laboratories. 
     An actinomycete, isolated from the Upjohn soils screen, was found to be similar in cultural characteristics to the cultures Streptomyces griseus var. purpureus, S. californicus and S. vinaceus. In 1955, Burkholder et al. [Burkholder, P. R. and S. H. Sun, L. E. Anderson, and J. Ehrlich. 1955. The identity of viomycin-producing cultures of Streptomyces Bull. Torrey bot. Cl. 82:108-117], proposed that the viomycin-producing cultures be brought to synonymy in a new variety of S. griseus designated S. griseus var. purpureus. The viomycin-producers are distinguished from S. griseus by their distinctive red-purple reverse and pigment on many media [Buchanan, R. E., and N. E. Gibbons. 1974. Bergey&#39;s Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore] [Burkholder, P. R., supra] [Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. II. Species descriptions from first study. Int. J. Syst. Bacteriol. 18:69-189][Shirling, E. B., and D. Gottlieb. 1969. Cooperative description of type cultures of Streptomyces. IV. Species descriptions from the second, third and fourth studies. Int. J. Syst. Bacteriol. 19:391-512]. They do not differ from S. griseus [Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. III. Additional species descriptions from first and second studies. Int. J. Syst. Bacteriol 18:280-399] in their temperature, carbon utilization, and general growth requirements, or in their spore chain or spore surface pattern. 
     In 1966, Buchanan et al. [Buchanan, R. E., J. G. Holt, and E. F. Lessel, Jr. 1966. Index Bergeyana. The Williams and Wilkins Co., Baltimore] declared S. griseus var. purpureus Burkholder et al. an illegitimate name. S. vinaceus (Mayer et al.) Waks. and Henrici was also declared illegitimate. S. californicus, S. floridae and S. puniceus were considered legitimate names. In Bergey&#39;s Manual, 8th ed. [Buchanan, R. E., and N. E. Gibbons, supra], the last three named cultures are cited as type cultures. In Shirling and Gottlieb [Shirling and Gottlieb, supra at 18:69-189 and at 19:391-512], S. californicus, S. puniceus and S. vinaceus are cited as type cultures. The cultural characteristics cited in Burkholder, supra, in Shirling and Gottlieb, supra at 18:69-189, and in Bergey&#39;s Manual, 8th ed. [Buchanan, R. E., and N. E. Gibbons, supra] for S. puniceus are in agreement with the characteristics noted for the cultures compared with the new soil isolate. Of these cultures, S. puniceus Patelski (1950) is the earliest described [Burkholder, P. R., supra]. The new isolate exhibits minor differences in color of growth and antibiotic production from the cultures cited. On the basis of these differences, we propose the designation Streptomyces puniceus subsp. doliceus subsp. nov. for this new culture. 
     The methods used were those cited by Dietz [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N. Y. Acad. Sci. 60:152-154] [Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94:2022-2026], Dietz and Mathews [Dietz, A., and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21:527-533], and Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340]. S. puniceus subsp. doliceus was compared with the following viomycin-producing cultures to which it appeared most similar on Ektachrome (Table 1): S. griseus var. purpureus NRRL 2423 (UC 2414), S. griseus var. purpureus CBS (UC 2468), S. vinaceus NRRL 2285 (UC 2920), and S. californicus ATCC 3312 (UC 5270). 
     Taxonomy. Streptomyces puniceus Finlay &amp; Sobin subsp. doliceus Dietz and Li subsp. nov. 
     Color characteristics. Aerial growth cream to cream pink to lavender-pink. Melanin negative. Appearance on Ektachrome is given in Table 1. Reference color characteristics are given in Table 2. The new culture and S. californicus UC 5270 may be placed in the Gray and Violet color groups of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338]; S. griseus v. purpureus UC 2414 in the Red and Violet color groups; and S. griseus v. purpureus UC 2468 and S. vinaceus UC 2920 in the Gray color group. 
     Microscopic characteristics. Spore chains long, flexuous (RF) in the sense of Pridham et al. [Pridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79]. Spore chains may be in tufts. Spores, examined with the scanning electron microscope, are rectangular, appressed, and have a smooth surface which depresses to give a ridged effect. 
     Carbon utilization. See Tables 3 and 4. 
     Cultural and biochemical characteristics. See Table 5. 
     Temperature. All the cultures grew poorly at 18° C., well at 24° C. and very well at 28°-37° C. in 48 hours. There was no growth at 4° C., 45° C. or 55° C. Plates were removed from incubation after 14 days. Plates showing no growth were then incubated at 24° C. All plates from 4° C. showed growth in 24 hours at 24° C.; plates from 45° C. and 55° C. showed no growth with the exception of the plates containing the new culture. This culture grew out from the plates previously incubated at 45° C. 
     Antibiotic-producing properties. The reference cultures produce the antibiotic viomycin [Burkholder, P. R., supra]. UC 2414 produces the Bacillus subtilis and Klebsiella pneumoniae activities of the new culture. The new culture produces antibiotic gougerotin and antibiotic 354. 
     
                                           Table 1__________________________________________________________________________Appearance of cultures of Ektachrome*   De-ter-    S. puniceus      S. griseus              S. griseus   mi-      subsp.  var.    var.Agar    na-      doliceus              purpureus                      purpureus                              S. vinaceus                                      S. californicusMedium  tion      NRRL 11160              NRRL 2423                      UC 2468 NRRL 2285                                      ATCC 3312__________________________________________________________________________Bennett&#39;s   S  Lavender-pink              Lavender-pink                      Lavender-pink                              Lavender-pink                                      Trace lavender-pink   R  Red-tan Red-tan Red-tan Red-tan Red-tanCzapek&#39;s   S  Pale pink              Pink    Pink    Pale Pink                                      Very slight trace pinksucrose R  Pale pink              Pink    Pale pink                              Pale pink                                      Pale pinkMaltose S  Lavender-pink              Lavender-pink                      Lavender-pink                              Lavender-pink                                      Very pale pinktryptone   R  Red-tan Red-tan Red-tan Red-tan Yellow-tanPeptone-iron   S  --      Trace   Trace   Trace   --              lavender-pink                      lavender-pink                              lavender-pink   R  Yellow-tan              Yellow-tan                      Yellow-tan                              Yellow-tan                                      Yellow-tan0.1% Tyrosine   S  Pale pink              Pale pink                      Pale pink                              Pale pink                                      Trace pale pink   R  Yellow-tan              Red-tan Pale pink                              Pale pink                                      Red-tanCasein starch   S  Lavender-pink              Lavender-pink                      Lavender-pink                              Lavender-pink                                      Very slight trace pink   R  Gray-tan              Gray-tan                      Gray-tan                              Gray-tan                                      Gray-tan__________________________________________________________________________ S = Surface R = Reverse *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60:152-154. 
    
     
                                           Table 2.__________________________________________________________________________ Reference Color Characteristics from ISCC-NBS ColorName Charts Illustrated with Centroid Colors*    S. puniceas            S.griseus                    S. griseusDe-     subsp.   var.    var.ter-    doliceus purpureus                    purpureus                             S. vinaceus                                      S. californicusmi-     NRRL 11160            NRRL 2423                    VC 2468  NRRL 2285                                      ATCC 3312Agar na-   Chip     Chip    Chip     Chip     ChipMediumtion   No.      Color No.               Color                    No.                       Color No.                                Color No.                                         Color__________________________________________________________________________Bennett&#39;sS  240      Light 32 Grayish                    63 Light 63 Light 229                                          Dark      reddish  yellowish                       brownish brownish grayish      purple to               pink    gray     gray     purple   93 Yellowish      grayR  17 Very dark            21 Blackish                    19 Grayish                             20 Dark  260                                         Very dark      red      red     red      grayish  purplish                                red      redP  57 Light 45 Light                    60 Light 60 Light  60                                         Light      brown    grayish grayish  grayish  grayish               reddish brown    brown    brown               brownCzapek&#39;sS  253      Grayish            253               Grayish                    93 Yellowish                             93 Yellowish                                      223                                         Moderatesucrose    purplish purplish                       gray     gray     purple      pink     pinkR  253      Grayish            237               Strong                    93 Yellowish                             244                                Pale  223                                         Moderate      purplish reddish gray     purple      pink     purple           purpleP  -- --    -- --   -- --    -- --    -- --MaltoseS  226      Very pale            32 Grayish                    93 Yellowish                             93 Yellowish                                      228                                         Grayishtryptone   purple   yellowish                       gray     gray     purple               pinkR  21 Blackish            17 Very dark                    62 Dark  81 Dark  257                                         Very deep      red      red     grayish  grayish  purplish                       brown    yellowish                                         red                                brownP  57 Light 45 Light                    60 Light 60 Light -- --      brown    grayish grayish  grayish               reddish brown    brown               brownHickey-S  63 Light 32 Grayish                    93 Yellowish                             93 Yellowish                                      227                                         Pale purpleTresner    brownish yellowish                       gray     gray      gray     pinkR  17 Very  260               Very 259                       Dark  20 Dark  260                                         Very dark      dark red dark    purplish grayish  purplish               purplish                       red      red      red               redP  57 Light 45 Light                    60 Light 60 Light  60                                         Light      brown    grayish grayish  grayish  grayish               reddish brown    brown    brown               brownYeast extract-S  63 Light 32 Grayish                    93 Yellowish                             93 Yellowish                                      234                                         Darkmalt extract      brownish yellowish                       gray     gray     purplish(ISP-2)    gray     pink                      grayR  73 Pale  73 Pale 73 Pale  73 Pale   17                                         Very dark      orange   orange  orange   orange   red      yellow to               yellow to                       yellow to                                yellow to   21 Blackish            46 Grayish                    46 Grayish                             20 Dark  -- --      red      reddish reddish  grayish               brown   brown    redP  57 Light 45 Light                    60 Light 60 Light -- --      brown    grayish grayish  grayish               reddish brown    brown               brownOatmealS  240      Light 226               Very pale                    63 Light 93 Yellowish                                      257                                         Very deep(ISP-3)    reddish  purple  brownish gray     purplish      purple to        gray              red   93 Yellowish      grayR  242      Dark  259               Dark 259                       Dark  262                                Grayish                                       17                                         Very dark      reddish  purplish                       purplish purplish red      purple   red     red      redP  57 Light 45 Light                    60 Light 60 Light  60                                         Light      brown    grayish grayish  grayish  grayish               reddish brown    brown    brown               brownInorganic-S  63 Light 32 Grayish                    93 Yellowish                             93 Yellowish                                      227                                         Palesalts      brownish yellowish                       gray     gray     purplestarch     gray     pink(ISP-4)R  242      Dark  257               Very 257                       Very  259                                Dark   21                                         Blackish      reddish  deep    deep     purplish red      purple   purplish                       purplish red               red     redP  57 Light 45 Light                    60 Light 60 Light  60                                         Light      brown    grayish grayish  grayish  grayish               reddish brown    brown    brown               brownGlycerol-S  63 Light 32 Grayish                    93 Yellowish                             93 Yellowish                                      227                                         Pale purpleasparagine brownish yellowish                       gray     gray(ISP-5)    gray     pinkR  21 Blackish            257               Very 257                       Very  259                                Dark  21 Blackish      red      deep    deep     purplish red               purplish                       purplish red               red     redP  57 Light 45 Light                    60 Light 60 Light 60 Light      brown    grayish grayish  grayish  grayish               reddish brown    brown    brown               brown__________________________________________________________________________ S = Surface R = Reverse P = Pigment *Kelly, K. L., and D. B. Judd. 1955. The ISCC-NBS method of designating colors and a dictionary of color names. U.S. Dept. of Comm. Circ. 553, Washington, D.C. 
    
     
                                           Table 3__________________________________________________________________________ Growth on Carbon Compounds in the SyntheticMedium of Pridham and Gottlieb*     S. puniceus            S. griseus                   S. griseus     subsp. var.   var.     doliceus            purpureus                   purpureus                         S. vinaceus                                S. californicus     NRRL 11160            NRRL 2423                   UC 2468                         NRRL 2285                                ATCC 3312__________________________________________________________________________  CONTROL (+)    (+)    (-)   (+)    (-)  D-Xylose     +      +      +     +      +  L-Arabinose     (+)    (+)    (+)   (+)    (+)  Rhamnose     (+)    (+)    -     (+)    -  D-Fructose     +      +      +     +      +  D-Galactose     +      +      +     +      +  D-Glucose     +      +      +     +      +  D-Mannose     +      +      +     +      +  Maltose +      +      +     +      (+)  Sucrose (+)    (+)    (+)   (+)    (+)10.  Lactose (+)    (+)    (-)   (+)    +  Cellobiose     +      +      +     +      +  Raffinose     (+)    (+)    (+)   (+)    (-)  Dextrin +      +      +     +      +  Inulin  (+)    (+)    (+)   (+)    (-)  Soluble Starch     +      +      +     +      +  Glycerol     +      +      +     +      +  Dulcitol     (+)    (+)    (-)   (+)    (-)  D-Mannitol     +      +      +     +      +  D-Sorbitol     (+)    (+)    (+)   (+)    (-)20.  Inositol     (+)    (+)    (+)   (+)    (-)  Salicin (+)    (+)    (+)   (+)    (+)  Phenol  -      -      -     -      -  Cresol  -      -      -     -      -  Na Formate     (-)    (-)    -     (-)    -  Na Oxalate     (-)    (+)    (+)   (+)    -  Na Tartrate     (+)    (+)    (-)   (+)    (-)  Na Salicylate     -      -      -     -      -  Na Acetate     +      +      +     +      (+)→+  Na Citrate     +      +      +     +      (-)30.  Na Succinate     +      +      +     +      (+)__________________________________________________________________________ += Good growth (+) = Fair growth (-) = Trace growth -= No growth *Pridham, T. G., and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56:107-114. 
    
     
                                           Table 4__________________________________________________________________________ Growth of Carbon Compounds in the SyntheticMedium of Shirling and Gottlieb*     S. puniceus            S. griseus                   S. griseus     subsp. var.   var.     doliceus            purpureus                   purpureus                         S. vinaceus                                S. californicus     NRRL 11160            NRRL 2423                   UC 2468                         NRRL 2285                                ATCC 3312__________________________________________________________________________Negative Control(Synthetic Medium-ISP-9)    ±   ±   ±  ±   -Positive Control(Synthetic Medium+D-Glucose     ++     ++     ++    ++     ++Carbon Compounds:L-Arabinose     ±   +      +     ±   +Sucrose   -      -      ±  -      -D-Xylose  ++     ++     +     +      ++Inositol  -      -      -     -      -D-Mannitol     ++     ++     ++    ++     ++D-Fructose     +      ++     +     ++     ++Rhamnose  ±   ±   ±  ±   ±Raffinose -      -      -     -      -Cellulose -      -      -     -      -__________________________________________________________________________ ++ Strong utilization + Positive utilization ± Doubtful utilization - Negative utilization *Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340. 
    
     
                                           Table 5__________________________________________________________________________Cultural and Biochemical Characteristics  De-  ter-     S. puniceus             S. griseus                     S. griseus  mi-     subsp.  var.    var.  na-     doliceus             purpureus                     purpureus                             S. vinaceus                                    S. californicusMedium tion     NRRL 11160             NRRL 2423                     UC 2468 NRRL 2285                                    ATCC 3312__________________________________________________________________________AgarPeptone-iron  S  Cream-pink             Pale cream-                     Cream   Pale cream-                                    Trace cream             pink            pink  R  Yellow-tan             Yellow-tan                     Yellow-tan                             Yellow-tan                                    Yellow-tan  P  --      --      --      --     --  O  Melanin Melanin Melanin Melanin                                    Melanin     negative             negative                     negative                             negative                                    negativeCalcium  S  Very slight             Cream   Trace cream                             Cream  Trace creammalate    trace cream-                   aerial     pink  R  Colorless             Colorless                     Colorless                             Colorless                                    Colorless  P  --      --      --      --     --  O  Malate not             Malate not                     Malate not                             Malate not                                    Malate     solubilized             solubilized                     solubilized                             solubilized                                    solubilizedGlucose  S  Cream-pink             Cream   Cream   Cream  Cream-pinkasparagine  R  Maroon  Maroon  Pale pink-red                             Pale pink-red                                    Maroon     center, tan             center, tan                     center, cream                             center, cream                                    center, tan     edge    edge    edge    edge   edge  P  Pale pink             Pale pink                     Pale pink                             Pale pink                                    Pale pinkSkim milk  S  Very slight             Cream   Cream   Cream-pink                                    --     trace cream-     pink  R  Yellow-tan-             Yellow-tan-                     Yellow-tan-                             Yellow-tan-                                    Yellow-tan-     orange  orange  orange  orange orange  P  Yellow-tan-             Yellow-tan-                     Yellow-tan-                             Yellow-tan-                                    Yellow-tan-     orange  orange  orange  orange orange  O  Casein  Casein  Casein  Casein Casein     solubilized             solubilized                     solubilized                             solubilized                                    solubilizedTyrosine  S  Cream   Cream   Cream   Cream  Trace cream  R  Light tan             Tan     Tan     Tan    Pale yellow  P  Tan     Tan     Tan     Tan    Pale yellow  O  Tyrosine             Tyrosine                     Tyrosine                             Tyrosine                                    Tyrosine     solubilized             solubilized                     solubilized                             solubilized                                    solubilizedXanthine  S  Cream   Cream   Cream   Cream  Trace cream  R  Pale cream-             Cream-yellow                     Cream-yellow                             Cream-yellow                                    Pale yellow     tan  P  Cream-tan to             Cream-yellow                     Cream-yellow                             Cream-yellow                                    Very pale     very pale             to pale tan                     to pale tan                             to pale tan                                    yellow     tan  O  Xanthine not             Xanthine not                     Xanthine                             Xanthine not                                    Xanthine     solubilzed             solubilized                     slightly                             solubilized                                    solubilized                     solubilizedNutrient  S  Cream   Cream   Cream   Cream  Trace creamstarch R  Pale cream-             Cream-yellow                     Pale cream-                             Pale cream-                                    Very pale yellow     tan             pink-tan                             pink-tan  P  None to very             Cream-yellow                     Very pale                             Very pale                                    --     pale tan             to very pale                     pink-tan                             pink-tan             tan  O  Starch  Starch  Starch  Starch Starch     solubilized             solubilized                     solubilized                             solubilizedYeast extract-  S  Pale    Pale    Cream with                             Cream with                                    Trace creammalt extract     lavender             lavender                     fish-net                             fish-net     with cream             with cream                     edge    edge     edge    edge  R  Maroon with             Maroon with                     Maroon with                             Maroon with                                    Pale reddish-     tan edge             tan edge                     tan edge                             tan edge                                    tan  P  Pale red-tan             Pale red-tan                     Very pale tan                             Very pale tan                                    Very pale red-tanPeptone-yeast  S  Colorless             Trace cream                     White   White  Colorlessextract-iron     vegtative             aerial on              vegtative(ISP-6)           colorless             vegetative  R  Colorless             Colorless                     Yellow-tan                             Yellow-tan                                    Yellow-tan  P  Trace tan             Trace tan                     Yellow-tan                             Yellow-tan                                    Yellow-tan  O  Melanin Melanin Melanin Melanin                                    Melanin     negative             negative                     negative                             negative                                    negativeTyrosine  S  Mottled Cream   Gray-cream                             Gray-cream                                    Lavender(ISP-7)   cream-     lavender  R  Maroon  Maroon  Maroon  Maroon Maroon  P  Trace pale             Trace pale                     Gray-pink                             Gray-pink                                    Pale red-tan     red-tan red-tan  O  Melanin Melanin Melanin Melanin                                    Melanin     negative             negative                     negative                             negative                                    negativeGelatinPlain  S  Trace white             Trace white                     Trace   Trace white                                    --     aerial  aerial  colorless                             aerial                     vegetative  P  Pale yellow             Pale yellow                     Pale yellow                             Pale yellow                                    Pale yellow  O  Liquefaction             Liquefaction                     Liquefactin                             Liquefaction                                    No liquefaction     1/3     1/3     1/3     1/3Nutrient  S  Trace white             Trace white                     Trace white                             Trace white                                    --     aerial  aerial  aerial  aerial  P  Yellow  Yellow  Yellow  Yellow YellowO      Liquefaction     Liquefaction             Liquefaction                     Liquefaction                             No liquefaction     1/3     1/3     1/3     1/3Nitrate BrothSynthetic  S  --      --      --      Cream-pink                                    --                             aerial on                             lavender-pink                             vegetative                             pellicle  P  --      --      --      --     --  O  Compact Trace bottom                     Compact Trace bottom                                    Compact     bottom growth             growth  bottom growth                             growth bottom growth     No reduction             No reduction                     No reduction                             Reduction                                    No reduction     Red with             Red with                     Red with       Red with     Zn dust Zn dust Zn dust        Zn dustNutrient  S  Cream aerial             Lavender                     Lavender                             Gray-cream                                    Gray-cream     on maroon             aerial on                     aerial on                             aerial on                                    aerial on     ring    surface surface maroon ring                                    maroon ring             pellicle                     pellicle  P  --      --      --      --     --  O  Trace bottom             Trace bottom                     Trace bottom                             Trace bottom                                    Trace bottom     growth  growth  growth  growth growth     No reduction             Reduction                     Reduction                             Reduction                                    Reduction     Red with     Zn dustLitmus Milk  S  Cream aerial             Gray aerial                     Gray aerial                             Gray aerial                                    Trace gray     on blue on blue on blue-gray                             on blue-gray                                    aerial on     vegetative             vegetative                     ring    ring   blue-gray     ring    ring                   red ring  P  Slight purple             Slight purple                     --      --     --  O  Trace   Trace   Peptonization                             Peptonization                                    Peptonization     peptonization             peptonization          good                                    Litmus reduced                                    in one     pH 7.07 pH 7.12 pH 7.3  pH 7.07                                    pH 7.07__________________________________________________________________________ S = Surface R = Reverse P = Pigment O = Other characteristics 
    
     The compounds of the invention process are produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer&#39;s yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers&#39; solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium. 
     Production of the compounds by the invention process can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18° and 40° C., and preferably between about 20° and 28° C. Ordinarily, optimum production of the compounds is obtained in about 3 to 15 days. The medium normally remains acidic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium. 
     When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the compounds and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N 2  agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the compounds, so long as a good growth of the microorganism is obtained. 
     A variety of procedures can be employed in the isolation and purification of the compounds produced by the subject invention from fermentation beers, for example, charcoal absorption, 1-butanol extraction, and adsorption on cellulose and cation exchange resins. 
     In a preferred recovery process the compounds produced by the subject process invention are recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. 
     The antibiotics are then recovered from the filtered or centrifuged broth by absorption on a charcoal column. The charcoal can be eluted by passing 10 to 50% acetone in water (v/v) to remove the antibiotics. 
     The eluates are pooled and concentrated to an aqueous solution. This solution then is passed over a weak cation exchange resin in the ammonium form, for example, IRC-50 which is supplied by Rohm &amp; Haas, Philadelphia, Pa. The resin can be eluted with an inorganic salt, for example, ammonium chloride, ammonium sulfate (preferred), calcium perchlorate, and the like. Collected fractions are assayed for antibacterial activity as herein described. Fractions showing antibacterial activity can be extracted with 1-butanol to remove impurities. The antibiotics remain in the aqueous phase. 
     The aqueous phase can be passed over a charcoal column which is then eluted with 25% acetone in water (v/v) and fractions are collected. These fractions are concentrated to an aqueous which is then lyophilized. The next step of the purification process will separate antibiotic 354 from gougerotin. 
     The lyophilized solid containing antibiotic 354 and gougerotin, described above, is dissolved in a minimum amount of water. This material is injected or layered onto a cellulose column. The column is eluted with methanol and fractions are collected. These fractions contain antibiotic 354. Gougerotin is removed from the cellulose column by eluting the column with water. 
     Antibiotic 354 is obtained in essentially pure form from the above-described methanol eluates by first concentrating the pooled eluates to a solid and then passing the solid, which has been dissolved in a minimum amount of water, over a strong cation exchange resin, for example, Dowex 50 (supplied by Dow Chemical Co., Midland, Mich.). The column is eluted with a solution of an inorganic salt (ammonium sulfate preferred) and fractions containing essentially pure antibiotic 354 are collected. 
     Essentially pure gougerotin can be obtained by taking the fractions containing gougerotin from the cellulose column, described above, and passing them over a strong cation exchange resin, as described above for antibiotic 354. 
     Since antibiotic 354 is a strongly basic compound, procedures involving adsorption on cationic ion exchange resins and elution by organic bases or ammonia can be used to purify crude preparations of antibiotic 354. Also, crude preparations of antibiotic 354 can be purified by transformation to a salt form by treatment with inorganic or organic acids. The base form of the antibiotic can be recovered by neutralization of the acid anion with ammonia or other inorganic or organic bases. 
     In order to make salts of antibiotic 354 with both inorganic or organic acids, as hereinafter disclosed in exemplary form, it is necessary that the acid be carefully added to an aqueous solution of antibiotic 354 in view of the instability of this antibiotic at acid pH&#39;s. Examples of inorganic and organic acids which can be used, but which examples should not be considered limiting, are hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic, cholic, palmitic, mucic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, 3-phenylsalicylic, 5-phenylsalicyclic, 3-methylglutaric, orthiosulfobenzoic, cyclohexanesulfamic, cyclopentanepropionic, 1,2-cyclohexanedicarboxylic, 4-cyclohexenecarboxylic, octadecenylsuccinic, octenylsuccinic, methanesulfonic, benzenesulfonic, helianthic, Reinecke&#39;s, dimethyldithiocarbamic, sorbic, monochloroacetic, undecylenic, 4&#39;-hydroxyazobenzene-4-sulfonic, octadecylsulfuric, picric, benzoic, cinnamic, and like acids. 
     Other procedures for making certain salts are as follows. The sulfate salts can be made by using ammonium sulfate elution from a cation exchange resin. Also, the acetate salts can be made by using pyridinium acetate to elute the antibiotic from cation exchange resins. Further, the chloride salts of antibiotic 354 can be made by using ammonium chloride to elute the antibiotic from a cation exchange resin. The sulfate salts can be converted to the chloride by passing them over an anion exchange resin, for example, Dowex 1 (Cl - ) and Dowex 2 (Cl - ). If the resin is used in the OH -  form, the free base of 354 is isolated. 
     The salts of antibiotic 354 can be used for the same biological purposes as the parent antibiotic. 
     Acylates of antibiotic 354 can be made as follows: A sample of antibiotic 354 is dissolved in an excess of a silylating reagent such as TMS-imidazole or bis-TMS-trifluoroacetamide. A catalyst such as trimethylchlorosilane and/or a base such as pyridine may be used but neither is necessary. An acylating reagent such as trifluoroacetyl-imidazole or acetic anhydride is then added. Acylation is rapid and quantitative as judged by combined gas chromatography-mass spectroscopy. The peaks corresponding to silylated antibiotic 354 (both mono and di-silylated derivatives can be present) disappear and a new one appears with longer retention time and with a mass spectrum indicative of an acylated and monosilylated antibiotic 354. This derivative can then be selectively hydrolyzed with methanol or water to give an acylated derivative of antibiotic 354. 
     Suitable acid-binding agents include amines such as pyridine, quinoline, and isoquinoline, and buffer salts such as sodium acetate. The preferred base is pyridine. Carboxylic acids suitable for acylation include (a) saturated or unsaturated, straight or branched chain aliphatic carboxylic acids, for example, acetic, propionic, butyric, isobutyric, tertbutylacetic, valeric, isovaleric, caproic, caprylic, decanoic, dodecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic, margaric, stearic, acrylic, crotonic, undecylenic, oleic, hexynoic, heptynoic, octynoic acids, and the like; (b) saturated or unsaturated, alicyclic carboxylic acids, for example, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentenecarboxylic acid, methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid, dimethylcyclohexanecarboxylic acid, dipropylcyclohexanecarboxylic acid, and the like; (c) saturated or unsaturated, alicyclic aliphatic carboxylic acids, for example, cyclopentaneacetic acid, cyclopentanepropionic acid, cyclohexaneacetic acid, cyclohexanebutyric acid, methylcyclohexaneacetic acid, and the like; (d) aromatic carboxylic acids, for example, benzoic acid, toluic acid, naphthoic acid, ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid, and the like; and (e) aromatic aliphatic carboxylic acids, for example, phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamic acid, phenylpropiolic acid, and naphthylacetic acid, and the like. Also, suitable halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and loweralkoxyhydrocarboncarboxylic acids include hydrocarboncarboxylic acids as given above which are substituted by one or more of halogen, nitro, hydroxy, amino, cyano, or thiocyano, or loweralkoxy, advantageously loweralkoxy of not more than six carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy, and isomeric forms thereof. Examples of such substituted hydrocarboncarboxylic acids are: 
     mono-, di- and trichloroacetic acid; 
     α- and β-chloropropionic acid; 
     α- and γ-bromobutyric acid; 
     α- and δ-iodovaleric acid; 
     mevalonic acid; 
     2- and 4-chlorocyclohexanecarboxylic acid; 
     shikimic acid; 
     2-nitro-1-methylcyclobutanecarboxylic acid; 
     1,2,3,4,5,6-hexachlorocyclohexanecarboxylic acid; 
     3-bromo-2-methylcyclohexanecarboxylic acid; 
     4- and 5-bromo-2-methylcyclohexanecarboxylic acid; 
     5- and 6-bromo-2-methylcyclohexanecarboxylic acid; 
     2,3-dibromo-2-methylcyclohexanecarboxylic acid; 
     2,5-dibromo-2-methylcyclohexanecarboxylic acid; 
     4,5-dibromo-2-methylcyclohexanecarboxylic acid; 
     5,6-dibromo-2-methylcyclohexanecarboxylic acid; 
     3-bromo-3-methylcyclohexanecarboxylic acid; 
     6-bromo-3-methylcyclohexanecarboxylic acid; 
     1,6-dibromo-3-methylcyclohexanecarboxylic acid; 
     2-bromo-4-methylcyclohexanecarboxylic acid; 
     1,2-dibromo-4-methylcyclohexanecarboxylic acid; 
     3-bromo-2,2,3-trimethylcyclopenanecarboxylic acid; 
     1-bromo-3,5-dimethylcyclohexanecarboxylic acid; 
     homogentisic acid, o-, m-, and p-chlorobenzoic acid; 
     anisic acid; 
     salicyclic acid; 
     p-hydroxybenzoic acid; 
     β-resorcylic acid; 
     gallic acid; 
     veratric acid; 
     trimethoxybenzoic acid; 
     trimethoxycinnamic acid; 
     4,4&#39;-dichlorobenzilic acid; 
     o-, m-, and p-nitrobenzoic acid; 
     cyanoacetic acid; 
     3,4- and 3,5-dinitrobenzoic acid; 
     2,4,6-trinitrobenzoic acid; 
     thiocyanoacetic acid; 
     cyanopropionic acid; 
     lactic acid; 
     ethoxyformic acid (ethyl hydrogen carbonate); and the like. 
     The above acylates of antibiotic 354 are useful to upgrade the parent compound, i.e. by acylating the parent compound, then removing the acyl group, the parent compound is isolated in a purer form. 
     Trimethylsilylation of antibiotic 354 gives a volatile di-TMS derivative (along with a little mono-TMS derivative) which is useful in vapor phase chromatography and mass spectroscopy work. This derivative can be prepared by heating a sample of antibiotic 354 at about 60° C. for about 30 minutes in tetrahydrofuran with an excess of bistrimethylsilylacetamide. 
     Also, the mono-TMS derivative can be prepared using either trimethylsilylimidazole or bistrimethylsilyltrifluoroacetamide. The monosilylated antibiotic 354 can be acylated in situ using trifluoroacetylimidazole, trifluoroacetic anhydride or acetic anhydride. These are also useful in vpc-mass spectroscopic work and represent a practical route to selective (O vs. N) protection of antibiotic 354. 
     The following examples are illustrative of the process and products of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. 
     EXAMPLE 1 
     A. Fermentation 
     A biologically pure culture of Streptomyces puniceus subsp. doliceus, NRRL 11160, is used to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml of sterile medium consisting of the following ingredients: 
     
         ______________________________________Glucose                 10 g/lYeast Extract           2.5 g/lPeptone                 10 g/lDeionized Water q.s.    1 liter______________________________________ 
    
     The seed medium presterilization pH is 6.5. The seed inoculum is grown for three days at 28° C. on a Gump rotary shaker operating at 250 r.p.m. Seed inoculum, prepared as described above, is used to inoculate 500-ml Erlenmeyer fermentation flasks containing 100 ml of sterile fermentation medium consisting of the following ingredients: 
     
         ______________________________________Brer Rabbit/Sucrest*     20 ml/1Yeast Extract/Brewer&#39;s Yeast**                     2 g/lDextrin***               10 g/lCerelose***              15 g/lProteose-Peptone #3**    10 g/lPeanut Meal               5 g/lWater q.s.                1 liter______________________________________ *RJR Foods, Inc., New York, NY **Difco Laboratories, Detroit, MI ***CPC International, Inc., Englewood Cliffs, NJ 
    
     The presterilization pH is 7.0. The fermentation flasks are inoculated at the rate of 5 ml of seed inoculum per 100 ml of fermentation medium. The fermentation flasks are grown for 3 days at a temperature of 25°-28° C. on a Gump rotary shaker operating at 250 r.p.m. 
     A representative shake flask fermentation harvested after 3 days shows the following assay pattern against Pseudomonas mildenbergii (UC 3029). 
     
         ______________________________________Day          Assay Results (BU/ml)______________________________________1             02            143            14______________________________________ 
    
     The assay is an agar disc plate assay using the microorganism P. mildenbergii. The agar medium is buffered with 0.1 M phosphate buffer at a pH of 7.4. A unit volume (0.08 ml) of solution containing the substance to be assayed is placed on a 12.7 mm paper disc which is then placed on an agar plate seeded with the assay organism. The agar plate is then incubated for 16-18 hours at 37° C. A biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the above assay conditions. Thus, if for example a fermentation beer, or other solution containing the antibiotic, needs to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer or solution is 100 BU per ml. 
     B. Recovery 
     (1) Carbon Sorption 
     Ten liters of shake flask-grown beer, as described above, is stirred with 4 l washed, granular charcoal for 15 minutes. The charcoal is allowed to settle for 10 minutes and the beer is decanted. The charcoal is washed with deionized water until the water remains clear. An overhead paddle stirrer is used in the first step and a 10 l pail in the second step. The charcoal is slurried into a chromatography tube with deionized water. The activities are eluted with 25% acetone in water using gravity flow at the highest possible flow rate. One liter fractions are collected until a yellow color emerges. When the yellow color is almost undectable small fractions are again collected. The yellow fractions (11 l) are concentrated to an aqueous at 35°-40° C./l mm and assayed. The data are presented in Table I below. It is seen that 80% of the UC 3029 biounits are recovered from the column. 
     
                                           Table I__________________________________________________________________________            Pseudomonas            Mildenbergii                   BacillusSample Volume       Dilution            (UC 3029)                   subtilis                        Conc. BU/mg__________________________________________________________________________Whole Beer  10 1 FS*  28 mm  24 mm                        --    --       1:2  23     21   --    --       1:4  trace  20   --    --       1:8  NZ**   19   --    --Spent Beer  11  1       FS   NZ     20   --    --1st Eluate  3 1  FS   NZ     --   --    --2nd Eluate  9 1  FS   25     --   --    --3rd Eluate  2 1  FS   NZ     --   --    --Aqueous Pool  8 1  FS   29     23   16.9  0.18                        mg/ml       1:2  23     19   (3                        BU/ml)       1:4  16     trace__________________________________________________________________________ FS* = Full Strength NZ** = No Zone 
    
     (2) IRC-50 Sorption 
     Carbon eluates from four runs (about 48 l of beer in all), as described above, are pooled to give 33 l of aqueous which assay for a total of 91,000 UC 3029 biounits. 
     This is passed over 2 pounds of IRC-50 (NH 4   + ) in a chromatography tube at 5-6 l/hour. The column is then washed with 4 l of deionized water and eluted with 1 M (NH 4 ) 2  SO 4  solution. The fractions are assayed by UV after diluting them 1:10 with water. On the basis of the UV data, the first two eluates are pooled. The third eluate is saved. The data are presented in Table II below. 
     
                       Table II______________________________________            Pseudomonas            mildenbergii                       BacillusSample  Volume   (UC 3029)  subtilis                              A     λ______________________________________Spent   33.4 1   NZ         NZ     1.4   265 nmWash     3.5 1   NZ         NZ     0.24  2651st Eluate    320 ml  21 mm      20 mm  0.62  2652nd Eluate   2000 ml  31         30     5.80  2603rd Eluate   2000 ml  23         20     2.18  2554th Eluate   1000 ml  18         NZ     1.29  2555th Eluate   1000 ml  NZ         NZ     1.13  255______________________________________ 
    
     (3) Desalting 
     Eluate #3 above (2000 ml) is passed over 200 ml granular charcoal in a chromatography tube. The column is washed with 500 ml deionized water. Neither the spent nor the wash has any UV absorbance. The activities are eluted with 700 ml 25% acetone in water. The eluate is concentrated to an aqueous amounting to 500 ml. A 1:10 dilution of this absorbs strongly at 255 nm. Bioautography on cellulose with methanol shows that gougerotin and antibiotic 354 are present. 
     (4) Ultrafiltration 
     The desalted aqueous from above is passed over an Amicon UM 2 ultrafilter (Amicon Corp., 21 Hartwell Avenue, Lexington, MA 02173). The retentate shows no activity and is discarded after a wash of one volume. The first filtrate and wash are pooled and lyophilized. The residue weights 4.5 gm. This shows a UV max at 252 nm with an inflection at 212 nm on a strong end absorption. 
     (5) Separation of Gougerotin From Antibiotic 354 
     A 68 gm preparation treated essentially as described above (except for the ultrafiltration), assays at 0.68 BU/mg vs. B. subtilis; it is labeled Prep. 216-4. Bioautography shows that it contains gougerotin and antibiotic 354. 
     A cellulose 300 column measuring 5.0 × 150 cm is flushed with methanol at 20 ml/minute (10 psi). The bed volume is 2.9 l. 
     Thirty grams of the above Prep. 216-4 is dissolved in 65 ml water and the solution is injected onto the column. Some of the solid precipitates when it contacts the methanol but this does not plug the column or otherwise interfere with the procedure. The column is eluted with methanol at 20 ml/minute. The eluate fractions are assayed by UV using 1:10 dilutions. The data are presented in Table III. Fractions 3-8 are pooled and concentrated to give 10.2 gm tan solid. Bioautography shows this to contain antibiotic 354 and no gougerotin. A second injection using the rest of the sample and similarly analyzed yields 12.0 gm of a like preparation. 
     
                       Table III______________________________________                              Pseudomonas                              mildenbergilFraction  Volume   Color       A.sub.255-265                              (UC 3029)______________________________________1      1300  ml Colorless   0.00   --2      1200  ml Colorless   0.21   --3      400 ml   Colorless   2.13   30 mm4      400 ml   Light Yellow                       9.30   405      425 ml   Light Yellow                       12.8   396      500 ml   Light Yellow                       12.4   367      500 ml   Trace Yellow                       8.4    278      500 ml   Colorless   1.12   239      500 ml   Colorless   --     2010     500 ml   Colorless   --     1911     500 ml   Colorless   --     trace______________________________________ 
    
     After fraction #11 the solvent is switched to water and the elution is continued at 20 ml/minute. The data are presented in Table IV below. Fractions 2 and 3 are pooled and lyophilized to give 17.9 gm tan solid. A second injection yields 22.1 gm. Bioautography shows this to contain only gougerotin. 
     
                       Table IV______________________________________Fraction  Volume      Color        A.sub.269______________________________________1         1000 ml     Colorless    0.302         1000 ml     Yellow       11.03          900 ml     Light Yellow 2.644         1400 ml     Colorless    0.08______________________________________ 
    
     The 68 gm sample (216-4) gives 22.2 gm (32.6%) antibiotic 354-containing solids and 40 gm (58.8%) gougerotin-containing solids. 
     The 22 gm of antibiotic 354 material assays at 0.35 BU/mg vs. B. subtilis and 0.83 BU/mg vs. UC 3029. 
     The 22 gm of gougerotin material obtained from the second run assays at 0.5 BU/mg vs. B. subtilis and 1.5 BU/mg vs. UC 3029. 
     Both preparations are grossly impure. 
     (6) Pure Gougerotin 
     A pool from two cellulose runs similar to that described above (but ultrafiltered) amounts to 5.8 gm. This is dissolved in 15 ml water and injected onto a 200-400 mesh Amberlite CG-120 (NH 4   + ) column (Rohm &amp; Haas, Philadelphia, Pa.) measuring 2.5 × 100 cm. This is eluted with a water to 1 M (NH 4 ) 2  SO 4  gradient at 12 ml/minute. 
     Fractions amounting to 25 ml are collected. Every fifth fraction is assayed (100 λ/12.7 mm pad) vs. UC 3029. There is no activity through tube #250. The UV assay is done with 1:10 dilutions of every tenth tube. This shows no 265 nm band through tube #250. The UV data for subsequent tubes is presented in Table V. The B. subtilis zones obtained for tubes 260-370 are very small. Tubes 280-340 are pooled to give 1.4 l solution. This is desalted over 200 ml charcoal in a chromatography tube. The charcoal is washed with water and eluted with 25% acetone in water. The fractions are monitored by UV absorbance at 268 nm. The aqueous eluate is lyophilized to give 1.0 gm of essentially pure gougerotin as a white solid. 
     
                       Table V______________________________________Fraction  A.sub.268  Fraction     A.sub.268______________________________________260       0.26       320          1.87270       0.27       330          --280       0.45       340          0.68290       1.30       350          0.32300       2.46       360          0.20310       2.55       370          0.15______________________________________ 
    
     (7) Pure Antiobiotic 354 As The Sulfate 
     A pool is made of various fractions which had been treated as described above through the cellulose column step. It amounts to 3.78 gm and is dissolved in 10 ml water. This is injected onto the CG-120 (NH 4   + ) column described above for gougerotin and eluted with the same gradient at the same rate. An aliquot of every tenth tube is diluted 1:10 with water and examined by UV and dioassayed. Nothing elutes up to tube #220. The data for subsequent tubes are presented in Table VI. Fractions 248-340 are pooled to give 2.5 l solution with A 252  = 1.27. This is desalted as described for gougerotin using a bed of charcoal measuring 3.5 × 28 cm (270 ml) and monitoring at the appropriate wavelengths. The desalted eluate is lyophilized to give 2.36 gm essentially pure antibiotic 354 as a tan solid. It assays at 8 BU/mg vs. UC 3029. 
     
                       Table Vi______________________________________                         Pseudomonas                         mildenbergil -Fraction A.sub.255 A.sub.212 (                         UC 3029)______________________________________230       0.23      1.08      --240       1.10      5.39      --250       1.60      8.20      --260       1.87      8.60        35 mm270       2.75      10.1      40280       2.05      9.00      39290       1.49      6.95      37300       1.03      4.60      33310       0.69      2.41      26320       0.46      1.88      NZ330       0.31      1.25      NZ340       0.20      0.82      NZ350       0.15      0.51      NZ______________________________________ 
    
     EXAMPLE 2 
     Acetylation Of Antibiotic 354 
     A sample of antibiotic 354 is stirred in tetrahydrofuran with pyridine and acetic anhydride. The solvent is removed in vacuo and the residue is partitioned between ethyl acetate and 0.01 N HCl. The aqueous is lyophilized. When the solids are redissolved in water, crystals form and are collected. These crystals are shown by mass spectroscopy to be the mono-N-acetyldehydrochlorinated derivative of antibiotic 354. 
     EXAMPLE 3 
     Preparation Of Antibiotic 354.HCl From Antibiotic 354.H 2  SO 4   
     A 14 mg sample of antibiotic 354.H 2  SO 4  is dissolved in 0.3 ml water and passed over a 0.4 × 8 cm column of 100-200 mesh Dowex 2 × 8 (Cl - ). Eluted with distilled water and assayed each 3.0 ml fraction with ninhydrin spray after spotting onto a cellulose tlc (thin layer chromatography) plate and by UV absorption (bands at 251 and 211 in a 1:4 ratio). The appropriate pool is lyophilized. The solid residue is judged to be the hydrochloride by mass spectroscopy. 
     EXAMPLE 4 
     Preparation Of Antibiotic 354.HOAc From A Mix Of Gougerotin And Antibiotic 354 
     An aqueous carbon eluate (4 l) containing gougerotin and antibiotic 354 is passed over a column of 200 g Dowex 50 W × 8 (H + ). The column is washed with deionized water and eluted with 2.0 M pyridinium acetate buffer at pH 5. Fractions 13-17 (45 ml each) are pooled on the basis of bioactivity (12.7 mm pads, agar tray) vs. UC 3029 an lyophilized. The solids give bioautographic patterns indicating that the main activity is antibiotic 354 which must be in the acetate form. The mixture of acetates so obtained is separated into gougerotin acetate and antibiotic 354 acetate by cellulose chromatography as described above.