Patent Abstract:
A new species of Nocardiopsis, designated Nocardiopsis atra Huang sp. nov. ATCC 31511, when aerobically propagated in an aqueous nutrient medium, produces nocardicin A in good yield.

Full Description:
BACKGROUND OF THE INVENTION 
     Among the best known and widely used class of antibacterial agents are the so-called β-lactam antibiotics. These compounds are characterized in that they have a nucleus consisting of a 2-azetidinone (β-lactam) ring. When the β-lactam is fused to a thiazolidine ring, the compounds are usually referred to generically as penicillins. When the β-lactam is fused to a dihydrothiazine ring, the compounds are referred to as cephalosporins. 
     The β-lactam antibiotics are synthesized, for no apparent reason, by only a few microorganisms. All of the organisms recognized to produce β-lactam antibiotics are filamentous microorganisms, but not all of these microorganisms are taxonomically related. Some are fungi (eukaryotes), whereas others are streptomycetes (prokaryotes). Thienamycin, U.S. Pat. No. 3,950,357, is an interesting new β-lactam antibiotic produced by a species of Streptomyces. 
     Nocardicin A, U.S. Pat. No. 3,923,977, is a novel β-lactam antibiotic produced by Nocardia uniformis var. tsuyamanensis ATCC 21806. Nocardicin A has an unusual β-lactam moiety which is uniquely different from all other β-lactam antibiotics in not being fused to another ring as shown in the following structure: ##STR1## 
     The unique structure of nocardicin A may account for its relative stability toward a variety of β-lactamases as well as for its negligible immunological cross-reactivity with penicillins and cephalosporins. 
     Unfortunately, the outstanding in vitro and in vivo antimicrobial properties of the penicillins and caphalosporins are not shared by nocardicin A. However, the antibiotic&#39;s nucleus, 3-aminonocardicinic acid, of the following structure has been prepared, J. Am. Chem. Soc. 100, 3933 (1978): ##STR2## 
     The present invention is concerned with the discovery that nocardicin A is produced by a new species of Nocardiopsis designated Nocardiopsis atra Huang sp. nov. ATCC 31511. The microorganism with its potential for cultural and mutational development offers promise of the production of large amounts of nocardicin A, subsequent cleavage to 3-aminonocardicinic acid and the synthesis of a variety of clinically useful semisynthetic nocardicins paralleling those of the semisynthetic penicillins and cephalosporins. 
     SUMMARY OF THE INVENTION 
     Nocardicin A, previously reported β-lactam antibiotic, is produced by the aerobic propagation in aqueous nutrient media of a new species of Nocardiopsis designated Nocardiopsis atra Huang sp. nov. ATCC 31511. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The microorganism useful for the production of nocardicin A was isolated from a coil sample from Nomozaki, Japan. This culture, designated Nocardiopsis atra Huang sp. nov., has been deposited in The American Type Culture Collection, Rockville, Md. as the type culture under their accession number ATCC 31511. The permanency of the deposit of this culture at The American Type Culture Collection at Rockville, Md. and ready accessibility thereto by the public are afforded throughout the effective life of the patent in the event the patent is granted. Access to the culture is available during pendency of the application under 37 CFR 1.14 and 35 USC 112. All restrictions on the availability to the public of the culture deposited will be irrevocably removed upon granting of the patent. 
     The culture was planted from a slant into liquid ATCC No. 172 medium and grown for 4 days at 28° C. on a shaker. It was then removed from the shaker, centrifuged for 20 minutes, washed three times with sterile distilled water and planted on media commonly used for identification of members of the Actinomycetales. 
     The inoculated plates were incubated at 28° C. and records of results were made after suitable incubation time with most of the final results recorded at 14 days. The colors were described in common terminology, but exact color was determined by comparison with color chips from the Color Harmony Manual, fourth edition. 
     The methods of whole-cell and sugar analyses are those described by Becker, B. et al., Appl. Microbiol., 12, 421-423 (1964) and by Lechevalier, M.P., J. Lab. Clin. Med., 71, 934-944 (1968). Mycolate analyses were performed by the method of Lechevalier, M.P. et al., J. Bacteriol., 105, 313-318 (1971). 
     The results of chemical tests of whole-cell and cell wall indicated the new culture belonged in the genus Nocardiopsis. For comparison purposes, therefore, culture studies included Nocardia uniformis subsp. tsuyamanesis ATCC 21806 and Nocardiopsis dassonvillei ATCC 23218. 
     Identification media used for the characterization of the culture and references for their composition are as follows: 
     1. Tryptone Yeast Extract Broth (ISP #1 medium, Difco). 
     2. Yeast Extract-Malt Extract Agar (ISP #2 medium, Difco). 
     3. Oatmeal Agar (ISP #3 medium, Difco). 
     4. Inorganic Salts--Starch Agar (ISP #4 medium, Difco). 
     5. Glycerol--Asparagine Agar (ISP #5 medium, Difco). 
     6. Glycerol--Asparagine Agar (prepared in our lab). 
     7. Peptone--Yeast Extract Iron Agar (#6 medium, Difco). 
     8. Gelatin Agar--R. E. Gordon and J. M. Mihm, Jr. Bact. 73: 15-27, 1957. 
     9. Starch Agar--Ibid. 
     10. Organic Nitrate Broth--Ibid. 
     11. Dextrose Nitrate Broth--S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1. p. 328, 1961, and 3 g dextrose substituted for 30 g sucrose and agar omitted. 
     12. Potato Carrot Agar--M. P. Lechevalier, Jr. Lab. and Clinical Med. 71: 934-944, 1968 but use only 30 g potatoes, 2.5 g carrots and 20 g agar. 
     13. 2% Tap Water Agar 
     14. Czapek-Sucrose Agar--S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1, p. 328, 1961. 
     15. Glucose Asparagine Agar--Ibid, medium no. 2, p. 328. 
     16. Glucose-Yeast Extract Agar--Ibid, medium no. 29, p. 331 
     17. Emerson&#39;s Agar--Ibid, medium no. 28, p. 331. 
     18. Nutrient Agar--Ibid, medium no. 14, p. 330. 
     19. Bennet&#39;s Agar--Ibid, medium no. 30, p. 331. 
     20. Gordon and Smith&#39; Tyrosine Agar R. E. Gordon and M. M. Smith, Jr. Bact. 69: 147-150, 1955. 
     21. Casein Agar--Ibid. 
     22. Calcium Malate Agar--S. A. Waksman, Bact. Rev. 21: 1-29, 1957. 
     23. Gauze&#39;s #1 Mineral Agar--G. F. Gauze et al. Problems in the Classification of Antagonistic Actinomycetes. English Ed., p. 13, 1957. 
     24. Gauze&#39;s #2 Organic Agar--Ibid. 
     25. Skim Milk--Difco 
     26. Cellulose utilization-- 
     (a) H. L. Jensen, Proc. Linn. Soc. N.S.W. 55: 231-248, 1930. 
     (b) M. Levine and H. W. Schoenlein, A Compilation of Culture Media, Medium 2511, 1930. 
     27. Utilization of Organic Acids--R. E. Gordon et al., Int. Jr. Syst. Bact. 24: 54-63, 1974. 
     28. Carbohydrate Utilization and Acid Production from Carbohydrates--Ibid. 
     29. Hydrolysis of Hippurate and Esculin--Ibid. 
     30. Decomposition of Adenine, Hypoxanthine, Xanthine, and Urea--Ibid. 
     31. Yeast Dextrose Agar for Studies of Temperature and Survival at 50° C.--Ibid. 
     32. Resistance to Lysozyme--Ibid. 
     33. Glucose Broth--Ibid. 
     
         __________________________________________________________________________                  Nocardia         Nocardiopsis                  uniformis subsp.                            Nocardiopsis   Cultural         atra     tsuyamanensis                            dassonvilleiAgar    Character*         ATCC 31511                  ATCC 21806                            ATCC 23218__________________________________________________________________________Yeast Extract-   G     Poor to moderate                  Good      ExcellentMalt Extract   SC    Brown to greyish                  Orange    White with paleAgar          brown    (4 ia to 4 lc)                            orange tint         (4 lg to 4 li)     (3 ea)   T     Thin, smooth to                  Thin to slightly                            Wrinkled with a         slightly Sc                  raised, wrinkled                            fluffy edge,   AM    --       --        White   RC    Same as SC                  Same as SC                            Yellowish orange                            (3 ia to 3 lc)   SP    --       --        Pale yellowishOatmeal Agar   G     Moderate Moderate  Moderate   SC    Dull white                  Pale brownish                            Colorless to                  (3 gc)    white   T     Thin, smooth,                  Submerged, smooth                            Thin, smooth with         with a few small                  with spreading                            spreading edge         white dots                  edge   AM    None to scant                  --        White, sparse   RC    Same as SC                  Same as SC                            Same as SC   SP    Pale greenish                  --        --Inorganic   G     Moderate Moderate to good                            ModerateSalts-Starch   SC    Pale grey to                  Yellowish orange                            White to paleAgar          greyish (near                  (3 ea, 3 ia to                            yellowish         grey series,                  3 la)     (2 ga)         2 dc to 2 fe)         with white tint   T     Thin, smooth                  Thin to slightly                            Thin to slightly                  raised    raised                            smooth to                            slightly                            wrinkled   AM    White    --        White   RC    Same as surface                  Same as surface                            Same as surface   SP    Pale greyish green                  --        --         (1 gc)Glycerol   G     Poor to moderate                  Poor to moderate                            PoorAsparagine   SC    Colorless to cream                  Colorless to pale                            Colorless toAgar                   yellowish brown                            pale cream(dehydrated            (near 2 ea)powder from   T     Thin, appearing as                  Submerged, smooth                            Thin, smooth,Difco)        confluent smear    with a fluffy         or very small      edge         isolated dots   AM    --       --        --   RC    Same as surface                  Same as surface                            Same as surface   SP    --       --        --Glycerol   G     Good     Moderate  Moderate to goodAsparagine   SC    Grey, dark grey to                  Orange    White to creamAgar          black (near grey)                  (3 ga to 3 ia)                            (1 1/2 ca)(Prepared)    series, 3 ih to         3 ml)   T     Raised, slightly                  Thin, smooth, with                            Raised, rough-         wrinkled a spreading                            ened to                  edge      wrinkled   AM    --       --        White   RC    Brown (4 pg to                  Same as surface                            Pale yellowish         4 pi) to black     to pale yel-                            lowish green                            (1 1/2 ea to                            1 1/2 gc)   SP    Greyish orange                  Very pale Pale yellowish         (near 3 gc) to                  orange         blackGelatin Agar   G     Good     Good      Good   SC    Grey to black                  Orange (3 ia)                            Cream to pale         (near grey         yellowish         series, 3 fe)      (2 ca to 2 ga)   T     Raised, wrinkled                  Thin, smooth but                            Thin, smooth but                  slightly wrin-                            slightly wrin-                  kled near the                            kled near the                  edge      end of streak   AM    --       --        White   RC    Same as surface                  Same as surface                            Yellowish (2 ia)   SP    Greyish  --        --Starch Agar   G     Good     Good      Good   SC    Greyish to black                  Orange (near 4 ia                            Yellowish brown         (near grey                  or 4 la)  (near 2 lc)         series 2 ih to     but white         7 ml               near edge   T     Raised, roughened                  Slightly raised,                            Slightly raised,                  wrinkled  wrinkled   AM    --       --        White   RC    Same as surface                  Same as surface                            Same as surface   SP    Greyish  --        --Potato Carrot   G     Moderate Poor to moderate                            Poor to moderateAgar    SC    Cream (1 1/2                  Colorless to pale                            Colorless, white         ca)      brown (2 ca)                            to pale brown                            (2 ca)   T     Thin, smooth,                  Submerged, thin,                            Thin, smooth,         with some small                  smooth, with a                            with a fluffy,         slightly raised                  spreading edge                            spreading edge         dots   AM    None to sparse                  --        White   RC    Same as surface                  Same as surface                            Same as surface   SP    --       --        --Tap Water   G     Poor to moderate                  Poor      PoorAgar    SC    Colorless to white                  Colorless Colorless   T     Thin, smooth, with                  Submerged, smooth,                            Thin, smooth         a few white dots                  with a spread-                  ing edge   AM    None to sparse                  --        --   RC    Same as surface                  Same as surface                            Same as surface   SP    --       --        --Czapek-Sucrose   G     Moderate Moderate  ModerateAgar    SC    Pale greyish green                  Yellowish to                            Cream (1 1/2 ca)         (1 1/2 ec) with                  yellowish with very pale         some small black                  orange (1 1/2 ea                            greenish tint         dots, greyish                  to 2 lc)         near the end of         streak (near grey         series, 2 fe)   T     Thin, smooth                  Submerged, smooth                            Slightly raised                  with a spread-                            and roughened                  ing edge   AM    --       --        White, sparse   RC    Same as surface                  Same as surface                            Same as surface   SP    Pale greenish                  --        Pale greyish         (1 1/2 gc)Glucose G     Moderate Moderate  ModerateAsparagine   SC    Pale brown (3 gc)                  Yellowish orange                            Cream (1 1/2 ca)Agar          to black (3 ga to 3 la)   T     Slightly raised,                  Thin, smooth, with                            Thin to slightly         smooth, with                  a few small                            raised appear-         small raised                  raised dots                            ing as small         black dots         isolated dots,                            slightly                            wrinkled   AM    --       --        --   RC    Brown to black                  Same as surface                            Same as surface   SP    Pale yellowish                  --        --         brownGlucose Yeast   G     Good     Good      GoodExtract Agar   SC    Black (near grey                  Orange    White to cream         series, 3 po)                  (near 4 ia)                            (2 ca)   T     Raised, wrinkled                  Slightly raised,                            Raised, strongly                  wrinkled  wrinkled   AM    --       --        White   RC    Black    Same as surface                            Yellowish brown                            (near 2 ic)   SP    Black    --        Brown (3 ic)Emerson&#39;s   G     Good to excellent                  Good      GoodAgar    SC    Black (near grey                  Orange    White, cream to         series, 3 ml)                  (4 ga to 4 ia)                            brown (2 ca to                            3 le)   T     Raised, wrinkled                  Slightly raised,                            Slightly raised,                  wrinkled, but                            roughened to                  smooth near                            wrinkled, with                  the edge  a fluffy,                            spreading edge   AM    --       --        White   RC    Black    Same as surface                            Brown   SP    Black    Yellowish brown                            Brown (4 ne)                  (3 lc)Nutrient Agar   G     Moderate to poor                  Moderate  Moderate   SC    Pale grey to                  Pale orange                            Cream (2 ca)         grey (near grey                  (between 3 ea         series, 2 dc,                  4 ea)         2 ih to 2 ml)   T     Thin, smooth, with                  Thin, smooth                            Thin, smooth or         raised black       appearing as         dots               small isolated                            dots, with a                            spreading edge   AM    --       --        White, sparse   RC    Same as surface                  Same as surface                            Cream to pale                            yellowish   SP    Pale greyish                  --        --Bennett&#39;s Agar   G     Good to excellent                  Moderate to good                            Good   SC    Black (near grey                  Yellowish orange                            Cream to yellow-         series, 3 ml)                  (3 ga to 3 la)                            ish (2 ca to                            2 ea) with a                            white edge   T     Raised, wrinkled,                  Thin, smooth to                            Moderately                  slightly wrin-                            raised, wrin-                  kled      kled, with a                            fluffy edge   AM    --       --        White   RC    Black    Same as surface                            Same as surface   SP    Black    --        --Gordon and   G     Moderate Moderate to good                            ModerateSmith&#39;  SC    Grey to dark grey                  Dark orange                            Dirty whiteTyrosine      (near grey                  (near 4 lc)Agar          series, 2 ih         to 2 ml)   T     Thin to slightly                  Thin, smooth                            Thin, smooth or         raised, slightly   appearing as         roughened to       small isolated         granular           dots, with a                            spreading edge   AM    --       --        White   RC    Same as surface                  Same as surface                            Pale yellowish   SP    Greyish black                  Brown (near 3 ic)                            Brown (between                            3 gc and 3 ic)Calcium G     Moderate Poor to moderate                            ModerateMalate  SC    Black    Yellowish to                            Dirty white toAgar                   yellowish brown                            cream                  (2 ga to 2 lc)                            (2 ca)   T     Thin, smooth, with                  Thin, smooth, with                            Appearing as         some raised dots                  a spreading sub-                            isolated dots                  merged edge   AM    --       --        White   RC    Black    Same as surface                            Yellowish brown                            (2 ic)   SP    Pale greenish                  --        --Gauze&#39;s #1   G     Moderate Moderate to good                            Moderate to goodMineral Agar   SC    Pale greenish                  Yellowish orange                            White         cream    (3 ea to 3 ia)   T     Thin, with small                  Thin, smooth, with                            Slightly raised         white to greyish                  a spreading edge                            and wrinkled         dots   AM    White, sparse                  --        White   RC    Same as surface                  Same as surface                            Greyish brown                            (2 ie)   SP    Pale greenish (1 ca)                  --        Pale greyish                            (betwen 2 ec                            and 2 ge)Gauze&#39;s #2   G     Moderate to good                  Good      Moderate to goodOrganic Agar   SC    Dark brown to                  Orange (3 ga,                            Cream         greyish (3 ni,                  3 ia to 3 gc)         near grey series         3 ih)   T     Raised, granular                  Slightly raised,                            Raised and         to flaky wrinkled, with                            wrinkled                  a moderately                  spreading edge   AM    --       --        --   RC    Dark brown to                  Same as surface                            Same as surface         black   SP    Greyish brown                  Pale yellowish                            Pale yellowish                  orange__________________________________________________________________________ *G-growth, SCsurface color, Ttexture, AMaerial mycelium, RCreverse color, SPsoluble pigment, &#34; means none. 
    
     
         ______________________________________                 NocardiaBiochemical and       N. atra   uniformis subsp.                             NocardiopsisPhysiological       ATCC      tsuyamanensis                             dassonvilleiProperties  31511     ATCC 21806  ATCC 23218______________________________________Gram stain  +         +           +Acid-fastness       -         -           -Decomposition of:Adenine     -         -           +Calcium malate       +         +           +Casein      +         +           +Hypoxanthine       -         -           +Tyrosine    +         -           -Urea        +         +           +Xanthine    -         -           +Hydrolysis of:Esculin     -         +           +Hippurate   +         -           +Starch      +         +           +Growth at:45° C.       -         -           -37° C.       very poor good        good28° C.       good      good        good21° C.       very poor good        goodSurvival at 50° C.       -         +           -8 hMelanin production       -         -           -H.sub.2 S production       -         +           +Gelatin liquefaction       +         +           +Cellulosedecomposition       -         -           -Growth incellulose broth:Jensen&#39;s    poor      good        poorLevine &amp;Schoenlein&#39;s       -         poor        -Skim milk:Coagulation -         +           -peptonization       -         +           -Acid from and(utilization of):Adonitol    + (+)     - (-)       - (-)Arabinose   + (+)     + (±)    + (+)Cellobiose  + (+)     + (+)       + (+)Dulcitol    - (-)     - (+)       - (-)Erythritol  - (+)     - (±)    - (-)Fructose    + (+)     + (+)       + (+)Galactose   + (+)     + (+)       + (+)Glucose     + (+)     + (+)       + (+)Glycerol    + (+)     + (+)       + (+)Inositol    + (+)     - (-)       - (-)Lactose     + (+)     + (+)       - (±)Maltose     + (+)     + (+)       + (+ )Mannitol    + (+)     + (+)       + (+)Mannose     + (+)     + (+)       + (+)Melezitose  - (±)  - (±)    - (±)Melibiose   + (+)     + (+)       - (-)α-Methyl-d-       + (+)     - (-)       - (±)glucosideRaffinose   + (+)     + (+)       - (±)Rhamnose    + (+)     + (+)       + (+)Ribose      + (+)     - (-)       + (+)Salicin     + (+)     + (+)       + (+)Sorbitol    - (+)     - (±)    - (-)Sorbose     - (±)  - (±)    - (-)Starch      + (+)     + (+)       + (+)Sucrose     + (+)     + (+)       + (+)Trehalose   + (+)     + (+)       + (+)Xylose      + (+)     + (+)       + (+)Utilization of:Acetate     +         +           +Benzoate    -         -           -Citrate     +         -           +Dextrin     -         -           -Lactate     +         +           +Malate      +         +           +Mucate      -         -           -Oxalate     -         -           -Phenol      -         -           -Propionate  -         -           -Pyruvate    +         +           +Succinate   +         +           +Nitrite fromNitrate:Dextrose nitrate       -         +           +brothOrganic nitrate       -         +           +brothResistance to       -         +           -lysozyme______________________________________ &#34;+&#34;, positive; &#34;-&#34;, negative; &#34;±&#34;, doubtfully utilized. 
    
     Morphological observations were made 1, 2, 3, 5, 7, 14 and 28 days after the inoculation of the new Nocardiopsis culture on Czapek sucrose agar. Substrate mycelium began to fragment into bacillary cells after 3 days of incubation; aerial mycelium appeared after 5 days of incubation; substrate and aerial mycelia fragmented after 14 days of incubation into rods of varying lengths which were smooth and measured 1.5-10 (or longer)×0.6-0.9 μm. 
     The cell wall contained meso-diaminopimelic acid but not diagnostic sugars. The cell wall contained no mycolates of any kind. 
     The new culture is characterized by gram-positive reaction, non-acid-fastness, black or greyish colonies, black or greyish colony reverse, black soluble pigment on some media and fragmentation of both aerial and substrate mycelia. These features plus the presence of mesodiaminopimelic acid in the cell wall but the absence of diagnostic sugars and mycolates place the new culture in the genus Nocardiopsis. 
     The biochemical and physiological properties distinguishing the new culture from Nocardia uniformis subsp. tsuyamanesis are the decomposition of tyrosine; hydrolysis of esculin and hippurate; growth at 21° and 37° C. survival at 50° C. for 8 hours; H 2  S production; growth in Jensen&#39;s cellulose broth; coagulation and peptonization of milk; nitrate reduction; resistance to lysozyme; acid production after utilization of adonitol, inositol, α-methyl-d-glucoside and ribose. The new culture differs from Nocardiopsis dassonvillei in hydrolysis of esculin; growth at 21° and 37° C.; H 2  S production, nitrate reduction; decomposition of adenine, hypoxanthine tyrosine and xanthine; acid production from adonitol, inositol, lactose, melibiose, α-methyl-d-glucoside and raffinose; and utilization of adonitol, erythritol, inositol, melibiose and sorbitol. 
     The new culture was considered to be a new species of Nocardiopsis and was designated as Nocardiopsis atra Huang sp. nov. 
     Cultivation of Nocrdiopsis atra Huang sp. nov. ATCC 31511 preferably takes place in aqueous nutrient media at a temperature of 24-36° C. and under submerged aerobic conditions with agitation. Nutrient media which are useful for such purposes include a source of assimilable carbon such as sugars, starches and glycerol; a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cottonseed meal, peanut meal, wheat gluten, soy flour, meat meal and fish meal. A source of growth substances such as grain solubles and yeast extract as well as salts such as sodium chloride and calcium carbonate and trace elements such as iron, zinc, cobalt and maganese may also be utilized with advantageous results. If excessive foaming is encountered during fermentation, antifoam agents such as vegetable oil or silicones may be added to the fermentation medium. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of about 1/2 to 2 volumes of free air per volume of broth per minute. Agitation may be maintained by means of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth. 
     Inoculum for the preparation of the antibiotic may be obtained by employing growth from a slant of the culture. The growth may be used to inoculate either shake flasks or inoculum tanks or the inoculum tanks may be seeded from the shake flasks. Growth in shaken flasks will generally have reached its maximum in 2 to 4 days whereas inoculum in submerged inoculum tanks will usually be at the most favorable period in 1.5-3 days. Substantial antibiotic activity is obtained in the final fermentor stage in approximately 2 to 5 days. 
     The process of antibiotic production is conveniently followed during fermentation by biological assay of the broth employing a sensitive strain of Comamonas terrigena ATTC 8461 or Micrococcus lutenus. Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with broth is used as a measure of antibiotic potency. 
     The antibiotic produced by Nocardiopsis atra Huang sp. nov. ATCC 31511 was recovered from the filtered fermentation broth and purified by a combination of carbon absorption and elution and column chromatography. The antibiotic was established to be identical with previously reported nocardicin A (U.S. Pat. No. 3,923,977) by comparison with the published physicochemical data and side by side comparisons with nocardicin A obtained from the fermentation broth of known producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806. 
    
    
     EXAMPLE 1 
     A sterile aqueous medium having the following composition is prepared: 
     
         ______________________________________Ingredient           Grams/liter______________________________________Glucose              25Corn steep liquor    15Distillers&#39; solubles 10Cottonseed meal      5Cobalt chloride      0.01Calcium carbonate    3______________________________________ 
    
     Cells from a slant culture of Nocardiopsis atra ATCC 31511 are transferred to each of a number of 300 ml shake flasks each containing 40 ml of the above medium and shaken for 3 to 4 days at 28° C. 
     Aliquots sufficient to provide a 5% v/v inoculum are transferred to fermentors each containing two liters of the above described sterile medium. The temperature is maintained at 30° C. The broth is stirred at 200 r.p.m. and aerated at the rate of about one volume of air per volume of broth per minute. The fermentation is maintained until substantial antibiotic activity is obtained (3-4 days). 
     Nocardicin A may be recovered and separated by the methods described in U.S. Pat. No. 3,923,977. 
     EXAMPLE II 
     The method of Example I may be repeated with comparable results employing a fermentation medium of the following composition: 
     
         ______________________________________Ingredient           Grams/liter______________________________________Glucose              30Corn steep liquor    6Glycine              3Cottonseed meal      6DL-methionine        2Calcium carbonate    5______________________________________ 
    
     EXAMPLE III 
     The method of Example I may be repeated with comparable results employing a fermentation medium of the following composition: 
     
         ______________________________________Ingredient           Grams/liter______________________________________Glucose              1Dextrin              24Yeast extract        5Beef extract         3Polypeptone          5Calcium carbonate    4______________________________________

Technology Classification (CPC): 2