SUMMARY OF THE INVENTION 
This invention provides two new strains of Actinoplanes missouriensis which 
operate together to cosynthesize a new glycopeptide antibiotic called 
CUC/CSV. In addition, each strain can be used separately to bioconvert 
actaplanin factor A to the new CUC/CSV antibiotic. Antibiotic CUC/CSV has 
formula 1: 
##STR1## 
wherein 
R is L-ristosaminyl; 
R.sub.1 is the disaccharide mannosyl-glycosyl; and 
R.sub.2 and R.sub.3 are mannosyl. CUC/CSV and its salts, particularly the 
pharmaceutically acceptable salts, are useful new antibiotics. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention relates to two Actinoplanes missouriensis strains which 
together co-synthesize a new glycopeptide antibiotic and which 
independently are useful in certain bioconversion reactions. More 
particularly, the Actinoplanes missouriensis strains of this invention 
have the ability to co-synthesize the formula 1 glycopeptide antibiotic 
which has been designated CUC/CSV. The formula 1 compound is the subject 
of a co-pending application of LaVerne D. Boeck, Gladys M. Clem, Charles 
L. Hershberger, Marie T. Anderson and Karl H. Michel entitled GLYCOPEPTIDE 
COMPOUND CUC/CSV AND PROCESS FOR ITS PRODUCTION, Ser. No. 544,338, now 
U.S. Pat. No. 4,537,715, filed herewith this even date. 
The novel Actinoplanes missouriensis strains of this invention have been 
arbitrarily designated CUC 014 and CSV 558. Cultures CSV 558 and CUC 014 
are strains obtained by chemical mutagenesis of Actinoplanes missouriensis 
ATCC 31683, a strain which produces the glycopeptide antibiotic 
actaplanin. A. missouriensis ATCC 31683 in turn was derived by a series of 
mutations from the wild type A. missouriensis ATCC 23342 strain. 
The glycopeptide antibiotic actaplanin (which is also known as antibiotic 
A4696) and the A. missouriensis strain ATCC 23342 are described by Hamill 
et al. in U.S. Pat. Nos. 3,952,095 and 4,115,552. The actaplanin-producing 
A. missouriensis strains ATCC 32680, ATCC 31682 and ATCC 31683 are 
described by Debono et al. in U.S. Pat. Nos. 4,322,406 and 4,375,513. 
The novel A. missouriensis strains CSV 558 and CUC 014 of this invention do 
not produce actaplanin in any measurable quantity. Instead, strain CUC 014 
secretes an intermediate which is then converted by strain CSV 558 to give 
the formula 1 compound. 
CHARACTERIZATION OF CULTURES CSV 558 AND CUC 014 
The following taxonomic study of the new CSV 558 and CUC 014 cultures were 
made together with the parent ATCC 31683 and wild type ATCC 23342 strains 
in order to provide a complete evaluation. 
Cultures CSV 558 and CUC 014 are also classified as strains of Actinoplanes 
missouriensis. In classifying the cultures as strains of A. missouriensis, 
the methods recommended for the International Streptomyces Project (ISP) 
for the characterization of Streptomyces species [E. B. Shirling and D. 
Gottlieb, "Methods of Characterization of Streptomyces species," Int. J. 
Syst. Bacteriol. 16(3), 313-340 (1966)] and certain supplementary tests 
were followed. 
Carbon utilization was determined with ISP No. 9 basal medium to which 
filter-sterilized carbon sources were added to equal a final concentration 
of 1.0 percent. Plates were incubated at 30.degree. C. and read after 14 
days. 
Melanoid pigment production (chromogenicity) was determined with ISP No. 1 
(tryptone-yeast extract broth), ISP No. 6 (peptone-yeast extract iron 
agar), ISP No. 7 (tyrosine agar) and modified ISP No. 7 (which lacks 
tyrosine). 
Hydrogen sulfide production was checked by inserting test strips (Bacto 
H.sub.2 S test strips) in ISP No. 6 agar slants. 
Starch hydrolysis was determined by testing for the presence of starch with 
iodine on ISP No. 4 (inorganic salts-starch agar) plates (D. J. Blazevic 
and G. M. Ederer, "Principles of Biochemical Tests in Diagnostic 
Microbiology," John Wiley and Sons, Inc., New York, 1975, p. 136). 
Temperature range, NaCl and sucrose tolerances, pH range, and antibiotic 
sensitivity were determined using ISP No. 2 (yeast-malt extract agar) 
plates. Plates were incubated at 30.degree. C. for 14 days. The 
temperatures tested were 5.degree., 10.degree., 15.degree., 20.degree., 
25.degree., 30.degree., 37.degree., 40.degree., 45.degree., 50.degree. and 
55.degree. C. 
NaCl tolerance was measured by adding NaCl to the agar to equal: 0, 2, 4, 
6, 8, 10, and 12%; sucrose tolerance was measured by adding sucrose to 
equal: 0, 2, 4, 6, 8, 10, 12, 15 and 20%. The pH range was determined 
using the following buffers at 0.05M: citric acid, pH 3, 4, 5; 
2(N-morpholino)ethanesulfonic acid (MES, Sigma Chemical Co.), pH 6; 
3(N-morpholino)propanesulfonic acid (MOPS, Aldrich Chemical Co.), pH 7; 
N-[tris-(hydroxymethyl)methyl]glycine (Tricine, CalBiochem), pH 8; 
2-(cyclohexylamino)ethanesulfonic acid (CHES, P-L Biochemicals, Inc.), pH 
8.5, 9.0, and 9.5; 3-cyclohexylamino-1,1-propanesulfonic acid (CAPS), pH 
10.0 and 10.5. The pH values of the agar plates were measured with a flat 
surface electrode prior to inoculation, and this pH was taken as the 
correct value. Some of the buffers failed to hold their adjusted pH. 
Toxicity was tested by adjusting all the buffers to pH 7.0 and checking 
for growth. All the mutants are sensitive to citric acid, but the 
wild-type strain was not sensitive to citric acid. 
Antibiotic sensitivity was determined using sensitivity discs padded on the 
surface of seeded agar plates. The following antibiotics were used: 
cephalothin (sodium) 30 .mu.g, erythromycin (estolate) 15 .mu.g, 
chloromycetin 30 .mu.g, novobiocin 30 .mu.g, penicillin (G) 10 units, 
rifampin 5 .mu.g, streptomycin 10 .mu.g, tetracycline 30 .mu.g and 
vancomycin 30 .mu.g. 
For enzyme assays the methods of Blazevic and Ederer were followed (D. J. 
Blazevic and G. M. Ederer, "Principles of Biochemical Tests in Diagnostic 
Microbiology," John Wiley and Sons, Inc., New York, 1975). 
Color names were assigned using ISCC-NBC Centroid Color Charts, standard 
sample No. 2106 (K. L. Kelly and D. B. Judd, U.S. Dept. of Commerce, 
National Bureau of Standards, Washington, D.C. 20234, 1976). 
Lysozyme resistance and decomposition of casein, esculin, hypoxanthine, 
tyrosine, and xanthine were measured by the procedure of Berg [David Berg, 
"Laboratory Identification of Clinically Important Aerobic Actinomycetes," 
Appl. Microbiol. 25:665-681 (1973)]. 
CULTURAL CHARACTERISTICS 
Growth and cultural features of the four strains are similar. Only 
substrate, or primary, mycelia are produced. Neither secondary mycelia nor 
sporangia were observed on any of the fourteen agar-plating media used in 
the study. Sporangical descriptions of the original isolate were 
previously reported by Dr. John N. Couch, University of North Carolina. 
The mutant strains (ATCC 31683, CSV 558 and CUC 014) produce a yellowish 
gray colony and are readily distinguished from the wild type (ATCC 23342), 
which produces a distinctive orange pigmentation ranging from moderate to 
brownish-orange to strong orange, depending on the medium. The mutant 
strains are indistinguishable on most agar plating media. Cultural 
differences between these strains are as follows: CUC 014 produces a 
strong reddish-brown soluble pigment on certain media. This is observed on 
yeast-malt extract agar (ISP No. 2), glycerol-asparagine agar (ISP No. 5) 
and tyrosine agar (ISP No. 7). CSV 558 is unable to grow on tomato 
paste-oatmeal agar (TPO), while ATCC 31683 and CUC 014 grow very well. 
This information is summarized in Table I. 
TABLE I 
______________________________________ 
Cultural characteristics of Actinoplanes missouriensis 
Strains.sup.a, b. 
______________________________________ 
ATCC 23342 ATCC 31683 CSV 558 CUC 014 
______________________________________ 
ISP 2 
G good good good good 
R 53.m.O 90.gy.Y 90.gy.y 90.gy.Y 
Am none none none none 
Sp none none none r brown 
ISP 3 
G poor good fair fair 
R 76.1.yBr 93.yGray 93.yGray 
93.yGray 
Am none none none none 
Sp none none none none 
ISP 4 
G abundant good fair fair 
R 50.s.O 79.1.gy.yBr 
93.yGray 
93.yGray 
Am none none none none 
Sp none none none none 
ISP 5 
G fair fair (dull) 
good good 
R 68.s.OY 90.gy.Y 90.gy.Y 63.1.brGy 
Am none none none none 
Sp none none none 1.r. gray 
ISP 7 
G poor fair good good 
R 54.brO 80.gy.yBr 80.gy.yBr 
80.gy.yBr 
Am none none none none 
Sp reddish br light brown 
none d.r. brown 
Bennetts 
G abundant abundant good good 
R 53.m.O 93.yGray 93.yGray 
93.yGray 
Am none none none none 
Sp none none none 1.r. brown 
calcium 
malate 
G good (shiny) 
good (shiny) 
fair fair 
R 50.s.O 93.yGray 93.yGray 
93.yGray 
Am none none none none 
Sp none none none none 
Czapek's 
G good abundant good good 
R 71.m.OY 93.yGray 93.yGray 
93.yGray 
Am none none none none 
Sp none none none none 
Glucose 
Aspara- 
gine 
G good good none none 
R 71.m.OY 93.yGray none none 
Am none none none none 
Sp none none none none 
TPO 
G good good none abundant 
R 55.s.Br 91.d.gy.Y none 91.d.gy.Y 
Am none none none none 
Sp faint rBr none none none 
Anio- 
Hensens 
G poor fair fair fair 
R 70.1.OY 91.d.gy.Y 93.yGray 
93.yGray 
Am none none none none 
Sp none none none none 
53H 
medium.sup.c 
G fair good fair fair 
R 54.brO 91.d.gy.Y 93.yGray 
93.yGray 
Am none none none none 
Sp none none none 1.r. brown 
Czapek's 
Peptone 
G abundant abundant abundant 
abundant 
R 54.brO 90.gy.Y 90.gy.Y 90.gy.Y 
Am none none none none 
Sp none none none none 
______________________________________ 
.sup.a G = growth; R = reverse; Am = aerial mycelium; Sp = soluble pigmen 
.sup.b Strains incubated 14 days at 30.degree. C. 
.sup.c Medium 53H has the following composition (pH adjusted to 7.0): 
Ingredient 
Amount 
Yeast extract 
2.0 g 
CaCO.sub.3 
3.0 g 
Na.sub.2 S.sub.2 O.sub.3.5H.sub.2 O 
0.5 g 
V-8 Juice 200.0 ml 
Deionized H.sub.2 O 
800.0 ml 
Agar 20.0 g 
Morphological Characteristics 
None of the cultures produce aerial hyphae or sporangia. Consequently, the 
only morphological comparisons were those made with substrate mycelia. The 
morphology of the primary hyphae is indistinguishable. 
Physiological Characteristics 
Cultures ATCC 23342, ATCC 31683, CSV 558 and CUC 014 were tested for 
sensitivity to nine antibiotics. They exhibited an identical pattern of 
sensitivity. Resistance was shown to novobiocin and penicillin G. 
Sensitivity was shown to cephalotin, chloromycetin, erythromycin, 
rifampin, streptomycin, tetracycline, and vancomycin. The test results are 
summarized in Table II. 
TABLE II 
______________________________________ 
Antibiotic Sensitivity of Actinoplanes missouriensis strains. 
ATCC ATCC 
Antibiotic 
Conc. 23342 31683 CSV 558 
CUC 014 
______________________________________ 
Cephalothin 
30 .mu.g + + + + 
Chloromycetin 
30 .mu.g + + + + 
Erythromycin 
15 .mu.g + + + + 
Novobiocin 
30 .mu.g - - - - 
Penicillin G 
10 units - - - - 
Rifampin 5 .mu.g + + + + 
Streptomycin 
10 .mu.g + + + + 
Tetracycline 
30 .mu.g + + + + 
Vancomycin 
30 .mu.g + + + + 
______________________________________ 
+ = sensitive (zone of inhibition) 
- = resistant (no zone inhibition) 
Carbon utilization is identical for CSV 558 and CUC 014. ATCC 23342 differs 
in salicin utilization, and ATCC 31683 differs in ribose utilization. 
These test results are summarized in Table III. 
TABLE III 
______________________________________ 
Carbon Utilization of Actinoplanes missouriensis Strains 
ATCC ATCC 
Carbon Source 
23342 31683 CSV 558 
CUC 014 
______________________________________ 
no carbon - - - - 
glucose ++ ++ ++ ++ 
L-arabinose 
++ ++ ++ ++ 
cellobiose ++ ++ ++ ++ 
D-fructose ++ ++ ++ ++ 
D-galactose 
++ ++ ++ ++ 
i-inositol - - - - 
D-mannitol ++ + ++ ++ 
melibiose + + + ++ 
raffinose - - - - 
D-rhamnose ++ + ++ ++ 
D-ribose - (+) - - 
salicin + - - - 
sucrose ++ (+) ++ ++ 
D-xylose ++ ++ ++ ++ 
______________________________________ 
++ = equal to or &gt;glucose control; positive utilization 
+ = &lt;glucose control, &gt;no carbon control; positive utilization 
(+) = growth questionable; doubtful utilization 
- = no growth; negative utilization 
Physiological differences between CSV 558 and CUC 104 are: tolerance to 
sucrose, temperature range, and the degree of gelatin liquefaction. CSV 
558 tolerates up to 12% sucrose, grows in a temperature range of 
5.degree.-30.degree. C., and liquefies gelatin about 50% after 14 days 
incubation 30.degree. C. CUC 014 tolerates up to 15% sucrose, grows in a 
temperature range of 5.degree.-37.degree. C., and liquefies gelatin about 
80% after 14 days incubation at 30.degree. C. These differences, as well 
as additional physiological characteristics for all four strains, are 
given in Table IV. 
TABLE IV 
______________________________________ 
Additional Physiological Characteristics 
of Actinoplanes missouriensis Strains 
ATCC ATCC 
Characteristic 
23342 31683 CSV 558 
CUC 014 
______________________________________ 
casein + + + + 
decomposition 
catalase reaction 
+ + + + 
esculin decomposition 
+ + + + 
gelatin +(40%) +(100%) +(50%) +(80%) 
liquefaction 
H.sub.2 S production 
tr tr tr tr 
hypoxanthine decomp. 
- - - - 
lysozyme resistance 
- - - - 
melanoid pigments 
- - - - 
NaCl tolerance 
&lt;2% &lt;2% &lt;2% &lt;2% 
nitrate reduction 
- - - - 
pH growth range 
6-7 6-8.4 6-8 6-8 
phosphatase + + + + 
production 
skim milk - - - - 
starch hydrolysis 
+ - + + 
sucrose tolerance 
15% 20% 12% 15% 
temp. growth range 
15-37.degree. C. 
5-40.degree. C. 
5-30.degree. C. 
5-37.degree. C. 
tyrosine - + - - 
decomposition 
urease production 
+ - + + 
xanthine - - - - 
decomposition 
______________________________________ 
The Actinoplanes missouriensis strains CUC 014 and CSV 558 of this 
invention have been deposited and made part of the stock culture 
collection of the Northern Regional Research Center, Agricultural 
Research, North Central Region, 1815 North University Street, Peoria, 
Ill., 61604, from which they are available to the public under the 
accession numbers NRRL 15647 (CUC 014) and NRRL 15646 (CSV 558). 
As is true with other organisms, the characteristics of the Actinoplanes 
missouriensis NRRL 15646 and NRRL 15647 strains are subject to variation. 
For example, recombinants, mutants or artificial variants of the strains 
may be obtained by treatment with various known physical and chemical 
mutagens, such as ultraviolet light, X-rays, gamma rays, and 
N-methyl-N'-nitro-N-nitrosoquanidine. All natural and artificial variants, 
mutants and recombinants of the A. missouriensis NRRL 15646 and NRRL 15647 
strains which retain the ability to cosynthesize CUC/CSV may be used in 
this invention. 
Cosynthesis of antibiotic CUC/CSV is achieved by fermenting the secretor 
culture CUC 014 (NRRL 15647) and the converter culture CSV 558 (NRRL 
15646) together under submerged aerobic conditions in a suitable culture 
medium until substantial antibiotic activity is produced. When fermented 
separately, neither culture CUC 014 nor CSV 558 produces antibiotic 
activity. 
As will be appreciated by those in the art, the culture media used to grow 
the cosynthesizing A. missouriensis strains can be any one of a number of 
media (see, for example, U.S. Pat. No. 4,322,406 for a description of the 
media variations useful for the parent A. missouriensis ATCC 31683 
strain). When cosynthesizing antibiotic CUC/CSV, the fermentation can be 
carried out by inoculating a common medium with the two cultures 
simultaneously. Alternatively, a growing culture of A. missouriensis CUC 
014 can be established, and then combined with a growing culture of the 
CSV 558 strain. 
Antibiotic production can be followed during the fermentation by testing 
samples of the broth against organisms known to be sensitive to this 
antibiotic. One useful assay organism is Bacillus subtilis. The bioassay 
is conveniently performed by paper-disc assay on agar plates. In addition, 
antibiotic production can be monitored by high performance liquid 
chromatography (HPLC) with UV detection. 
Following its production under submerged aerobic fermentation conditions, 
antibiotic CUC/CSV can be recovered from the fermentation medium by 
methods recognized in the art, e.g. adsorptive and extractive procedures. 
Antibiotic CUC/CSV can also be prepared by bioconversion of actaplanin 
factor A using either culture CUC 014 or culture CSV 558 (see the 
co-pending application of Gladys M. Clem, LaVerne D. Boeck, Marie T. 
Anderson, and Karl H. Michel entitled GLYCOPEPTIDE BIOCONVERSION PRODUCTS, 
Ser. No. 544,332, now abandoned, filed herewith this even date). 
Antibiotic CUC/CSV inhibits the growth of pathogenic bacteria, especially 
gram-positive bacteria. Table V summarizes the minimal and inhibitory 
concentrations (MIC's) at which CUC/CSV inhibits certain organisms, as 
determined by standard agar-dilution assays. 
TABLE V 
______________________________________ 
In Vitro Activity of CUC/CSV 
Organism MIC (mg/ml) 
______________________________________ 
Staphylococcus aureus NRRL B313 
8 
Staphylococcus aureus V41 
8 
Staphylococcus aureus X400 
16 
Staphylococcus aureus S13E 
8 
Staphylococcus epidermidis EP11 
16 
Staphylococcus epidermidis 222 
8 
Streptococcus pneumoniae Park 1 
0.5 
Streptococcus faecium ATCC 9790 
4 
Streptococcus sp. group D 9960 
4 
______________________________________ 
Antibiotic CUC/CSV also inhibits the growth of anaerobic bacteria. Table VI 
summarizes the susceptibility of various anaerobic isolates to CUC/CSV. 
TABLE VI 
______________________________________ 
Susceptibility of Anaerobic Bacterial Isolates to CUC/CSV 
Anaerobic Bacteria MIC (.mu.g/ml).sup.a 
______________________________________ 
Clostridium difficile 2994 
1 
Clostridium perfringens 81 
4 
Clostridium septicum 1128 
4 
Eubacterium aerofaciens 1235 
2 
Peptococcus asaccharolyticus 1302 
4 
Peptococcus prevoti 1281 
8 
Peptostreptococcus anaerobius 1428 
2 
Peptostreptococcus intermedium 1264 
4 
Propionibacterium acnes 79 
1 
Bacteroides fragilis 111 
&gt;128 
Bacteroides fragilis 1877 
&gt;128 
Bacteroides fragilis 1936B 
&gt;128 
Bacteroides thetaiotaomicron 1438 
&gt;128 
Bacteroides melaninogenicus 1856/28 
&gt;128 
Bacteroides melaninogenicus 2736 
16 
Bacteroides vulgatis 1211 
&gt;128 
Bacteroides corrodens 1874 
&gt;128 
Fusobacterium symbiosum 1470 
&gt;128 
Fusobacterium necrophorum 6054A 
2 
______________________________________ 
.sup.a MIC's were determined by the agardilution method; endpoints were 
read after 24hours incubation. 
CUC/CSV has also shown in vivo antimicrobial activity against 
experimentally-induced bacterial administered to experimentally infected 
mice, the activity observed was measured as an ED.sub.50 value [effective 
dose in mg/kg to protect 50% of the test animals: see Warren Wick et al., 
J. Bacteriol. 81, 233-235 (1961)]. ED.sub.50 values observed for CUC/CSV 
are given in Table VII. 
TABLE VII 
______________________________________ 
ED.sub.50 Values for CUC/CSV in Mice 
Infecting Organism 
ED.sub.50 (mg/kg/2).sup.a 
______________________________________ 
Staphylococcus aureus 
1.59 
Streptococcus pyogenes 
1.09 
Streptococcus pneumoniae 
0.84 
______________________________________ 
.sup.a administered subcutaneously 1 and 4 hours postinfection 
The following examples illustrate this invention.

EXAMPLE 1 
Production of Antibiotic CUC/CSV by Cofermentation of Cultures CUC 014 and 
CSV 558 
A. Shake-Flask Fermentation of Cultures CUC 014 and CSV 558 
A lyophilized pellet of Actinoplanes missouriensis strain CUC 014 (NRRL 
15647) or strain CSV 558 (NRRL 15646) is dissolved in 1-2 ml of 
sterilized water. This suspension is used to inoculate an agar slant 
having the following composition: 
______________________________________ 
Ingredient Amount (%) 
______________________________________ 
Precooked Oatmeal 6.0 
Yeast 0.25 
K.sub.2 HPO.sub.4 0.1 
Czapek Mineral Stock.sup.a 
0.5 
Agar.sup.b 2.5 
Deionized H.sub.2 O 
q.s. to 100% 
Unadjusted pH = 6.2; adjust to pH 7.3 with 5N NaOH; 
after sterilization pH = 6.7. 
______________________________________ 
.sup.a Czapek Mineral Stock: 
Ingredient 
Amount (%) 
KCl 10.0 
MgSO.sub.4.7 H.sub.2 O 
10.0 
FeSO.sub.4.7 H.sub.2 O 
0.2 (dissolved in 2 ml of Conc. HCl) 
Deionized 
q.s. to 100% 
water 
.sup.b Difco Laboratories 
The inoculated slant is incubated at 30.degree. C. for about eight to ten 
days. The mature slant culture is scraped with the serrated edge of a 
sterile loop to mascerate and loosen the mycelial mat. About one-fourth of 
the loosened mat is used to inoculate 50 ml of a vegetative medium having 
the following composition: 
______________________________________ 
Ingredient Amount (%) 
______________________________________ 
Glucose 2.0 
Tryptone.sup.a 0.5 
Yeast Extract 0.5 
Tap H.sub.2 O q.s. to 100% 
Before sterilization, pH = 6.5; adjust to pH 7.2 with 5N 
NaOH; after sterilization, pH = 6.9; 
______________________________________ 
.sup.a Bacto Tryptone, Difco 
The inoculated vegetative medium is incubated in a 250-ml Erlenmeyer flask 
at 30.degree. C. for about 72 hours on a rotary shaker with a two-inch 
throw at 250 RPM. 
Vegetative cultures can be initiated with agar-slant cultures, with 
lyophilized pellets of the culture (one lyophile per 50 ml of media in a 
250-ml flask) and with cultures preserved in liquid nitrogen (0.8% 
inoculum). 
Incubated vegetative medium (5%, volume/volume) is used to inoculate 50 ml 
of a production medium having the following composition: 
______________________________________ 
Ingredient Amount (%) 
______________________________________ 
Glucose 2.5 
Corn Starch 3.5 
Blackstrap Molasses 
1.5 
Glycerol 1.5 
Yeast 2.0 
K.sub.2 HPO.sub.4 
0.05 
(NH.sub.4).sub.2 SO.sub.4 
0.025 
CaCO.sub.3 0.2 
Tap H.sub.2 O q.s. to 100% 
Before sterilization pH = 6.5; adjust to 6.8; 
after sterilization pH = 6.5. 
______________________________________ 
The inoculated production medium in incubated in a 250 ml Erlenmeyer flask 
at 30.degree. C. for 72 hours on a 2-inch rotary shaker at 250 RPM. 
B. Cosynthesis of Antibiotic CUC/CSV 
After cultures CUC 014 and CSV 558 have fermented for 72 hours separately, 
equal volumes of whole broth from each fermentation are combined 
aseptically in a sterile flask. The flasks are incubated at 30.degree. C. 
on a rotary shaker for an additional 96 hours. 
C. Assay for Antibiotic CUC/CSV 
Whole broth (adjusted to pH 10.5) is centrifuged. The supernatant is 
readjusted to pH 7.0. Samples thus prepared are assayed by a Bacillus 
subtilis plate assay and by thin-layer chromatography using silics-gel 
plates (Merck, pre-coated plastic sheets; silica gel 60, without 
fluorescent indicator) and an acetone:water:ammonia (160:40:1) solvent 
system. Detection way by bioautography using B. subtilis in a minimal 
growth medium and incubating plates at 37.degree. C. for about 18 hours. 
EXAMPLE 2 
Isolation of Antibiotic CUC/CSV 
Three lots of whole fermentation broth, prepared using procedures like that 
of Example 1, were combined (total volume=45 L.). This broth was 
centrifuged using a Cepa centrifuge. The mycelia were extracted twice with 
water which had been adjusted to pH 10.5 with sodium hydroxide. The 
extracts were combined (24 L.), adjusted to pH 7.0 with hydrochloric acid 
and applied to a column containing 4.0 L. of Diaion HP-20 (Mitsubishi 
Chemical Industries, Limited, Tokyo, Japan) at a flow rate of 160 
ml/minute. The column was washed successively with 8 L. of water and 12 L. 
of methanol:water (1:3) and then was eluted with 8 L. of methanol:water 
(1:1), 8 L. of methanol:water (3:1), and 20 L. of methanol, collecting 4-L 
fractions. Each fraction was analyzed for biological activity. The 
bioassay was performed by a paper-disc assay on agar plates seeded with 
Bacillus subtilis. Fractions containing the desired activity were 
combined, concentrated under reduced pressure and lyophilized to give 10.0 
g. of crude material. 
A portion of this material (0.5 g) was dissolved in 10 ml of methanol:waer 
(3:2) and filtered. The filtrate was applied to a 5.2-.times.41-cm 
Michel-Miller HPLPLC glass column packed with 590 ml of 25-40 micron 
Lichroprep RP-18 reversed-phase silica gel from MC/B Manufacturing 
Chemist, Inc., Cincinnati, OH. The column was eluted with (35:65) 
methanol:potassium dihydrogen phosphate buffer (0.05M adjusted to pH 3.5 
with phosphoric acid) at a rate of 10 ml/minute, collecting 20-ml 
fractions. The eluate was monitored at 280 nm using an Isco Model UA-5 UV 
monitor with a Type 6 optical unit (Instrumentation Specialties Co., 
Lincoln, NE). All fractions were analyzed by padding paper disks on agar 
plates containing a minimal media seeded with Bacillus subtilis. Fractions 
having the desired activity were combined, adjusted to pH 7.0 with sodium 
hydroxide and concentrated under reduced pressure. The concentrated pool 
(100 ml) was applied to a column packed with 90 ml of Diaion HP-20. The 
column was washed with 400 ml of water and then eluted with 
acetonitrile:water (4:1). The first eluate (29 ml) was discarded, and the 
next eluate (15 ml) was collected, concentrated under reduced pressure and 
lyophilized to give 27 mg of pure antibiotic CUC/CSV. CUC/CSV has the 
following characteristics: 
______________________________________ 
Elemental Analysis 
Calc..sup.a 
Found 
______________________________________ 
C--90 49.46 49.28 
H--98 5.63 4.35 
N--7 4.49 4.55 
O--41 38.80 39.82 (by difference) 
Cl--1 1.62 2.00 
______________________________________ 
.sup.a For C.sub.90 H.sub.98 N.sub.7 O.sub.41.12H.sub.2 O 
Ultraviolet Absorption (in methanol): 
.lambda..sub.max 278 nm, acid (.epsilon..about.17,000) 
.lambda..sub.max 277 nm, 361 nm, neutral (.epsilon..about.18,000, 9,000) 
.lambda..sub.max 295 nm, 340 nm, base (.epsilon..about.21,000, 14,500) 
Calculated on a molecular weight of 1200. The compound shows end-absorption 
at 230 nm. 
Solubility: soluble in dimethyl sulfoxide, dimethylformamide, 
acetonitrile:water, and alcohol:water mixtures. 
Mass Spectrometry (Fast Atom Bombardment): FAB MS indicates a molecular 
weight of 1968. 
EXAMPLE 3 
Preparation of CUC/CSV by Bioconversion of Actaplanin Factor A with Culture 
CSV 558 
A. The Bioconversion 
Actaplanin factor A (100 mg) is dissolved in water, sterilized by 
filtration, and added (final conc. of 0.3 mg/ml) to a five-day-old, 
one-liter fermentation of the convertor culture A. missouriensis CSV 558 
(NRRL 15646). The fermentation is incubated an additional 48 hours. The pH 
of the whole broth is adjusted to 10.5 with NaOH; the broth is 
centrifuged, and the centrate is neutralized with HCl. 
B. Isolation of CUC/CSV 
A bioconversion was carried out using the procedure of Sect. A. The broth 
was removed by filtration, and the mycelia were extracted with water at pH 
10.5. This extract (550 ml) was purified over a column packed with 100 ml 
of HP-20 as described in Example 2 to give a lyophilized crude product 
(190 mg). A portion of this product (100 mg), dissolved in 5 ml of 
CH.sub.3 CN:pyrOAc (36:64) at pH 3.6, was applied to a 300-ml glass column 
packed with Lichroprep RP-8 resin (25-40 .mu.m). The column was eluted 
with CH.sub.3 CN:0.05% pyrOAc (1:4) at pH 3.6 at a flow rate of 8 ml/min. 
Product was detected by UV absorbance at 280 nm, by B. subtilis bioassay 
and by analytical HPLC. Fractions containing the desired activity were 
combined, adjusted to pH 6.5 with N NaOH, then concentrated to remove 
CH.sub.3 CN. The resulting aqueous solution (50 ml) was applied to a 40-ml 
column filled with 12 ml of LP1-C18 resin in water. The column was washed 
with water (100 ml) to remove the salt, and the active material was eluted 
with CH.sub.3 CN:H.sub.2 O (7:3). The eluate was concentrated and 
lyophilized to give 10 mg of purified antibiotic CUC/CSV. 
EXAMPLE 4 
Preparation of CUC/CSV by Bioconversion of Actaplanin Factor A with Culture 
CUC 014 
Following the procedure of Example 3, but using culture CUC 014 (NRRL 
15647) instead of culture CSV 558, actaplanin factor A is converted to 
antibiotic CUC/CSV. 
EXAMPLE 5 
Analytical HPLC System For Antibiotic CUC/CSV 
Column: 4.6-.times.250-mm stainless steel 
Packing: Shandon ODS Hypersil-5 micron 
Solvent: CH.sub.3 CN:0.05M KH.sub.2 PO.sub.4 adjusted to pH 3.2 with 
H.sub.3 PO.sub.4 (21:79) 
Flow Rate: 1.0 ml/min. 
Detection: UV at 220 nm 
Chart speed: 20 cm/hr. 
Retention time: 9.3 minutes