P R O D U C T I O K O F T R O P O L O S E BY X PSE UDOA!,?OnilS G. D. LIXDBERG and J. hI. LARKIS Departnieiit of Pleiti Pathology aitd C r o p Physiology, Agricultiiral Experz?iie~ifStation atid Departnient of dfzcrobiology, respectiLely, Loiizsiaiia State L-mi ersity, B ~ t o i iR o u g e , Louisiana 70808
H.-4.ITHALEY Injectioiis Disease Research, T h e Gpioliii Coinpmty, Kalamaroo, N i c h i g a n ,$9001 !LBSTRACT.--8 bacterium n i t h strong antimicrobial activity n-as characterized as a member of the genus Pseudonionas. The mass spectroscopy spectrum of the crystalline antibiotic obtained from the Pseudomonas filtrate was identical t o that of tropolone (l),as m-ere the uv, ir, pmr and cmr spectra.
Dewar (2) reported the structure of stipitatic acid, a metaboliye of Peizicillium stipitatztm. He suggested that stipitatic acid and colchicine, a natural product of certain Liliaceae, n ere members of a new system of “nonbenzenoid aromatic compounds.” He then predicted the parent cycloheptatrienolone and proposed the term tropolone for it. Several other tropolones are produced by fungi (3) besides DeTvar’s stipitatic acid. Other tropolones that have been reported to occur in nature are those prepent in western red cedar (5) of which the isopropyl tropolone, p-thujaplicin (4-isopropyltropolone), is probably the best knou-n (7). Such tropolones presuniably are responsible for the durability of red cedar. X bacterium (Pseudomonas ATCC 31099) with strong antimicrobial activity was found among colonies of Helmiitthosporium cyizodoiatis Marig. isolated from bermuda grass (Cynodoiz dactylon (L.) Pers.). This report is concerned with the identification of tropolone as the antibiotic agent produced by the Pseudomonas. Tropolone, 2-hydroxy-2,4,G-cycloheptatriene-l-one(1), seems not to have been reported from nature (5, G), but the antibacterial activity of tropolone has been reported (6).
EXPERIMEXTAL AKD RESULTS PRODCCTIOK OF Ah-TIBIOTIC.-The Pseudomoitas and its antimicrobial activity were first observed on potato dextrose agar (300 g peeled. diced potatoes, 23 g dextrose, and 15 g Difco agar in 2 liters of distilled water). Production trials of the antibiotic in liquid culture, therefore, were first made in potato dextrose broth (PDB, ingredients were the same as above except that the agar was deleted). The Pseudomonas v a s seeded to 600 ml of PDB in 2 liter Erlenmeyer flasks, placed under fluorescent light for 72 hrs and thereafter incubated without added light for 11 days at 22’. Stock cultures of the Pseudomonas were maintained by lyophilization or as a suspension in sterile distilled n-ater at Go. ISOLATIOK OF TROPOLONE (1) .-Eight liters of concentrated (ca 4 fold) fermen592
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tation filtrate of the Psezitiomorias, sent from the laboratories of Louisiana State University to the Upjolin Company. were used for the isolation and identification of tropolone as the antibacterial component. dfter adju-tment to p H 3 n i t h 6 -Y HCl, the fermentation concentrate i\a. extracted tu-ice TI itli four liter portions of dichloroniethane. These combined extracts contained essentially all the antimicrobial actix-it>-as determined by paper di-c-agar diffusion a=ay again-t Pciricilliitw oxaIicu?u. Concentraticn of these extracts 111 i o c i ~ ogave 3.06 g of crude extract aolids nhich n a - purified by c1iromatograph;i- over a 30 g column (1.9x 43 em) of silica gel-GO (E. Aierck =TT34) prepared in dichloroniethane. Dichloromethane containing 3°C metbanol ( Y v) eluted the antibiotic a< s h o i ~11 b j bioa-aj-. The chomatographic product appeared as a -ingle component. Ri 0 85. by tlc on AIS-polygram si1 S-HK (Brinkman 1nstrume:ita.. Inc.) developed in dichloromethane-methanol (9 :1 v v) and bioautographed on anp of several microorganism.: BaciIlzis siibtilis. Sarcina littea, Sacclrarontyces pastoriati its. Concentration of the active fraction3 gave an unn eighed dark, gummy solid n liich resiqted further purification by d i c a gel chromatography. -4portion of the chromatographic product n-as further purified by auhlimation under reduced pressure: a dry ice cooled, cold finger trap was used to collect the colorless qublimate. A portion of this ,ubliniate, n hen crystallized from aqueous acetone, produced colorless plate.. nip 30-31O. The crystalline antibiotic obtained from the Psezrdomoitas fermentation n as identified as tropolone first by mas3 spectroscopy. A molecular ion at Ai+122.0372 (C7HsO2= 122.0368) TI as obtained n hich displayed strong fragmentation peaks at 94.0418 (C6H60=94.0418).66 and 155. The spectrum n a s identical to tllat of tropolone (1). UT-,ir, pnir and cmr ipectra nere either identical to that reported for tropolone or identical to that obtained from a known sample of tropolone.' TABLE 1. Additional characteristics of the organism. Characteristics
1
Reaction
-
..I
S i t r a t e reduction.. . . B-galactosidase. . . .Irginine dihydrolase. . . . . . Lysine decarboxylase. . I Ornithine decarboxylase. . . , Citrate utilization. . . .. H?3 production. Crease production Phenylalanine deaminase Indole production. . . . T-oges-Proskauer (acetoin) . . l l a l o n a t e ut iliz 2.t i o n , Gelatin h>--drol:-sis.. .. Esculin h:--drol>-sis. . . '
'
-
-
~
-
-
-
THE BACTERIK~I.-The bacteriuni is a Gram negative. straight rod. and iq motile n ith approximately six polar flagella. h bright red-to-orange pigmeni i q produced in potato dextrose broth standing culture under fluorescent light Its metabolism is respiratory; it doe3 not produce acid from the carbohydrate. glucose. rlianinose, sucrose, melibiose. amygadalin or ar2bino.e. or from the alcohols mannitol, ino-itol, or sorbitol (4). It is catalase and oxidase positi7-e. These 'Aldrich Chemical Company, =T8, 9iO-2.
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characteristics warrant its placement in the genus Pseudomonas. Additional characteristics of the organism were determined either with the RIinitek (BBL, Cockeysville, MD) or Path-o-teck (Warner-Chilcott) systems and are given in table 1. ACKIiOWLEDGMEIiT The authors wish t o thank L. Bacznj-skyj for the ms determination and D. R . Wait for technical assistance. Recetoed 15 January 1980.
LITERATCRE CITED 1. H. Bud;ikiewicz, C. Djerassi, and D . H. Williams, "Mass Spectroscopy of Organic Compounds, Holden-Day, San Francisco, 1967, p. 540. 2. 11.J . S. Dewar, ATature (London), 155, 50 (1945). 3. B. I-.Divekar, H. Raistrick, T. A . Dobson, and L. C. 1-ining, Canad. J . Ciieni., 43, 1835 (1965). 4. R. Hugh and E. Leifson, J . B u c f e r z o l . , 66, 24 (1953). 5. P. L. Pauson, Ciienz. Ren., 55, 9 (1955). 6. T. J . Trust, Antimicrobial A g e n t s and Chemotherapy, 7 , 500 (19i5). 7 . T. J. Trust and R. W. Coombs, Camdzan Jour. Jl?crobzology, 19, 1341 (1973).
