Isolation and characterization; of α -santonin assimilating Pseudomonad

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1 J. Biosci., Vol. 4, Number 1, March 1982, pp Printed in India, Isolation and characterization; of α -santonin assimilating Pseudomonad U. M. X. SANGODKAR and S. MAVINKURVE Department of Microbiology, Centre of Post-graduate Instruction and Research University of Bombay, Panaji, Goa MS received 10 August 1981; revised 24 October 1981 Abstract. Pseudomonas cichorii strain S, isolated by soil enrichment technique, utilized santonin as the sole source of carbon, forming chromatographically destinguishable transformation products. One of the intermediary transformation products was identified as 1,2-dihydro α -santonin. Keywords. α -santonin. α -santonin; microbial transformation; Pseudomonas cichorii; 1, 2-dihydro Introduction α-santonin (santonin) (figure 1,1), a pharmaceutically important sesquiterpenoid, has recently assumed significance as a potential parent compound for antitumour drugs (Fujimoto et al., 1979). Its chemical conversions are being worked out extensively. The microbial transformation, though viewed as a useful tool for obtaining newer derivatives, has not yet yielded successful results, as santonin itself is not easily amenable to microbial attack (Fujimoto et al., 1979). The transforma- Figure 1. Structure of α-santonin (1)and its transformation products, 1-2-dihydro α-santonin (II). tion products which are detected by using mixed cultures are in small quantities and hence, pose difficulty during purification (Hikino et al., 1970). Resistance of santonin to microbial conversion led Fujimoto et al. (1979) to use chemical analogues for microbial transformation. We report our findings on the enrichment, isolation and chracterization of a single bacterial strain, capable of utilizing santonin as the sole source of carbon. 79

2 80 α-santonin-assimilating Pseudomonad Materials and methods Media Santonin medium used for enrichment, isolation and other growth studies, consisted of mineral salts medium (Mahtani and Mavinkurve, 1979) supplemented with 0.5% (w /v) α-santonin I.P. Santonin agar was prepared by solidifying the above medium with 2% bacto-agar (Difco). Media were sterilized at 121 C for 10 min. Enrichment, isolation and characterization Garden soil was enriched by regular additions of santonin (Lonsane et al., 1974). This soil was then used as inoculum in santonin medium. The enriched cultures thus obtained were inoculated on nutrient agar or santonin agar for isolation and purification of the organisms using santonin as the carbon source. The isolates were characterized morphologically and biochemically (Society of American Bacteriologists, 1957). The G+C content of the DNA was determined as reported by Mandel (1966) and cultures were further identified (Buchanan and Gibbons, 1974; Stanier et al., 1966 and Sands et al., 1970). Utilization and transformation of santonin The isolate capable of utilizing santonin was inoculated into the santonin medium and incubated on a rotary shaker at room temperature (28-32 C). The growth was followed turbidimetrically using a Klett Summerson Photoelectric Colorimeter. One ml aliquots of the culture broth were withdrawn at intervals and extracted twice with 0.5 ml chloroform. Individual chloroform extracts were analyzed by thin layer chromatography and for ultraviolet absorbance profiles. Characterization of one of the transformation products The culture was grown in 3 litres of santonin (0.3%) medium for 24 h. The broth, after removing the cells by centrifugation, was extracted with chloroform. The extract was concentrated by vacuum evaporation and the products were separated by multiple run ( 4) preparative thin layer chromatography using petroleum ether o C and ethyl acetate (9:1) solvent systems. The separated bands were eluted with ethyl acetate. The chromatographically pure product obtained was characterized spectroscopically. Results Isolation and identification of santonin utilizing culture The soil enrichment technique yielded a mixed bacterial culture capable of utilizing santonin as the sole source of carbon, which could be maintained by regular transfers in the santonin medium. The enriched culture on plating on nutrient agar, gave consistently two isolates, identified subsequently as Pseudomonas putida and P. solanacearum which either individually or both together failed to grow in santonin medium. The mixed culture plated on santonin agar gave rise to minute colonies of slow growing organism. The purified isolate

3 Sangodkar and Mavinkurve 81 showed a growth pattern similar to that of parent mixed culture in santonin medium. The isolate consisted of strictly aerobic, gram negative, non-sporulating 0.9 to 1.26 µm long coccobacilli, arranged singly or in pairs and actively motile with polar multitrichous flagella. The characteristics of the isolate including G +C content (table 1) confirm its identity as Pseudomonas cichorii (Buchanan and Gibbons, 1974; Sands et al., 1970) and is designated as strain S. Table 1. Physiological characteristics of the strain S. Santonin utilization by Pseudomonas cichorii strain S The growth of Pseudomonas cichorii strain S on santonin agar showed a clear halo around the colonies due to dissolution of santonin crystals (figure 2). Each of the chloroform extracts of the culture broth, withdrawn at intervals and scanned individually, showed a single λ max at 242 nm, the absorbance being decreased as a function of increase in bacterial growth in santonin medium. Transformation pattern of santonin during the growth of Pseudomonas cichorii strain S The spots visualized on thin layer chromatograms either by iodine vapours or by spraying with 50% sulphuric acid or under ultraviolet light after spraying with rhodamine B gave identical results indicating sequential formation of the intermediate product during the growth of isolate designated as L, M, N, O, and P (figure 3). None of these products however, could be detected when the organism was grown on acetate, glucose or benzoate.

4 82 α-santonin-assimilating Pseudomonad Figure 2. Colonies of P. Cichorii strain S on santonin agar Figure 3. Thin layer chromatograms of the chloroform extracts of the culture medium, during the growth of P. cichorii strain S in santonin medium. Supports: Silica gel C; Solvent system: Benzene and ethyl acetate (1:1); Visualizing agent: Iodine vapours; S: Santonin; L,M, N, O, P: Transformation products O, O, : intensities of the spots in increasing order.

5 Microbially transformed product of α-santonin Sangodkar and Mavinkurve 83 The transformation product M which appeared as a single substance initially, resolved into two components during preparative multiple run thin layer chromatography. One of the purified components M 1, analysed spectroscopically has the molecular formula C 15 H 20 O 3 (M + 248). Its infrared and nuclear magnetic resonance spectra indicated the absence of C 1- C 2 unsaturation with the remaining structural features intact. Spectral comparison and co-migration on chromatogram with authentic 1,2 dihydro santonin prepared by partial hydrogenation of santonin over 10% palladised charcoal established the identity of M 1 with 1,2-dihydro santonin (figure 1, II). The santonin utilizing Pseudomonad grows luxuriently on the authentic derivative (figure 1, II) as sole source of carbon forming similar intermediates as those from α-santonin. The santonin grown washed cells of P. cichorii strain S showed slightly higher oxygen uptake with 1,2-dihydrosantonin (6.08 ppm/h/mg dry wt) than that with santonin (5.03 ppm/h/mg). Discussion The soil enrichment technique yielded the santonin utilizing mixed bacterial culture, in which two strains of Pseudomonas proliferated consistently, apparently by utilizing some of the products formed from santonin by strain S. Synergism of this kind has been reported earlier during metabolism of quarternary ammonium compounds (Dean-Raymonds and Alexander, 1977). The elusiveness of strain S during initial isolation from the mixed enriched culture may be attributed to its slow growth, forming barely visible colonies on nutrient agar after 48 h ; a characteristic uncommon for Pseudomonas but a distinctive feature of Pseudomonas cichorii (Buchanan and Gibbons, 1974). The only inconsistency of strain S from the other reports for P. cichorii was with respect to it lack of hypersensitivity towards Nicotiana tobacum (Klement, 1963), and plant of Cichorium sp. Variations of this type are known to occur in cultures continuously maintained in the laboratory (Buddenhagen and Kelman, 1964). The dissolution of santonin crystals in agar around the growing colonies (figure 2) implies the release of certain extracellular factors for solubilization or transport of santonin, perhaps similar to those described for n-alkanes (Hisatsuka et al., 1977). The reduction of santonin concentration during growth is unequivocally established from the disappearance of santonin (figure 3) and the steep decrease in absorbance at 242 nm (λ max of α-santonin) of chloroform extracts of culture broth. Some of the transformation products appear to retain the conjugated carboxyl grouping intact which evidently contribute to the residual absorbance at 242 nm even after the disappearance of santonin after 72 h. Spectral features of the transformation product M 1, and growth and oxygen uptake by Pseudomonas cichorii strain S with authentic 1,2-dihydrosantonin, confirm the latter to be one of the initial metabolites in the pathway of utilization of santonin by the culture. It is of interest to note that the same product was also reported as.a cometabolite of santonin, formed by Cunnhinghamella blakesleeana

6 84 α-santonin- assimilating Pseudomonad and Streptomyces aureofaciens (Hikino et al., 1970). The reduction of the double bond at 1-2 position appears to be an initial step of microbial attack on santonin as well as its analogues (Fujimoto, et al., 1979). The sequential appearance followed by disappearance of the other transient metabolites during the growth indicate the potential usefulness of the system in elucidating the pathway of santonin catabolism. To our knowledge, the utilization of santonin as a sole source of carbon and energy by a single organism is not yet reported. The elucidation of the structures of the transformation products is expected to be rewarding not only in the biodegradation studies but may also prove fruitful in emergence of some of the pharamaceutically important products. Acknowledgements The financial assistance by the University Grants Commission for the research and Research Fellowship to U. M. X. Sangodkar is gratefully acknowledged. The authors are thankful to Dr. S. K. Paknikar for his interest and constant encouragement throughout the work. References Buchanan, R. E. and Gibbons, N. E. (1974) Bergey's manual of determinative bacteriology, 8th edn: Williams and Wilkins, Baltimore. Buddenhagen, I. W. and Kelman, A. (1964) Ann. Rev. Phytopath., 2, 203. Dean-Raymond D. and Alexander, M. (1977) Appl. Environ. Microbiol. 33, Fujimoto, Y., Shimizu, T., Ishimoto, T. and Tatsuno, T. (1979) Yakugaku Zasshi, 98, 230. Hikino, H., Tokuoka, Y. and Takemoto, T. (1970) Chem. Pharma. Bull. (Tokyo), 18, Hisatsuka, K., Nakahara, T., Minoda, Y. and Yamada, K. (1977) Agric. Biol. Chem., 41, 445. Klement,Z. (1963), Nature (London), 199, 299. Lonsane, B. K., Baruah, P. K., Singh, M.D., Baruah, J.N. and Iyengar, M.S.(1974)Ind.J.Exp.Biol., 12, 158. Mahtani, S. and Mavinkurve, S. (1979) J. Ferm. Technol, 57, 529. Mandel, M. (1966) J. Gen. Microbiol., 43, 273. Society of American Bacteriologists (1957) Manual of Microbiological Methods, McGraw-Hill Book Company, New York. Stanier, R. Y., Palleroni, N. J. and Doudoroff, M. (1966)J. Gen, Microbiol., 43, 159. Sands, D. C, Schroth, M. N. and Mildebrand, D. C. (1970) J. Bacteriol, 101, 9.