STUDIES ON PIGMENTATION OF SERRATIA MARCESCENS

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STUDIES ON PIGMENTATION OF SERRATIA MARCESCENS I. SPECTRAL AND PAPER CHROMATOGRAPHIC PROPERTIES OF PRODIGIOSIN' ROBERT P. WILLIAMS, JAMES A. GREEN2 AND DONALD A.

1 STUDIES ON PIGMENTATION OF SERRATIA MARCESCENS I. SPECTRAL AND PAPER CHROMATOGRAPHIC PROPERTIES OF PRODIGIOSIN' ROBERT P. WILLIAMS, JAMES A. GREEN2 AND DONALD A. RAPPOPORT Departments of Microbiology, Biochemistry, and Radiology, Baylor University College of Medicine, Houston, Texas Received for publication June 23, 1955 The red pigment of Serratia marcescens, prodigiosin, has interested chemists because it has been reported to be the only naturally occurring tripyrrylmethene and as such its biosynthesis might be related to porphyrin biosynthesis (Hubbard and Rimington, 1950). Wrede and Hettche (1929) extracted the pigment from S. marcescens using a procedure involving sodium hydroxide treatment. By degrading the alkaliextracted pigment, Wrede and Rothhaas (1934) defined the structure of prodigiosin as a tripyrrylmethene, a single compound. Hubbard and Rimington (1950) extracted prodigiosin by means of acid and alkali and compared the spectral properties of the pigment to a synthetic tripyrrylmethene. Since the spectral properties of the two substances were similar, these authors concluded that the tripyrrylmethene structure of prodigiosin was probably correct, and implied that the pigment was a single substance. However, Weiss (1949) showed that prodigiosin extracted by a mild organic solvent, n-butyl alcohol, separated into several bands when chromatographed on columns of magnesium oxide or a mixture of calcium carbonate and celite. This fact suggested that Hubbard and Rimington (1950) might have dealt with a mixture of pigments which they assumed to be a single substance. Since Weiss (1949) indicated the mixed nature of extracted prodigiosin, we felt further study of mildly extracted prodigiosin was warranted. Unfortunately, n-butyl alcohol extraction did not give satisfactory results in our hands. As a consequence we have developed a different method for pigment extraction and for the sepal This investigation was supported by research grant RG 4183 from the National Institutes of Health, Public Health Service. 2 Candidate for degree of Doctor of Philosophy, Biology Department, The Rice Institute, Houston, Texas. 115 ration of the individual components. The procedure and results of our method are presented. EXPERIMENTAL METHODS Pigment was extracted from cultures of S. marcescens strain Nima (from Dr. E. D. Weinberg, Department of Bacteriology, University of Indiana, Bloomington, Indiana). The cultures were grown on a modification of the medium developed by Bunting (1940), containing yeast extract, 0.1 per cent; Sheffield Farms "N-Z" enzymatic casein hydrolyzate, 0.2 per cent; glycerol, 1.0 per cent; ammonium citrate, 0.5 per cent; magnesium sulfate, 0.05 per cent; dipotassium phosphate, 1.0 per cent; sodium chloride, 0.5 per cent; ferric ammonium citrate, per cent; agar, 2.0 per cent; made up in water double distilled from glass; ph 7.1 : The medium was distributed in 5-L Povitsky diphtheria toxin bottles and sterilized by autoclaving. Inoculation was made by spreading on the surface 5 ml of a 1: per cent saline dilution of a 48-hr broth culture of the organisms. The inoculated bottles were kept at room temperature in the dark. Cultures were harvested 5 days after inoculation by washing the cells off the agar with 100 ml of deionized water. The pigment was extracted by adding 4 volumes of acetone to the cell suspension. The acetone mixture was shaken for 3 hr at room temperature, then centrifuged. The sedimented cell debris was washed twice by resuspension in 50 ml. of acetone, shaking for 30 min, and centrifuging. The washings were combined with the supernatant from the original centrifugation, and the solution was filtered. Pigment was extracted from small portions of the filtrate by mixing thoroughly 1 volume of the acetone solution with 2 volumes of petroleum ether in a separatory funnel. The acetone was removed by adding 10 volumes of water to the funnel, then drawing off

2 *: : :~~~~~~~~~~~~~~~~~~~~~~~. w;cs j,,, ~~~~~~~~~~~~~~~~~~~~~~.. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ~~~~~~~~~~~~~~~~~.!: 116 WILLIAMIS, GREEN AND RAPPOPORT [VOL. 71 the acetone-water phase. This procedure was 2 parts petroleum ether, was applied to the paper repeated until the entire filtrate was extracted, through a hole in the center of the top glass plate and the pigment was in the petroleum ether using a tightly fitted 5- or 10-ml syringe as a phase. To obtain dry pigment, the petroleum dispenser. The chiromatograms were made at ether extract was evaporated in vacuo at 30 to room temperature, and development was complete after 20 oi 30 min. 40 C. The dry pigment was used for chiomatographic or spectral analysis. Spectral analyses were made on approximately Chromatography was done on paper using the 5,ug of dried pigment dissolved in 10 ml of circular method developed by Rappoport et al. absolute ethanol. Acid oi alkaline conditions for (1955). Whatman no. 3AMIM paper was cut into spectral analysis w-ere obtainedl by adding 1 ml 13-in squares. Enough dried pigment was dissolved in 2 ml of chloroform to make a saturated hydroxide, respectively, to 10 ml of the ethanol of 1 N hydrochloric acid or 1 ml of 1 N sodium solution, and placed on the paper in a circle 1 in extract. Neutral spectral analyses were (lone on from the center. Since the paper as purchased was 10 ml of the ethanol extract to which 1 ml of somewhat acidic, it was necessary to pretreat the distilled water was added. All spectral analyses paper with ammonia vapors for a short time in were made on a Beckman spectrophotometer, order to obtain good separation of the pigment Model DU. components. Subsequently this paper was placed between two heavy plate glass squares further RESULTS weighted by 125 lb of lead bricks. The freshly Chromnatographic analysis. The circular chr o- mixed solvent, composed of 1 part ethyl ether to matogram, obtained as described above, is shown...: ;^.: :.... T l... ix-.. i A~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ '. 4.ie l^,,. 90 t..,,s.<~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ::... ~~~~~~~~~~~~~~~~~~~~~~ Figur2ze 1. Paper chromatogratin of acetone extracted prodigiosin.

1956] PIGMENTATION OF SERRATIA MARCESCENS. I 117 in figure 1, in which acetone-extracted prodigiosin sev&ates into at least four bands, at Rf 0.18, 0.48, 0.70, and 0.89.

3 1956] PIGMENTATION OF SERRATIA MARCESCENS. I 117 in figure 1, in which acetone-extracted prodigiosin sev&ates into at least four bands, at Rf 0.18, 0.48, 0.70, and The color designations in figure 1 refer to the visible colors of the various bands. The orange band at Rf 0.89 has only an evanescent orange color which rapidly turns red when exposed to air. The orange color can be returned temporarily by exposing the paper chromatogram to ammonia vapor. Thus this 2.0r X * * * I band is not the same red as the red bands at Rf 0.48 and A blue band at Rf 0.18 is consistently present. This component has not been reported previously. The red band at Rf 0.48 is always present, but only in very small amounts, which accounts for its faint appearance in figure 1. The relative amount of the combined red and orange pigments at Rf 0.48, 0.70, and 0.89 as compared to the blue component, Rf 0.18, is approximately 3:2 in a 5-day-old culture. The amounts of pigment were measured colorimetrically after establishing a standard curve using purified pigment. The total weight of pigment in the culture was about 750,ug per g of dry weight of cells. Spectral analysis. Figure 2 presents the acid, alkaline, and neutral spectral curves of prodigiosin extracted by the procedure outlined. Comparison of the acid and alkaline curves with those of Hubbard and Rimington (1950) indicates that our acetone-extracted pigment is similar in spectral properties to their pigment (table 1). Exact identity cannot be ascertained from their curves because the plots were published on a small scale. Prodigiosin can exist in two distinct forms, depending upon the hydrogen ion concentration of the solution. In an acid medium the pigment is red and exhibits a sharp spectral peak at 535 m,u (figure 1). The shoulder on the low wavelength limb of the acid curve at about 510 m,u is persistent in the whole pigment. In an alkaline medium the pigment is colored orangeyellow and possesses a broader spectral curve centered at 470 m,. TABLE 1 Comparison of spectral properties of prodigio8in Data from Points for Comparison Hubbard and Ti netgto Rimington (1950) This investigation -& i6p %P W~ N %- --m --% P Figure B. Spectral properties of acetone extracted prodigiosin. - -O - - curve when dissolved in absolute ethanol + H20; -v - curve when dissolved in absolute ethanol + HCI; -* - curve when dissolved in absolute ethanol + NaOH. The alkaline curve is displaced upward 0.1 optical density unit to avoid conffict with the neutral curve. Acid curve Maxima , , 275 Minima , 335, , 330, 260 Alkaline curve Maxima , , 275 Minima , , 260 Isosbestic point O.D. ratio of acid maximum to alkaline maximum

4 118 WILLIAMS, GREEN AND RAPPOPORT [VOL. 71 Downloaded from a lfigure Sa. Spectral properties of the blue fraction (Rf 0.18) of prodigiosin. Figure Sb. Spectra properties of the combined red fractions (Rf 0.48 and Rf 0.70) of prodigiosin. The legend is the same as in figure 2. b on April 14, 2019 by guest Spectral characterization of the bands separated by chromatography (figure 1) was attempted by eluting the individual bands from the paper with ethanol. Figure 3a shows the spectral properties of the blue band at Rf The red band, at Rf 0.48, was present in quantities too small to allow effective spectral characterization by itself, therefore the red bands, at Rf 0.48 and Rf 0.70, were eluted together. The spectral curves of the two red fractions are shown in figure 3b. The spectral properties of the orange band, at Rf 0.89, are shown in figure 4. Examination of figures 3 and 4 reveals that the spectral properties of the components of prodigiosin are similar to those of the whole pigment as shown in figure 2. Although spectral curves of the fractions were determined on unknown amounts of pigment, the spectral characteristics are qualitatively comparable. Figure 3a shows that the blue fraction has broad spectral peaks and low absorbency when compared to either the red fraction or the orange fraction as shown in figures 3b and 4. The ratios of the optical density maxima under acid, alkaline, or

$19561 PIGMENTATION OF SERRATIA MARCESCENS. I 119 m- Figure 4. Spectral properties of the orange fraction (Rf 0.89) of prodigiosin. The legend is the same as in figure 2.$

5 19561 PIGMENTATION OF SERRATIA MARCESCENS. I 119 m- Figure 4. Spectral properties of the orange fraction (Rf 0.89) of prodigiosin. The legend is the same as in figure 2. neutral conditions differ in the fractions. The orange band has a marked peak in the acid curve at 500 m,u, and none at 545 m,u. This fact distinguishes the orange band, at Rf 0.89, from the other fractions and the whole pigment. DISCUSSION The results presented indicate that acetoneextracted prodigiosin possesses spectral properties similar to those of the pigment of Hubbard and Rimington (1950). Our investigations have shown that prodigiosin can be separated into several fractions by chromatography as reported by Bunting (1940) and Weiss (1949). Although the spectral properties of the fractions, as eluted from paper chromatograms, are similar to the spectral properties of the whole pigment, there are differences. The orange fraction possesses a distinct spectral curve with a sharp peak in acid solution at 500 m,u. It is interesting to note that this peak occurs at approximately the same point at which a shoulder occurs in the spectral curve of the whole pigment. The shoulder does not exist in the acid solutions of either the red or the blue components. Thus it seems that the shoulder in the whole pigment is due to the presence of the orange fraction. Our studies show that the orange fraction is a consistent component of prodigiosin. This view is contrary to that expressed by other authors (Rizki, 1954). These findings establish that the prodigiosin described by Wrede and Rothhaas (1934) and Hubbard and Rimington (1950) is not a single substance, but is made up of at least four components. The presence of the blue component discovered during these investigations was suggested in previous reports (Bunting, 1940; Weiss, 1949) since they had observed a slow-moving purple band in their chromatograms of prodigiosin. The purple band was probably the same blue component reported here, but admixed with red fractions. Further studies on the isolated components will be reported in a subsequent publication. SUMMARY The pigment of Serratia marcescens can be extracted by a simplified procedure employing acetone. By means of circular paper chromatography prodigiosin is separated into four fractions: one blue, two red, and one orange, each possessing different Rf values. The blue fraction has not been reported previously. The spectral properties of acetone-extracted pigment are similar to those of pigment extracted by other investigators. These results establish that prodigiosin is not a single compound as suggested by others. REFERENCES BuNTING, M. I A description of some color variants produced by Serratia marcescens, strain 274. J. Bacteriol., 40, HUBBARD, R. AND RIMINGTON, C The biosynthesis of prodigiosin, the tripyrryl-

120 WILLIAMS, GREEN AND RAPPOPORT [VOL. 71 methene pigment from Bacillus prodigiosus (Serratia marcescens). Biochem. J. (London), 46, 220-225. RAPPOPORT, D. A., CALVERT, C. R., LOEFFLER, R. K.

6 120 WILLIAMS, GREEN AND RAPPOPORT [VOL. 71 methene pigment from Bacillus prodigiosus (Serratia marcescens). Biochem. J. (London), 46, RAPPOPORT, D. A., CALVERT, C. R., LOEFFLER, R. K., AND GAST, J. H Chromatographic separation and determination of porphyrin methyl esters. Anal. Chem., 27, RIzKI, M. T. M The nature of the pigment induced by chromogenic inductors of Serratia marcescens. Proc. Natl. Acad. Sci. U. S., 40, WBIss, C. M Spectrophotometric and chromatographic analyses of the pigment produced by members of the genus Serratia. J. Cellular Comp. Physiol., 34, WREDE, F. AND HETTCHE, tjber das Prodigiosin, den roten Farbstoff des Bacillus prodigiosus. I. Ber. deut. chem. Ges., 62, WREDE, F. AND ROTHHAAS, A tyber das Prodigiosin, den roten Farbstoff des Bacillus prodigiosus. VI. Hoppe-Seyler's Z. physiol. Chem., 226,

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glucose, acid from maltose and mannitol, but STUDIES ON PIGMENTATION OF SERRA TIA MARCESCENS III. THE CHARACTERISTICS OF AN ORANGE VARIANT1 ROBERT P. WILLIAMS AND JAMES A. GREEN Department of Microbiology, Baylor University College of Medicine, Houston,