Biosynthesis of Prodigiosin by White Strains of Serratia

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1983, p. 476-480 0095-1137/83/030476-05$02.00/0 Copyright 1983, American Society for Microbiology Vol. 17, No. 3 Biosynthesis of Prodigiosin by White Strains of Serratia marcescens Isolated from Patients MING-JER DING AND ROBERT P. WILLIAMS* Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030 Received 10 November 1982/Accepted 13 December 1982 Serratia marcescens isolated from infected adults generally does not synthesize prodigiosin. Other investigators have reported that most clinical strains form a pigment if furnished with 4-methoxy-2,2'-bipyrrole-5-carboxyaldehyde (MBC), a precursor of prodigiosin. To determine whether the pigment was prodigiosin, we studied 65 white strains of S. marcescens isolated from patients. On the basis of response to MBC, we assigned the strains to one of three classes: class 1 (14 strains), strains remaining white; class 2 (48 strains), strains becoming gray or pink; and class 3 (3 strains), strains becoming blue. Ethanol extracts of bacteria of classes 2 and 3 did not behave like prodigiosin when acidified or alkalinized, and the pigment spectra were not similar to prodigiosin spectra. If strains of class 3 were furnished with MBC plus 2-methyl-3-amylpyrrole (MAP), the other immediate precursor of prodigiosin, the pigment synthesized was characteristic of prodigiosin. Strains of classes 1 and 2 responded identically to MBC plus MAP and MBC alone. Although the majority of S. mar(escens white strains from patients formed pigments in the presence of MBC, the pigments were not prodigiosin. A few strains did synthesize prodigiosin, but only if furnished with both MBC and MAP. A prominent characteristic of Serratia marcescens is production of the red pigment prodigiosin. However, strains of S. marcescens isolated from adult patients usually do not synthesize the pigment (1, 2, 5). The reason for the defect in synthesis is unknown. Katz and Sobieski (6) have done experiments to determine whether white strains isolated from patients synthesize prodigiosin when exposed to precursors of the pigment. They found that 113 of 114 white strains produce a pink or violet color when exposed to 4-methoxy-2-2'-bipyrrole-5-carboxyaldehyde (MBC), one of the two immediate precursors of prodigiosin (see Fig. 1). No colors are formed when the white strains are exposed to 2-methyl-3-amylpyrrole (MAP), the other immediate precursor of prodigiosin. Although the white strains produce a pink or violet color in response to MBC, no experiments were done to determine if these pigments are prodigiosin. We have carried those studies to the next step to determine whether the pigments produced in response to MBC are prodigiosin. White strains of S. marcescens isolated from patients were exposed to MBC, MAP, or both, and the pigments formed were analyzed chemically. On the basis of the results, we determined that only a small number of white strains could synthesize prodigiosin in response to precursors, although the majority of strains produced a gray or pink color in response to MBC. In addition, we attempted to locate the white strains in the prodigiosin biosynthesis scheme constructed on the basis of the syntrophic responses of various mutants (7, 10 [Fig. 1]). However, these white strains did not respond in a fashion that enabled us to locate them in a position corresponding to any of the known mutants blocked in prodigiosin biosynthesis. MATERIALS AND METHODS Media. Stock cultures were maintained on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.). Peptone-glycerol agar (PGA) was used to grow bacteria used in assays for prodigiosin biosynthesis. This medium contained the following (per liter of glass-distilled water): 5 g of Bacto-Peptone (Difco Laboratories, Detroit, Mich.), 10 ml of glycerol, and 15 g of agar (6). The ph of this medium was approximately 7.0 and required no adjustment. Bacteria. A total of 65 white strains of S. marcescens were obtained from adult patients at Ben Taub General Hospital and The Methodist Hospital, Houston, Tex. The strains were identified as S. marcescens by the City of Houston Health Department Laboratory. S. marcescens wild-type strain Nima was used as a source of prodigiosin (4). Several mutants of S. marcescens produced in our laboratory and by other workers were used as sources of precursors for prodigiosin biosynthesis (Table 1). Assay for prodigiosin biosynthesis. The method of syntrophic formation of pigment (9) was used in the 476

VOL. 17, 1983 PRODIGIOSIN SYNTHESIS BY CLINICAL STRAINS 477 OCH3 ICH214CH3 I*BC MBC 83-- OF---- 3 E c ;;CH3 H H H prodigiosin C- -t- OCH 3 CHO OH OH ICH214CH3 FIIja7I2LCHIi~2CH H H H Norprodigiosin ICH21CH3 CHO H HH 3 NBC MAP 82 - - N82 M2 --- 81 --I-- WF Ml --1-- 933 FIG. 1. Prodigiosin biosynthesis scheme developed on the basis of the syntrophic responses of various mutants (7, 10). The classes of mutants and the locations of their blocks in synthesis are indicated. The locations of the blocks of the mutants listed in Table 1 are also shown. assay for prodigiosin biosynthesis. Petri dishes containing PGA were divided into halves or thirds. For plates divided in half, one-half of the agar was heavily. inoculated with a mutant of S. marcescens on a sterile cotton swab. The other half of the plate was inoculated in the same fashion with one of the white strains so that one growth was no more than 0.5 cm from the other. For plates divided into thirds, a white strain was inoculated onto one-third of the agar and two different mutant strains were inoculated onto each of the other thirds on either side of the white strain. Cultures for use in the assay were grown overnight at 22 to 24 C on slants of PGA. Growth was then taken up on a cotton swab for inoculation of the agar. The assay is based upon the discovery that prodigiosin precursors produced by appropriate mutant strains of S. marcescens diffuse through agar and cause prodigiosin to be synthesized by other appropriate mutants that produce another precursor (7, 9, 10). For example, mutants WF, N82, and OF (Table 1) are blocked at different points in MBC synthesis, but they all synthesize MAP (Fig. 1). These mutants produce prodigiosin if furnished with MBC synthesized by mutants such as 933. The latter mutant will also form prodigiosin if furnished with MAP synthesized by mutants such as WF, N82, and OF. MBC is a nonvolatile, stable compound that diffuses through agar, and mutant 933 must grow closely adjacent to recipient strains to be an effective donor of MBC. MAP is volatile, and growth of donor mutants in a closed system, such as a petri dish, permits appropriate MBC-producing recipients to form prodigiosin. Assay plates were incubated at 22 to 24 C for 4 days and observed daily for color formation. White strains alone were inoculated onto PGA as controls and incubated under the same conditions to determine if spontaneous color changes occurred. Control plates of PGA were also inoculated with mutant strains of S. marcescens to determine that the proper syntrophic feeding responses occurred for synthesis of prodigiosin. In all cases, the mutants responded correctly (Table 1 and Fig. 1). Presumptive color tests for prodigiosin. Pigmented areas of growth that were seen on assay plates after incubation for 4 days were scraped from the agar and suspended overnight in 95% ethanol at 22 to 24 C. Debris was removed from the suspension by centrifugation at 5,000 x g for 15 min. The clear solution was then divided into two portions. One part was acidified with a drop of concentrated HCl; the other part was alkalinized with a drop of concentrated ammonia solution. A red or pink color in the acidified solution and a

478 DING AND WILLIAMS J. CLIN. MICROBIOL. TABLE 1. S. marcescens mutants studied Designation Origina classn Product Reference 933 White mutant from wild-type Ml Stable MBC, prodigiosin if 8 strain HY furnished with MAP OF Orange mutant from wild- B3 Stable norprodigiosin and volatile 9 type strain Nima MAP, prodigiosin if furnished with MBC N82 White mutant from wild-type B2 Volatile MAP, prodigiosin if C strain Nima furnished with MBC WF White mutant from wild-type Bi Volatile MAP, prodigiosin if 4 strain Nima furnished with MBC a HY and Nima are strains of S. marcescens. b See Fig. 1 for explanations of mutant classes and locations of blocks in the prodigiosin biosynthetic pathway. c -, S. M. H. Qadri, C. W. Nichols, and R. P. Williams, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, 1130, p. 102. yellow or tan color in the alkaline solution indicated a positive, presumptive test for prodigiosin (3). Determination of adsorption spectra. Pigmented bacteria were treated with ethanol, and the debris in the suspensions was removed by centrifugation as described above. Samples (1 ml) of the ethanol extracts were acidified or alkalinized with 0.1 ml of 1 N HCI or 1 N NaOH, respectively. The absorbance spectrum of each sample was measured with a model 240 spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio) in the range of 300 to 700 nm. Acidified or alkalinized 95% ethanol was used as a blank. Prodigiosin extracted with ethanol from S. marcescens Nima showed characteristic maxima of 535 and 470 nm in acid and alkaline solutions, respectively (10). RESULTS None of the white strains produced color when grown separately on PGA. When patterns of syntrophic pigment formation were determined, the white strains were assigned to one of three classes (Table 2). Class 1 strains (14 strains) remained white and showed no response to any of the precursors synthesized by various mutants of S. marcescens. Class 2 strains (48 strains) did not respond to MAP produced by mutants OF, N82, and WF but formed a gray or pink color in response to MBC produced by mutant 933. Class 3 strains (3 strains) did not respond to MAP but formed a blue color in response to MBC. A red color was formed by class 3 strains in response to both MAP and MBC. All colors were formed in the growth regions directly adjacent to growth of the acceptor and donor strains. When grown with mutants 933 and WF on a single PGA plate, the class 3 white strains produced the most intense red TABLE 2. Patterns of syntrophic pigment formation by strains of S. marcescensa Syntrophic pattern with indicated donor strainb Acceptor strain Clinical white Mutant' Class 1 Class 2 Class 3 933 OF N82 WF 933 + WF Clinical white Class 1 ND ND ND - Class 2 ND ND ND g/p - - - g/p Class 3 ND ND ND b - - - + Mutant 933 - - - ND + + + ND OF - - - + ND - - ND N82 - - - + + ND - ND WF - - - + + + ND ND a See the text for details of assay. All cultures were incubated for 4 days at 22 to 24 C before examination for color changes. b ND, Not determined; -, no response detected-cultures retained their original color; g/p, formation of gray or pink pigment; b, formation of blue pigment; +, formation of red pigment. c See Table 1 for characteristics of mutant strains and Fig. 1 for locations of blocks in the prodigiosin biosynthetic pathway.

VOL. 17, 1983 TABLE 3. PRODIGIOSIN SYNTHESIS BY CLINICAL STRAINS 479 Presumptive color tests and absorption maxima for ethanol extracts of syntrophic pigments produced by S. marcescens strains Color produced by addition Maximal absorbance Pigment extracted from Color of of concentrated: (nm) of ethanol extract strain: extract in: HCI NH3 Acid Alkali Nima Red Pink Yellow 535 470 933 Purple Purple Brown 375 360 OF Brown-gold Red Orange 530 430 N82 Pink Pink Brown 325 b WF Yellow Brown Yellow 500 933 + OF, N82, or WF' Red Pink Yellow 535 470 Class 2 + 933c Purple Purple Yellow 535 Class 2 + 933 and WFC Brown Brown Yellow Class 3 + 933c Blue Blue Pink 595 350 Class 3 + 933 and WFC Pink Pink Yellow 535 470 a Pigment syntrophically produced by each mutant (Table 2) was analyzed. The colonies of each mutant were red. b, No maxima were observed in the range of 300 to 700 nm. c Pigment produced in response to adjacent growth of class 2 or 3 clinical white strains and mutant 933 or mutants 933 and WF (Table 2). color in the areas where they grew adjacent to the mutants. An ethanol extract of an S. marcescens Nima culture was visibly red (Table 3). The extract became bright pink when acidified and yellow when alkalinized. Maximal absorbance of the extract was at 535 nm in acid and at 470 nm in alkali. These characteristics are typical of prodigiosin (10). When prodigiosin was formed by mutants in response to the appropriate precursor, the same characteristics were present (Table 3), but they were not seen when the mutants were grown separately. Ethanol extracts of the gray or pink color produced by class 2 white strains grown adjacent to mutant 933 or mutants 933 and WF did not have the characteristics of prodigiosin (Table 3). Likewise, ethanol extracts of the blue color formed by class 3 white strains grown adjacent to mutant 933 did not show characteristics of prodigiosin. However, the acidified or alkalinized ethanol extracts of the red color produced by class 3 strains grown adjacent to mutants 933 and WF were identical in color and maximal absorbance to prodigiosin (Table 3). DISCUSSION Katz and Sobieski (6) reported that 99% of the white strains of S. marcescens that they studied respond to MBC by formation of a pink or violet color. Our class 2 strains responded in a similar fashion, although these strains constituted only 74% of our sample group. The class 1 strains, which constituted 22% of our sample group, showed no response to any of the precursors of prodigiosin. Less than 5% of our strains were assigned to class 3. These bacteria formed prodigiosin but only in response to MAP plus MBC. Our data and those of Katz and Sobieski (6) suggest that the majority of clinical white strains of S. marcescens produce pigments in response to MBC. However, the pigments produced by our class 2 strains were not prodigiosin, as established by the chemical reactions of ethanol extracts in acidic or alkaline solutions and by absorption spectra. We do not know whether clinical strains produce prodigiosin precursors that cannot be identified. However, the failure of the strains to form prodigiosin in response to, or to evoke prodigiosin formation by, any of the common mutants used to outline the prodigiosin biosynthetic pathway (Table 1 and Fig. 1) suggests that the block(s) in prodigiosin biosynthesis is at an early step, before formation of compounds that will respond to MAP and MBC to form prodigiosin. The location of the block(s) must await identification of the relevant biosynthetic steps and characterization of the enzymes responsible for them or development of a genetic system to analyze prodigiosin biosynthesis. Class 3 white strains responded to MAP plus MBC to form prodigiosin. These strains did not belong to any class of mutants blocked in the MAP or MBC pathway of prodigiosin biosynthesis (Fig. 1). However, Morrison (7) described mutants of an X class that respond to MAP plus MBC. These mutants were isolated infrequently, and Morrison suggested that they are blocked in an early prodigiosin biosynthesis step common to both MAP and MBC pathways. Our class 3 strains responded in a similar fashion, but they were not identical to the X class mutants because they did not form prodigiosin in response to products from mutants of classes B2 and B3 (Fig. 1), as did the mutants reported by

480 DING AND WILLIAMS Morrison (7). Although class 3 strains apparently had the enzyme required for conjugation of MAP with MBC to form prodigiosin, they were not similar to mutants of class C (Fig. 1). The latter mutants furnish both MAP and MBC, and mutants of classes Bi, B2, B3, Ml, and M2 respond by formation of prodigiosin. None of the mutants we tested responded to products from class 3 strains (Table 2). A blue color was produced by class 3 strains in response to products from mutant 933 (Table 2). This pigment was not prodigiosin. Identification of the block(s) in prodigiosin biosynthesis by class 3 strains must await further studies of these bacteria and a more complete analysis of the pathway for prodigiosin biosynthesis. None of the clinical white strains synthesized a product that evoked formation of prodigiosin by any of the mutants used to elucidate the pathway for pigment biosynthesis. Thus, apparently neither MAP nor MBC was produced by the clinical strains. Strains of classes 1 and 2 might have a defect in the enzyme that conjugates MAP with MBC to form prodigiosin because the bacteria failed to form the pigment, even when furnished with both precursors. The same failure would be observed if the bacteria were impermeable to one or both of the precursors. Why do strains of S. marcescens isolated from adult patients not synthesize prodigiosin? Does the failure contribute to the pathogenicity of the strains? Our present knowledge does not permit either question be be answered, but further J. CLIN. MICROBIOL. studies to establish the relationship between clinical white strains and prodigiosin biosynthesis are warranted. ACKNOWLEDGMENTS This investigation was supported in part by U.S. Public Health Service training grant Al 07145 from the National Institute of Allergy and Infectious Diseases. M.J.D. was supported by a grant from the National Science Council and the Ministry of Defense, Taipei, Taiwan, Republic of China. LITERATURE CITED 1. Ball, A. P., D. McGhie, and A. M. Geddles. 1977. Serratia marcescens in a general hospital. Q. J. Med. N. Ser. 46:63-71. 2. Clayton, E., and A. von Graevenitz. 1966. Nonpigmented Serratia marcescens. J. Am. Med. Assoc. 197:111-116. 3. Gerber, N. N., and M. P. Lechevalier. 1976. Prodiginine (prodigiosin-like) pigments from Streptomyces and other Actinomyces. Can. J. Microbiol. 22:658-667. 4. Green, J. A., and R. P. Williams. 1957. Studies on pigmentation of Serratia marcescens. IV. Analysis of syntrophic pigment. J. Bacteriol. 74:633-636. 5. Grimont, P. A. D., and F. Grimont. 1978. Biotyping of Serratia marcescens and its use in epidemiological studies. J. Clin. Microbiol. 8:73-83. 6. Katz, D. S., and R. J. Sobieski. 1979. Detection of pigment precursors in white clinical strains of Serratia marcescens. J. Clin. Microbiol. 9:301-303. 7. Morrison, D. A. 1966. Prodigiosin synthesis in mutants of Serratia marcescens. J. Bacteriol. 91:1599-1604. 8. Santer, U. V., and H. J. Vogel. 1956. Prodigiosin synthesis in Serratia marcescens: isolation of a pyrrole-containing precursor. Biochim. Biophys. Acta 19:578-579. 9. Williams, R. P., and J. A. Green. 1956. Studies on pigmentation of Serratia marcescens. III. The characteristics of an orange variant. J. Bacteriol. 72:537-542. 10. WilLiams, R. P., and S. M. H. Qadri. 1980. The pigment of Serratia, p. 31-75. In A. von Graevenitz and S. J. Rubin (ed.), The genus Serratia. CRC Press, Boca Raton, Fla.