JOURNAL OF BACTERIOLOGY, Dec. 1973, p. 1191-1198Copyright 0 1973 American Society for Microbiology

Vol. 116, No. 3Printed in U.S.A.

Role of Methionine in Biosynthesis of Prodigiosinby Serratia marcescens

S. M. HUSSAIN QADRI AND ROBERT P. WILLIAMS

Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77025

Received for publication 23 July 1973

Methionine alone did not allow biosynthesis of prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) in nonproliferating cells (NPC) of Serratiamarcescens strain Nima. However, when methionine was added to NPCsynthesizing prodigiosin in the presence of other amino acids, the lagperiod for synthesis of prodigiosin was shortened, an increased amount of thepigment was formed, and the optimal concentrations of the other amino acidswere reduced. Less prodigiosin was synthesized when addition of methionine wasdelayed beyond 4 h. The specific activity of prodigiosin synthesized by additionof 14CH,-methionine was 40 to 50 times greater than that synthesized frommethionine-2-14C or "COOH-methionine. NPC of mutant OF of S. marcescenssynthesized norprodigiosin (2-methyl-3-amyl-6-hydroxyprodigiosene), and thespecific activity of this pigment synthesized in the presence of "CH,-methioninewas only 5 to 13 times greater than that synthesized from methionine-2-14C or

"4COOH-methionine. A particulate, cell-free extract of mutant WF of S.marcescens methylated norprodigiosin to form prodigiosin. When the extract wasadded to NPC of mutant OF synthesizing norprodigiosin in the presence of14CH8-methionine, the prodigiosin formed had 80% greater specific activity thanthe norprodigiosin synthesized in the absence of the extract. The C6 hydroxylgroup of norprodigiosin was methylated in the presence of the extract andmethionine. Biosynthesis of prodigiosin by NPC of strain Nima also wasaugmented by addition of S-adenosylmethionine. Various analogues of methio-nine such as norleucine, norvaline, ethionine, and a-methylmethionine did notaffect biosynthesis of prodigiosin by NPC either in the presence or absence ofmethionine.

The pyrrole groups of prodigiosin (2-meth-yl-3-amyl-6-methoxy-prodigiosene; Fig. 1) aresynthesized by growing cultures of Serratiamarcescens from amino acids rather than from6-aminolevulinic acid (13, 17). Six amino acidspresent in casein hydrolysate, alanine, asparticacid, glutamic acid, histidine, proline, andserine caused synthesis of prodigiosin in suspen-sions of nonproliferating cells (NPC) (15), and"IC-label from these same amino acids wasincorporated into the pigment (S. M. H. Qadriand R. P. Williams, Can. J. Microbiol., submit-ted for publication). Although nothing is knownconcerning the pathway followed by theseamino acids to synthesize prodigiosin, prolinemay be incorporated intact (17, 18). Methioninealone did not induce biosynthesis of prodigiosinin NPC, but, when added together with aninducing amino acid such as alanine or proline,the amount of prodigiosin synthesized increased

about four times (14). We describe in this paperinvestigations of the role of methionine in thebiosynthesis of prodigiosin by NPC of S. mar-cescens.

MATERIALS AND METHODSOrganisms. The red, wild-type Nima strain, an

orange mutant (OF), and a white mutant (WF) of S.marcescens were carried in stock cultures and exam-ined periodically for mutants from Nima or revertantsfrom OF or WF, as described previously (21). StrainNima formed prodigiosin, OF produced the orangepigment, norprodigiosin (2-methyl-3-amyl-6-hydrox-yprodigiosene), and WF synthesized 2-methyl-3-amylpyrrole (MAP), the colorless, monopyrrole por-tion of prodigiosin (Fig. 1). When OF and WF aregrown together, prodigiosin is formed because WF canenzymatically methylate the hydroxyl group of nor-prodigiosin (5, 23). In addition to strain WF, stockculture strains of Escherichia coli and Pseudomonasaeruginosa also were used as sources of methylatingenzyme.

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9 8 7 6 4 3 potassium phosphate buffer, pH 7.0. The dialyzedextracts were used as the transmethylase systems toinvestigate enzymatic conversion of norprodigiosin,

/\\/1\\5 2\ produced by strain OF of S. marcescens, to prodigi-

\N N N N; 2 osin. In a typical experiment, 2 ml (4 to 6 mg of

1 12 protein) of one of the dialyzed extracts was added to aH H 250-ml Erlenmeyer flask containing L-proline (42.5

mM) and L-methionine (0.27 mM) in 20 ml (40 to 45mg of protein) of NPC of strain OF. The cells of strain

PRODIGIOSIN: at 2, CH3; at 3, C5HI; at 6, OCH3 OF were not treated to enhance their permeability.CRGIaCH3H a6H Conversion of norprodigiosin to prodigiosin.

)RPRODIGIOS IN: at 2, OH3; at 3, C5H, at 6, OH Norprodigiosin, produced by NPC of strain OF, wasconverted to prodigiosin either enzymatically, as

[G. 1. Chemical structure of prodigiosene, the described above, or by chemical means. Conversion

,nt compound of pprodiand prodigiosin-like shifted the spectral absorption peak from 525 nm for

cents (18). The appropriate substitutions are norprodigiosin to a single peak at 534 nm that iscated for prodigiosin and norprodigiosin. Mutant characteristic of prodigiosin (22), and no other pig-synthesizes only the monopyrrole portion of these mented substances were detected in the methylatedp,ounds, 2-methyl-3-amylpyrrole. norprodigiosin (Fig. 2). For chemical methylation,

norprodigiosin was extracted from bacteria with ace-ulture media and growth conditions. Bacteria tone (5). After centrifugation to remove the colorlessgrown on the minimal medium of Bunting (1) or cells, pigment was extracted from the acetone solutionnomnlete medium of Williams et al. (22) under the into chloroform (3). The chloroform extract was

tallu uVILllIUt" I&1vAILs>I1 V1 TVLIIGL&O 'Ut as. Ul ussuL-1XV

conditions we described previously (15, 21).Preparation of NPC. Suspensions of NPC of

strains Nima or OF were prepared by growth at 39 Caccording to the procedure of Qadri and Williams(15). Although these cells did not contain prodigiosinor norprodigiosin, biosynthesis of either pigmentcould be caused by addition of L-proline (42.5 or 85mM) or L-alanine 55 mM), with or without L-methio-nine (0.27 mM), to suspensions ofNPC of Nima or OFincubated at 25 C. After addition of the amino acids,the suspensions were incubated for the appropriatelength of time on a rotary shaker (New BrunswickScientific Co., New Brunswick, N.J.) operating at 200rpm.

For amino acid incorporation studies, 14C-labeledamino acids were added to NPC 1 to 2 h aftercommencement of prodigiosin or norprodigiosin syn-thesis. Samples for analysis and isotope counting weretaken after the time periods indicated in the figuresand text. No contamination by other organisms wasdetected during incubation.Preparation of cell-free extracts for trans-

methylase activity. Strains of WF, E. coli, or P.aeruginosa were grown in complete medium at 25 Cfor 24 h. Bacteria were harvested by centrifugation at27,000 x g at 2 C, and then were washed three timesby additional centrifugations in 0.85% (wt/vol) saline.Washed organisms (5 g wet weight) were suspended in15 ml of saline with 10 g of washed, fine glass beads(Superbrite type 110, 3 M Co., St. Paul, Minn.). Aftertreatment with a sonic disintegrator (Biosonik, Bron-will Scientific Co., Rochester, N.Y.) for three periodsof 3 min each at 2 C, the disintegrated bacteria werecentrifuged for 10 min at 7,900 x g in a Sorvall RC2-Brefrigerated centrifuge at 2 C. The residue (Rj) wasdiscarded because it contained intact cells. Thesupernatant fluid (Sj) again was centrifuged at 7,900x g for 20 min, yielding a clear supernatant fluid (S2)and a residue (R). Fraction R, was suspended insaline and dialyzed for 24 h at 2 C against 20 mM

0. 70

0. 60

0. 50

LI

0

0. 40

0. 30

0. 20

0. 10

0

475 500 550

WAVELENGTH (nm)600

FIG. 2. Absorption spectrum in acidic methanol ofnorprodigiosin before and after conversion to prodigi-osin by methylation.

10/

NC

FiEpare,pigrrindicWF.comi

Ciwerethe- X

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BIOSYNTHESIS OF PRODIGIOSIN

evaporated to dryness in a vacuum rotoevaporatorand then was treated with an excess of diazomethane(9). After 10 min, the diazomethane was evaporated,and the methylated pigment (prodigiosin) was puri-fied as described below.

Purification of prodigiosin for isotope counting.The procedure was similar to the method of Hubbardand Rimington (10) as modified by de Medina (Ph.D.thesis, Iowa State Univ., Ames, 1969), in whichprodigiosin was extracted from 10 ml of culture byshaking for 30 min with 20 ml of acetone and 20 ml ofacidic methanol (4.0 ml of 1 N HCl added to 96.0 ml ofmethanol). After the mixture was centrifuged at10,000 rpm for 20 min in a Sorvall refrigeratedcentrifuge, the sediment was used for protein determi-nations, and the supernatant fluid was diluted withan equal volume of distilled water in a separatoryfunnel. NaOH (3 N) was added to this mixture untilthe color changed from deep red to yellow-orange. Themixture was extracted repeatedly with 30 ml ofhexane (Mallinckrodt Chemical Works, St. Louis)until no more prodigiosin was removed. The hexaneextracts were pooled and dried for 2 h over anhydrousNa,SO4. The Na,SO4 was removed by filtration, andthe hexane solution was extracted twice with 30 ml of85% (vol/vol) aqueous ethanol containing 10% (voWwt) aqueous NaOH. These extracts were pooled,diluted with an equal amount of water, and thenextracted repeatedly with more hexane. Prodigiosinwas then extracted from the hexane solution with 85%aqueous ethanol containing 1% (vol/vol) aqueousglacial acetic acid until no more of the pigment wasremoved. The deep-red ethanolic solution was dilutedwith an equal amount of distilled water, treated with3 N NaOH until the color changed to orange, andagain extracted with hexane. The hexane extractswere treated for 2 h over anhydrous Na,SO4 and driedon a rotary evaporator at -20 C. The dried pigmentwas dissolved in 3 ml of hexane and further purifiedby column chromatography on diatomaceous earth(Hy Flo Super-cel, Johns Manville Co.). A red and anorange band developed, representing the acid andbase forms, respectively, of prodigiosin (8). The twobands were eluted from the column with hexane and95% aqueous ethanol, sequentially, and were com-bined for counting. When examined by visible andultraviolet spectrophotometry, the purified prodigi-osin showed a single peak at 534 nm.

Analytical procedures. Purified prodigiosineluted from the columns was concentrated to dry-ness on a rotary evaporator. The dried material wasdissolved in 10 ml of Fluoralloy TLA mix in toluene(Beckman Instruments, Inc., Fullerton, Calif.), andthen was counted in a Beckman scintillation counter,model CPM-100. Protein obtained from cells was pre-

pared for radioactive counting by the procedure ofGreenberg (6). These samples also were counted inthe Beckman scintillation counter.

Protein was measured by the method of Lowry et al.(12) with bovine serum albumin as a standard. Afterextraction with acidic methanol (4.0 ml of 1 N HCladded to 96.0 ml of methanol), the total amount ofprodigiosin in cultures was determined spectrophoto-

metrically in a Hitachi-Perkin-Elmer spectropho-tometer, model 139, according to the procedure ofGoldschmidt and Williams (4).Chemicals. Unlabeled and -4Clabeled amino acids

were obtained from Nutritional Biochemicals Co.,Cleveland, Ohio, and from New England NuclearCorp., Boston, Mass., respectively. Various methio-nine analogues, S-adenosylmethionine, and N-5-methyltetrahydrofolic acid were purchased fromSigma Chemical Co., St. Louis, Mo.

RESULTSEffect of methionine on biosynthesis of

prodigiosin by NPC. Methionine alone did notcause biosynthesis of prodigiosin in NPC of S.marcescens, but, when added to suspensionsinduced by the effective amino acids, a greateramount was synthesized than by addition oftheamino acids alone (Table 1). In addition, methi-onine shortened the lag period for biosynthesis.The optimal concentration of proline for synthe-sis of the maximal amount of prodigiosin wasreduced from 85 to 42.5 mM (Fig. 3). A similar

TABLE 1. Effect of methionine on biosynthesis byNPC of prodigiosin for amino acidsa

Lagperiod Prodig-Concn of before ii

Aminoacd Concn methio- prodig- (n/mgAminO aCid C(mncn) (Mnine josm(mM) (MM) first of pro.observed tein)

(h)

None 0 00.27 or 0

85

L-Alanine 55 0 14 7255 0.27 7 244

L-Aspartic acid 75 0 12 6775 0.27 8 201

L-Glutamic acid 70 0 15 870 0.27 10 19

DL-Histidine 95 16 17395 0.27 10 352

L-Proline 85 0 12 10985 0.27 6 346

L-Sermne 95 0 14 2095 0.27 6 71

Suspensions ofNPC of S. marcescens strain Nima(36.8 mg of protein) were incubated at 25 C on arotary shaker. All amino acids were added at 0 h, andprodigiosin was measured after incubation for 36 h.

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400

300

0

0E

:- 2001

on

85 170PROLINE ImM)

FIG. 3. Effect of L-methionine (0.27 mM) on bio-synthesis of prodigiosin in the presence of variousconcentrations of L-proline. Amino acids were addedat zero time to suspensions of NPC (36.8 mg ofprotein) of S. marcescens strain Nima. Cultures wereincubated on a rotary shaker at 25 C for 36 h, andthen prodigiosin was measured.

reduction occurred with the other amino acids.The optimal concentration of methionine for

maximal synthesis of prodigiosin in the pres-ence of 42.5 mM proline was 0.27 mM (Fig. 4).This concentration is about two logs less thanthe optimum for amino acids that can causebiosynthesis of prodigiosin in NPC (15). Methi-onine must be added within the first 4 h ofincubation for maximal synthesis of prodigiosin(Fig. 5). Although data for proline are shown inFig. 3 through 5, the same effects were obtainedwhen methionine was added to NPC induced tosynthesize prodigiosin by addition of any of theother five effective amino acids contained incasein hydrolysate (Table 1).Incorporation of the methyl group of me-

thionine into prodigiosin. We previously es-tablished that "CH8-methionine was incorpo-rated into prodigiosin more than "COOH-methionine or methionine-2-"1C when NPCwere caused to synthesize the pigment by addi-tion of proline (14). Incorporation of the methyl-labeled compound paralleled biosynthesis ofprodigiosin, and methionine apparently fur-

nished a methyl group that could be incorpo-rated into prodigiosin at carbons C2, C6, andpossibly C5 (Fig. 1).By use of mutant OF that synthesizes nor-

prodigiosin, we investigated the contribution bymethionine of a methyl group of C6. Norprodigi-osin was not synthesized when mutant OF wasgrown at 39 C, but, when these nonpigmented

400

300

E

200

20

5

CL00j2

0 0. 17 0 34 0 51METHIONINE ImNl'

FIG. 4. Effect of various concentrations of methio-nine on biosynthesis of prodigiosin by NPC of strainNima caused to synthesize the pigment by addition ofproline (42.5 mM). Other conditions were identical tothose described in legend of Fig. 3.

400

z

3000

ik 200z

o 100

tL

0 _g0 16 24 36 40

HOURS

FIG. 5. Effect of time of addition of methionine(0.27 mM) on biosynthesis of prodigiosin by NPC ofstrain Nima caused to synthesize the pigment byaddition of proline (42.5 mM). Other conditions wereidentical to those described in legend of Fig. 3.

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bacteria were harvested, washed, and incubatedat 25 C in the presence of proline, the pigmentwas produced just as in the case of formation ofprodigiosin by strain Nima (Table 2). Sincenorprodigiosin is difficult to purify, we firstconverted the pigment into prodigiosin bychemical methylation using diazomethane (9).The conversion was necessary to obtain purifiedpigment for comparison of isotope incorpora-tion. Some norprodigiosin undoubtedly was lostduring methylation, but comparison betweenexperiments using mutant OF should be valid.The amount of norprodigiosin measured as

prodigiosin was consistent in different experi-ments (Tables 2 and 3). Conditions used in theexperiments were optimal for synthesis of pro-digiosin, not of norprodigiosin, and this fact, aswell as the chemical manipulations, could ac-count for the lesser amount of pigment formedby NPC of mutant OF than by NPC of thewild-type strain Nima.

Isotope from "CH,-methionine was incorpo-rated into prodigiosin synthesized by NPC ofwild-type strain Nima 40 to 50 times more thanwas isotope from methionine-2-14C or 14COOH-methionine (Table 2). In contrast, lack of the

TABLE 2. Incorporation of proline and methionine into norprodigiosin synthesized by mutant OF andprodigiosin synthesized by wild-type strain Nima by NPCa

Norprodigiosin5 Prodigiosinc

Total Sp act Sp actIsotope added activity Total Total (counts per Utiliz- Total Total (counts per

added (pig/ml activity min per pg a d (pg/mg activity min per pg(counts/min) of pro- (counts/ of prodigio- ation of pro- (counts/ of prodigio-

tein) min) sin per mg tein) min) sin per mgof protein) of protein)

14C-proline (UL) 1,160,000 167 3,600 2,160 0.31 350 15,800 4,51014C-methionine (UL) 1,310,000 169 4,080 2,410 0.31 346 24,300 7,020"4COOH-methionine 15,800,000 172 2,600 1,500 0.017 355 6,400 1,800Methionine-2-14C 16,000,000 170 6,600 3,900 0.04 350 7,500 2,1404CHs-methionine 14,500,000 164 32,240 19,660 0.222 347 301,600 86,920

a Biosynthesis of norprodigiosin and prodigiosin was effected in NPC (38.4 mg of protein) of mutant OF andof wild-type strain Nima, respectively, by addition of proline (42.5 mM) and methionine (0.27). Suspensions ofNPC were incubated at 25 C on a rotary shaker for 30 h, at which time samples were analyzed.

b Measured as prodigiosin after methylation of norprodigiosin by diazomethane.c Data from Qadri and Williams (14).d Amount of 14C incorporated x 100.

TABLE 3. Incorporation of proline and methionine into norprodigiosin synthesized by NPC of mutant OF inpresence of methylating enzyme from Serratia marcescens mutant WF'

Norprodigiosin after enzymatic methylationb Cell protein

Total activity Sp actIsotope added added Total Total (counts per Utiliz- Total Utiliz-

(counts/min) (pg/mg of activity min perdgi ationc activity ationprotein) (counts/min) sin per mg (%) (counts/min) (%)

of protein)

4C-proline (UL) 1,190,000 164 4,330 2,640 0.36 211,000 17.74C-methionine (UL) 1,250,000 161 7,660 4,760 0.61 60,700 4.9"COOH-methionine 11,900,000 167 2,900 1,740 0.024 506,000 4.3'4C-methionine (UL) 15,300,000 162 8,060 4,980 0.05 716,000 4.714CH3-methionine 12,100,000 164 110,800 67,560 0.92 391,000 3.2

a Biosynthesis of norprodigiosin was effected in NPC (42.5 mg of protein) of mutant OF by addition of proline(42.5 mM) and methionine (0.27 mM). Cell-free extract (4.8 mg of protein) from mutant WF was present in allexperiments. Conditions of incubation and analysis were identical to those described in Table 2.

b Presence of extract from mutant WF converted norprodigiosin to prodigiosin. Analyses were made onpurified prodigiosin isolated from the suspensions.

c Amount of 14C incorporated x 100.

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methyl group on C6 of norprodigiosin reducedthe difference in incorporation to factors of 5and 13, respectively, for methionine-2-14C and4COOH-methionine when compared with4CH,-methionine. When incorporation of"CH,-methionine into norprodigiosin is com-pared with incorporation into prodigiosin, it isevident that loss of the methyl group from theformer pigment reduced incorporation by about80%. Both of the pigments incorporated isotopefrom proline-U-14C and methionine-U-14C al-though the specific activities for norprodigisionwere less.The colorless mutant WF can enzymatically

methylate norprodigiosin to form prodigiosin (5,23). When suspensions of NPC of mutant OFwere incubated at 25 C along with a particulate,cell-free extract prepared from mutant WF, thespecific activity of the prodigiosin isolated fromsuspensions containing "4CH,-methionine wasincreased about 80% (Table 3) as comparedwith suspensions not containing the extract(Table 2). Presence of the methylating enzymealso doubled the incorporation of methionine-U-14C but did not appreciably affect incorpora-tion of proline-U-14C, "4COOH-methionine, andmethionine-2-14C (Table 3). Adenosine triphos-phate probably served as the energy source forthe reaction and was generated by whole cells ofmutant OF because addition of proline to theNPC stimulated oxygen uptake (S. M. H. Qadriand R. P. Williams, unpublished data). Similarextracts prepared from E. coli or P. aeruginosadid not increase incorporation of "4CH3-methio-nine into norprodigiosin, although the extractswere active in transmethylation for other sys-tems.The data in Table 3 show that isotope from

proline and from the various forms of labeledmethionine was taken up by the bacteria andincorporated into protein. Proline was utilizedmore efficiently than methionine, the variousforms of which were incorporated into protein toabout the same extent.

Effect of various methyl donors upon bio-synthesis of prodigiosin by NPC of wild-typestrain Nima. Data shown in Table 4 suggestedthat transmethylation of prodigiosin was medi-ated by the high energy compound S-adenosyl-methionine rather than by tetrahydrofolic acidcompounds. One of the latter compounds hadno effect upon biosynthesis of prodigiosin byNPC of strain Nima, whereas, in the presence ofS-adenosylmethionine, about as much of thepigment was formed as with methionine. Theoptimal concentration of S-adenosylmethioninefor maximal synthesis of prodigiosin in the

presence of 42.5 mM of proline was 0.5 mM(Fig. 6).Effect of analogues of methionine upon

biosynthesis of prodigiosin by NPC of wild-type strain Nima. None of several analogues ofmethionine affected biosynthesis of prodigiosinby suspensions of NPC of strain Nima. Whennorleucine (2.5 mM), norvaline (2.5 mM),ethionine (1.5 mM), or 6-CH,-methionine (1.5mM) were added under the conditions shownin Table 4, biosynthesis of prodigiosin wasunaffected in the presence or absence ofmethionine (0.27 mM) plus proline (42.5 mM).

DISCUSSIONThe role of methionine in biosynthesis of

prodigiosin was unsuspected until our previousreport (14). Certain other amino acids causedbiosynthesis of the pigment in nonproliferatingbacteria (20), and the "C-label from some wasincorporated into prodigiosin formed by growingcells of S. marcescens (13, 17, 18).Methionine probably is converted into the

high-energy sulfonium compound, S-adenosyl-methionine, that then serves as the methyldonor. Energy for the conversion apparently isgenerated by utilization of the amino acidadded to cause biosynthesis of prodigiosin inNPC because only when such amino acids (Ta-ble 1) are added to NPC are oxygen uptake and

TABLE 4. Effect of methyl group donors onbiosynthesis of prodigiosin by NPC of Serratia

marcescens strain Nimaa

Amino acid Prodig-iosin

L-Pro- L-Me- Methyl donor (ag/mgline thionine of pro-

(mM) tein)

42.5 0 None 10842.5 0.27 None 34742.5 0 N-5-CH,-tetrahydrofolate 107

(0.50 mM)42.5 0.27 N-5-CH3-tetrahydrofolate 344

(0.50 mM)42.5 0 S-adenosylmethionine 312

(0.50 mM)42.5 0.27 S-adenosylmethionine 348

(0.50 mM)a Methyl donors were added at zero time to suspen-

sions of NPC along with the amino acids. Sampleswere taken every 6 h to measure prodigiosin. Figuresin the table represent amount of prodigiosin synthe-sized after incubation at 25 C for 36 h on a rotaryshaker. NPC contained 38.8 mg of protein.

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40C

300

C_

200

° 100

0 0. 25 0. 5 0. 75 1. 0

S-ADENOSYL METHIONINE mM'

FIG. 6. Effect of various concentrations of S-adenosylmethionine on biosynthesis of prodigiosin byNPC of strain Nima caused to synthesize the pigmentby addition of proline (42.5 mM). Other conditionswere identical to those described in legend of Fig. 3.

formation of prodigiosin stimulated (S. M. H.Qadri and R. P. Williams, unpublished data).When added by itself, S-adenosylmethioninedoes not cause biosynthesis of prodigiosin inNPC. Since analogues of methionine that are

known to inhibit utilization of the amino acidfor growth of bacteria by repressing enzymesfor its synthesis or by feedback inhibition (7,11) did not affect biosynthesis of prodigiosin,probably little methionine is synthesized byNPC. The cellular amino acid pool of NPC ofstrain Nima probably contains little methioninebecause small amounts of the amino acidcaused synthesis of more prodigiosin within a

relatively short time after addition.Our experiments with mutant OF indicate

that the methyl group on C6 of prodigiosincomes from methionine. Biosynthesis of nor-

prodigiosin by NPC of mutant OF in the pres-ence of "CH,-methionine and of a cell-freeextract containing the methylating enzymefrom mutant WF yielded prodigiosin with a

specific activity almost as great as prodigiosinsynthesized by NPC of wild-type strain Nima inthe presence of the specifically labeled aminoacid. Without the enzyme from mutant WF,norprodigiosin after chemical methylation toprodigiosin had about 70% less specific activity.The cell-free extract from mutant WF could notsynthesize the monopyrrole moiety (MAP) ofprodigiosin, although transmethylase activitywas present. Since the mutant cannot produce

the bipyrrole portion of prodigiosin (4, 5, 16) theonly way labeled prodigiosin can be synthesizedfrom norprodigiosin by the extract and "CH3-methionine is by methylation. The enzyme frommutant WF has some specificity for methylat-ing norprodigiosin because similar extracts fromE. coli and P. aeruginosa were unable to convertnorprodigiosin to prodigiosin in the presence ofmethionine, although the enzymes of the latterbacteria may have been unable to enter the cellsof S. marcescens.The methyl group on C2 of prodigiosin may

come from methionine as well as from themethene carbon at C5 (Fig. 1). The considerablespecific activity of norprodigiosin labeled by14CH,-methionine (Table 2) suggests that themethyl group, in addition to C6, is incorporatedelsewhere in the molecule. Investigations withmutants that synthesize only MAP may deter-mine whether the methyl group on C2 derivesfrom methionine. Such experiments may nowbe feasible since the technical difficulties forisolating MAP apparently have been solved (2).An important role of methionine in biosyn-

thesis of prodigiosin is methylation of the pig-ment. However, this function alone seems una-ble to explain the sparing effect of the aminoacid upon other amino acids that cause biosyn-thesis of prodigiosin in NPC (Table 1) or uponformation of greater amounts of pigment morerapidly. Methionine may function in other waysin the biosynthesis of prodigiosin, but, untilmore is learned about the pathway and enzy-mology of prodigiosin synthesis, a completeunderstanding of the function(s) cannot beobtained.

ACKNOWLEDGMENTSThis study was supported by grant Q-359 from the Robert

A. Welch Foundation, Houston, Texas. S. M. H. Q. is apostdoctoral fellow supported by the Foundation.

LITERATURE CITED

1. Bunting, M. I. 1940. A description of some color variantsproduced by Serratia marcescens strain 274. J. Bacte-riol. 40:57-68.

2. Deol, B. S., J. R. Alden, and J. L. Still. 1972. Theisolation and characterization of monopyrroles fromSerratis marcescens. Biochem. Biophys. Res. Com-mun. 46:1378-1385.

3. Efimenko, 0. M., G. A. Kuznetsova, and P. A. Yamikov.1956. Prodigiosin-an antibiotic from Bacterium prodi-giosum. Biokhimiya 21:416-421.

4. Goldschmidt, M. C., and R. P. Williams. 1968. Thia-mine-induced formation of the monopyrrole moiety ofprodigiosin. J. Bacteriol. 96:609-616.

5. Green, J. A., and R. P. Williams. 1957. Studies onpigmentation of Serratia marcescens. IV. Analysis ofsyntrophic pigment. J. Bacteriol. 74:633-636.

6. Greenberg, D. M. 1957. Methods for isolation and degra-dation of labeled compounds, p.-708-710. In S. P.

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Colowick and N. 0. Kaplan (ed.), Methods in enzymol-ogy, vol. 4. Academic Press Inc., New York.

7. Gross, D., and H. Tarver. 1955. Studies on ethionine. IV.The incorporation of ethionine into the proteins ofTetrahymena. J. Biol. Chem. 217:169-182.

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