APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1976, p. 70-77Copyright C 1976 American Society for Microbiology

Vol. 31, No. 1Printed in U.S.A.

Macromolecular Syntheses During Biosynthesis of Prodigiosinby Serratia marcescens

ROBERT P. WILLIAMS,* RANDOLPH H. SCOTT,' DANIEL V. LIM, AND S. M. HUSSAIN QADRI2

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

Received for publication 18 September 1975

Amino acids that were utilized as sole sources of carbon and nitrogen forgrowth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin innon-proliferating bacteria. Addition of alanine, proline, or histidine to non-pro-liferating cells incubated at 27 C increased the rate of protein synthesis and alsocaused biosynthesis of prodigiosin. No increase in the rate of protein synthesiswas observed upon the addition of amino acids that did not stimulate prodigiGsinbiosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of de-oxyribonucleic acid (DNA) (a small amount) also occurred after addition of aminoacids that resulted in biosynthesis of prodigiosin. After incubation for 24 h, thetotal amount of protein in suspensions of bacteria to which alanine or prolinewas added increased 67 and 98%, respectively. Total amounts of DNA and ofRNA also increased before synthesis of prodigiosin. The amounts of these macro-

molecules did not increase after addition of amino acids that did not induce bio-synthesis of prodigiosin. However, macromolecular synthesis was not relatedonly to prodigiosin biosynthesis because the rates of DNA, RNA, and proteinsynthesis also increased in suspensions of bacteria incubated with proline at39 C, at which temperature no prodigiosin was synthesized. The quantities ofDNA, RNA, and protein synthesized were lower in non-proliferating cells than ingrowing cells. The data indicated that amino acids causing biosynthesis of pro-

digiosin in non-proliferating cells must be metabolized and serve as sources ofcarbon and of nitrogen for synthesis of macromolecules and intermediates.Prodigiosin was synthesized secondarily to these primary metabolic events.

Non-proliferating Serratia marcescens incu-bated at 27 C can synthesize prodigiosin afteraddition of certain amino acids such as alanine,aspartate, glutamate, histidine, hydroxypro-line, ornithine, proline, and serine (9, 10, 17).Biosynthesis of prodigiosin occurs in such asystem, independent of bacterial proliferation(15).

Concentrations of 55 to 95 mM of the aboveamino acids are optimal for prodigiosin syn-thesis (10). Although under optimal conditionsmore prodigiosin is formed by non-proliferatingcells than by growing cultures (10), the quantityof pigment synthesized is small compared tothe amount of amino acid added. In addition,a lag period of 4 h or more occurs between timeof addition of amino acid and appearance ofprodigiosin (10). These observations suggestthat biosynthesis of prodigiosin is preceded by

I Present address: Department of Biochemistry andMolecular Biology, University of Texas Medical School atHouston, Houston, Tex. 77025.

2Present address: Department of Pathology, Ufiiversityof Texas Medical School at Houston, Houston, Tex. 77025.

70

other metabolic activities and that the aminoacids are utilized to form macromolecules andmetabolic intermediates, as well as for syn-thesis of prodigiosin. This article describes in-vestigations on the synthesis of deoxyribo-nucleic acid (DNA), ribonucleic acid (RNA), andprotein after addition of amino acids to non-proliferating suspensions of S. marcescens ascompared with growing cells.

MATERIALS AND METHODS

Organism, growth media, and preparation ofnon-proliferating cells. S. marcescens Nima wascarried as a stock culture on Trypticase soy agar(BBL). Bacteria were grown either in liquid minimalmedium containing (wt/vol) 1.0% glycerol, 0.5% am-monium citrate, 0.05% MgSO4-7H20, 1.0% K2HPO4,0.5% NaCl, and 0.005% ferric ammonium citrate (1),or in liquid complete medium containing the sameingredients plus 0.1% yeast extract (Difco) and 0.2%casein hydrolysate (Sigma Chemical Co., St. Louis,Mo.). Preparation of non-proliferating bacteria wasdescribed previously (10). These bacteria containedno prodigiosin. A saline suspension of bacteria at anoptical density of 2.5 at 600 nm represented 1.5 mg

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MACROMOLECULAR SYNTHESES AND PRODIGIOSIN 71

of protein per ml. Prodigiosin biosynthesis in thesenon-proliferating cells was effected by addition ofL-alanine, .-proline, or .-histidine (0.1 M) to suspen-sions incubated at 27 C on a rotary water bathshaker (model G76, New Brunswick Scientific Co.,Inc., New Brunswick, N.J.) set at 200 rpm (17).Since addition of L-lysine (0.1 M) to suspensions ofnon-proliferating bacteria did not effect formation ofprodigiosin (17), this amino acid was used as a nega-tive control. L-Methionine (0.27 mM) was added toall suspensions. Although methionine alone did noteffect synthesis of prodigiosin, the amino acid re-duced the amount of other amino acids needed,increased the amount of prodigiosin formed, anddecreased the time necessary for the appearanceofprodigiosin when added in conjunction with aminoacids required for pigment formation (11).

Utilization of amino acids as a carbon source forgrowth was tested in a modified minimal mediumcontaining 1% (wt/vol) of the appropriate amino acidin the place of glycerol, and with the substitution offerric chloride and ammonium chloride for ferricammonium citrate and ammonium citrate, respec-tively. When amino acids were tested as a source ofnitrogen for growth, the modified minimal mediumcontained 1% of the amino acid; ferric chloride wassubstituted for ferric ammonium citrate, and sodiumcitrate was substituted for ammonium citrate. Allmedia were adjusted to pH 7.2. From a 24-h culturein minimal medium, approximately 105 bacteriawere inoculated into 2 ml of the modified media.Growth was measured as optical density at 600 nmon a Gilford model 300-N microsample spectropho-tometer (Gilford Instrument Laboratories, Inc.,Oberlin, Ohio) after incubation for 48 h at 27 C onthe rotary shaker at 200 rpm.

Labeling procedures. Rates of DNA, RNA, andprotein synthesis were determined at various timesduring incubation by removing 0. 1-ml samples fromthe cell suspensions and mixing these samples with0.1 ml of labeling medium kept at the same tempera-ture as the cell suspension (3). The labeling mediumcontained [6-3H]thymidine, [5-3Hluridine, or i[1-4C]ileucine, and the same concentration of aminoacids or medium as in the suspension of cells sam-pled. Triplicate samples were removed at each timeinterval for the measurement of DNA, RNA, orprotein synthesis. At 5-, 10-, and 15-min intervals,1 ml of cold 5% trichloroacetic acid was added im-mediately to each sample, and the sample wasmixed. After 1 h at 0 C, acid-precipitable materialwas collected on glass fibre filters (Whatman GF/C),and the precipitate was washed with 10 ml of cold5% trichloroacetic acid. Filters were dried for 15 hin an oven at 60 C and then counted in a Beckmanmodel LS 250 liquid scintillation system (BeckmanInstruments, Inc., Fullerton, Calif.). The rate ofsynthesis of DNA, RNA, or protein was determinedby expression of the counts obtained as picomolesper 10 min of incorporation of precursor.

Chemical analyses. Samples for DNA and RNAwere prepared by the method of Neidhardt andMagasanik (8). Two milliliters of cell suspension wasadded to 2 ml of 10% trichloroacetic acid and storedat 4 C until analyzed. Before analysis for DNA and

RNA, samples were centrifuged, and the pelletswere washed with cold 5% trichloroacetic acid. Thewashed pellets then were resuspended in 4 ml of 5%trichloroacetic acid and were heated at 100 C for 30min. The cooled mixtures were centrifuged, and thesupernatant fluids were carefully decanted. Thepellets were resuspended again in 4 ml of 5% tri-chloroacetic acid and heated at 100 C. After centrifu-gation, the supernatant fluids from both hydroly-sates were combined and assayed directly for DNAand RNA. DNA was measured by the diphenyl-amine procedure (2), and RNA was measured bythe orcinol procedure (12). To determine the con-centration of protein, the method of Lowry et al.(5) was followed, except that the cell pellet waswashed also once with acidic methanol (4) to removeprodigiosin that might interfere with protein de-termination. Prodigiosin was extracted from cellswith acidic methanol and spectrophotometricallymeasured by the procedure of Goldschmidt andWilliams (4).

Materials. All chemicals used were of reagentor analytical grade unless otherwise noted. Acetal-dehyde, orcinol, and diphenylamine were obtainedfrom Fisher Scientific Company (Houston, Tex.).Amino acids were purchased from Sigma ChemicalCompany. [6-3Hlthymidine, [5-3H]uridine, and L-[1_-4C]leucine were obtained from Amersham/SearleCorp. (Arlington Heights, Ill.).

RESULTSPigment formation. Addition of L-alanine, L-

proline, or L-histidine plus L-methionine tosuspensions of non-proliferating cells resultedin formation of prodigiosin after differentperiods of incubation at 27 C (Fig. 1). Additionof ilysine plus methionine did not effect pro-digiosin formation in non-proliferating cells.Non-proliferating cells incubated at 39 C did notsynthesize prodigiosin in the presence of any ofthese amino acids (10).

Rates of macromolecular synthesis in non-proliferating cells. (i) DNA synthesis at 27 C.The amino acids that caused biosynthesis ofprodigiosin also increased the rate of [3H]thymi-dine incorporation; conversely, lysine causedno increase in the rate of incorporation (Fig.2). An increase in rate of DNA synthesis, asmeasured by labeled thymidine incorporation,occurred before the onset of pigmentation insuspensions containing alanine, proline, orhistidine. In each instance, the peak of in-corporation corresponded closely to the appear-ance of pigment, with the rate of DNA syn-thesis increasing first in the presence of alanine,followed by proline and, finally, by histidine.

(ii) RNA synthesis at 27 C. The rate of [3H]-uridine incorporation, as a measurement ofRNA synthesis, increased only in suspensionsof non-proliferating cells containing alanine,proline, or histidine, and not in suspensions

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2000 I I I RNA synthesis increased before an increase inpigmentation and in the rate of protein syn-thesis occurred. These data suggested a re-quirement for DNA and RNA synthesis beforethe beginning of protein synthesis.

(iv) DNA, RNA, and protein synthesis at1600 _ / _ 39 C. Prodigiosin is not synthesized at 39 C by

S. marcescens Nima (10, 18). If the increasedrates of macromolecular synthesis shown inFig. 2 through 4 were related only to biosyn-thesis of pigment, such increases would not

-1200 _ / _ occur in suspensions of non-proliferating cells1200 incubated at the higher temperature. Figure 5

0/ shows that there is an increase in the rates of

DNA, RNA, and protein synthesis at 39 C inE suspensions containing proline and methionine.

800_ As would be expected at the higher tempera-ture, during which metabolic processes occur

V) /more rapidly, the maximal rate was reachedo / earlier, and the slopes of increase and decrease

for all three parameters were sharper than at27 C. However, no prodigiosin was synthesizedat 39 C. Again, in the presence of lysine, nosignificant increases occurred in the rates ofDNA, RNA, or protein synthesis.

0 >= 0.16 1 I I I I

0 2 4 6 810 12 14 16 18 20 22 24 .0.14 -

TIME (HRS.)FIG. 1. Prodigiosin formation in suspensions of ° 0.12-

non-proliferating cells incubated at 27 C on a rotaryshaker. Amino acids (0.1 M) plus L-methionine (0.27mM) were added at zero time, and the amount of o 0.10prodigiosin formed was measured spectrophotometri- zcally (4). All suspensions contained 1.5 mg of pro- a; \tein per ml. Symbols: 0, proline; *, alanine; A, z 0.08histidine; 0, lysine.

0.06 -

containing lysine (Fig. 3). Like DNA synthesis,the peak ofRNA synthesis corresponded closely . \to the appearance of pigment. o

(iii) Protein synthesis at 27 C. Addition of\alanine, proline, or histidine to non-proliferat- 0.02ing cells caused a linear increase in the rate of[14C]leucine incorporation at the beginning of 0 l l l l l l Epigmentation (Fig. 4). In contrast to DNA and 0 2 4 6 8 10 12 14 16 18 20 22 24RNA synthesis, however, the rate of protein TIME 1HRS.)synthesis declined gradually after reaching a T2M (HR

of Ipeak. As with RNA synthesis, the greatest in- FIG. 2. Rate of DNA synthesis in suspensions ofcrease in protein synthesis occurred in the non-proliferatingcells incubated at27 C on a rotarysuspension containing proline. No increase in shaker. Rate of [3H]thymidine (specific activity, 26rate of labeled leucine incorporation occurred Ci/mmol) incorporation into DNA was determined

as described in the text. Other conditions were identi-in the presence of lysine. cal to those described in the legend of Fig. 1. NoAs shown in the preceding three figures, in prodigiosin was detected in suspensions containing

non-proliferating cell suspensions containing lysine. Symbols: 0, proline; *, alanine; A, histidine;alanine, proline, or histidine, rates ofDNA and 0, lysine.

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MACROMOLECULAR SYNTHESES AND PRODIGIOSIN

700 1 1 1 1 thesizing prodigiosin increased to a peak andthen declined over a 24-h period (Fig. 7). Al-though the total amounts of nucleic acids in-

/M\creased earlier in suspensions containing ala-nine than in suspensions containing proline,DNA and RNA remained at high levels longerin suspensions containing proline. There was

- 500 / \ no increase in either DNA or RNA content inZE _00 E _ suspensions containing lysine. In suspensions2 O \ containing proline or alanine, the amount of

protein increased continuously during a 24-h400 _ j \ _ period, with no increase occurring in suspen-

sions containing lysine. Similar results were

obtained with [3H]thymidine, [3H]uridine, andI[4C]leucine used to follow DNA, RNA, and

_ 300 _ / \ _ protein synthesis over a 24-h period in non-

proliferating cells.Amino acids as sources of carbon or nitrogen

OE0 I2 - 1400

10oo 1200-

02 4 6 8 0 12 14 16 18 20 22 24TIME (HRS.)

FIG. 3. Rate of RNA synthesis in suspensions of Xnon-proliferating cells incubated at 27 C on a rotary ° 800shaker. Rate of incorporation of[3H]uridine (specific zactivity, 407 t,Cil,tmol) into RNA was determinedas described in the text. Other conditions were identi- Zcal to those described in the legend of Fig. 1. No - 600-prodigiosin was detected in suspensions containing/lysine. Symbols: 0, proline; U, alanine; A, histidine;0, lysine.

> 400DNA, RNA, and protein synthesis in grow-

ing cells. Rates of DNA, RNA, and proteinsynthesis increased before the appearance ofprodigiosin in Serratia growing on minimal 200medium at 27 C (Fig. 6). While RNA and pro-tein increased at a similar point in growth,the increase in DNA occurred several hoursearlier. When measured on a per cell basis, 0 2 4 6 8 10 12 14 16 18 20 22 24the rates of macromolecular synthesis for non-proliferating cells were much lower for RNA T ME H R S.)and protein and almost negligible for DNA. FIG. 4. Rate of protein synthesis in suspensionsSmall amounts of RNA and protein synthesis, of non-proliferating cells incubated at 27 C on aand practically no DNA synthesis, occurred in rotary shaker. Rate of ['4C]leucine (specific activity,thenonproicsa11 pCiltL/mol) incorporation into protein was de-

the non-proliferating cells as compared with termined as described in the text. Other conditionscells growing on minimal medium. were identical to those described in the legend of

Total amounts of DNA, RNA, and protein Fig. 1. No prodigiosin was detected in suspensionsin suspensions of nonproliferating cells. The containing lysine. Symbols: *, proline; U, alanine;amounts of DNA and RNA in suspensions syn- A, histidine; 0, lysine.

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74 WILLIAMS ET AL.

I I I

1800 F-

0.15

0.00

0 2 4 6 8 10 12 14 16 18 20 22 24TIME (HRS. )

FIG. 5. Comparison of rates of DNA (-), RNA(U), and protein (A) synthesis in suspensions ofnon-proliferating cells incubated at 27 C (solid lines)or at 39 C (dashed lines) on a rotary shaker. Ratesof [3H]thymidine (specific activity, 26 Ci/mmol) in-corporation into DNA, [3H]uridine (specific activity,407 ,uCi/l,mol) incorporation into RNA, and ['4C]-leucine (specific activity, 11 ,uCi/l,mol) incorporationinto protein were determined as described in thetext, after addition of 0.1 M L-proline and 0.27 mML-methionine to cell suspensions. Data for rates ofsynthesis at 27 C are taken from Fig. 2 through 4.Heavy arrow indicates time prodigiosin first was

measurable in suspensions incubated at 27 C. Nopigmentation and no macromolecular syntheses were

observed in suspensions incubated with L-lysine andL-methionine at 27 or 39 C (data not shown).

for growth. Only those amino acids that couldbe used as sources of both carbon and nitrogenfor growth effected biosynthesis of prodigiosinin non-proliferating cells (Table 1). If the bac-teria were provided with a source of carbon,glycine or threonine served as sources of nitro-gen for growth, and prodigiosin also was syn-thesized in the cultures. However, these aminoacids alone did not result in the biosynthesisof prodigiosin in cultures of non-proliferating

bacteria. Ornithine supported poor growth as asource of carbon, with no prodigiosin synthe-sized, but the pigment was formed when theamino acid was added to suspensions of non-proliferating cells.

DISCUSSIONOnly amino acids that were utilized as

sources of both carbon and nitrogen for growthresulted in biosynthesis of prodigiosin in non-proliferating cells of S. marcescens. After addi-tion of the amino acids, and before the appear-ance of prodigiosin, the rates of synthesis ofDNA, RNA, and protein increased. The datasuggested that significant metabolism occurredin the bacteria and that the added amino acidswere used for other cellular activities, proba-bly as sources of energy, of intermediates, andfor synthesis of enzymes, in addition to biosyn-thesis of prodigiosin. Such utilization of aminoacids probably accounted for the high concen-trations of effective amino acids required forsynthesis of prodigiosin by non-proliferatingcells (17).The lag period of 4 to 6 h after addition of

alanine or proline before appearance of pro-digiosin indicated the need for degradation ofthe amino acids and initiation of antecedentmetabolism before biosynthesis of the pigment.The prolonged lag after the addition ofhistidinesuggested that metabolism of this amino acidwas more complex, but reinforced the hy-pothesis that antecedent metabolism mustoccur before prodigiosin was synthesized. For-mation of prodigiosin was apparently a sec-ondary manifestation of active metabolism bythe bacteria because at 39 C, where pigmentwas not synthesized (10, 16, 18), increases inDNA, RNA, and protein synthesis also oc-curred.Although increased rates of synthesis of

macromolecules did occur, addition of the aminoacids neither caused bacterial multiplication(15) nor increased dry weight (R. Scott, Ph.D.thesis, Baylor College of Medicine, Houston,Tex., 1974). Furthermore, although there wasa significant increase in the rates of RNA andprotein synthesis in non-proliferating cells be-fore the appearance of prodigiosin, the rate ofDNA synthesis in non-proliferating cells wasinsignificant compared to growing cells (Fig. 6).These data suggested the absence of bacterialproliferation, but the presence of transcriptionand translation, during biosynthesis ofprodigio-sin. After catabolism, amino acids such as ala-nine, proline, or histidine, which were presentin large concentrations, probably supplied ma-jor pools of intermediates for metabolic pro-cesses.

a

a-c.

0C-,z

014

C-)

LA-

acrLJw

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MACROMOLECULAR SYNTHESES AND PRODIGIOSIN

0 2 4 68

10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24

TIMEIHRS. ) 4 TIME (HRS. )

FIG. 6. Rates of macromolecular synthesis in growing cells and in non-proliferating cells. Growing cells(in minimal medium) and non-proliferating cells (containing proline and methionine) were incubated at 27 Con a rotary shaking water bath. Rates of DNA, RNA, and protein synthesis were observed and are expressedon the basis of viable cell count. The growing cell suspension contained 6.6 x107 viable cellslml at zero time,and 3.7 x 1010 viable cellslml at 24 h. The non-proliferating cell suspension contained 1.2 x 10'0 viable cellslml at zero time, and 8.0 x 109 viable cellslml at 24 h. Specific activities of label in growing cells:[3H]thymi-dine, 242 IACiliAmol;[3H]uridine, 244MuCilAmol; and['4C]leucine, 6.6 ACiIumol. Specific activities of label innon-proliferating cells:[3H]thymidine, 26 Cilmmol; [3H]uridine, 407~XiIumol; and['4C]leucine, 11uCipumol. Heavy arrow indicates time when pigment was first detected. Symbols: 0, rate of DNA synthesis; U,

of synthesis; A, ofprotein synthesis. Note 10-fold difference in vertical scales of the two parts of

thefigure.

210 l l l l l The events occurring in the suspensions of200- DNA _ non-proliferating cells might be termed a

190 _ / _ "pseudo shift-up" in metabolism. Addition ofalanine, proline, or histidine provided thewashed bacteria with a source of carbon and

160 - _ nitrogen after the shift-down from 39 C. How-ever, metabolism could not be sustained be-

RNA cause ions such as magnesium or phosphorus360_Athat are required for ribosomal integrity and=

330_s _ for bacterial proliferation were not added (7).

E1 300~>\-_Increased rates of synthesis of RNA, protein,a \M and DNA are typical of a shift-up in growth(6). These higher rates of synthesis also ac-

240 _ count for increases in the total amounts ofllll l l l l l l l l l DNA, RNA, and protein (Fig. 7). The pseudo2400PROTE N

- shift-up of the non-proliferating S. marcescens

was maintained for 5 to 17 h, long enough forE 2ooo initiation of primary metabolism. However, thez 1600_ bacteria remained under the stress of growth°1200;- _ limitation and could not initiate cellular divi-

800-FEsion or continue synthesis of DNA. Multiplica-

________________________________ tion of the bacteria was not required for bio-0 2 4 6 8 10 12 14 16 18 2022 24 26 synthesis of prodigiosin.

TIMEI

HR S. Prodigiosin is a secondary metabolite and isofDNA, RNA, and protein

synthesized after the primary metabolic ac-present suspensions of non-proliferating cells that tivities of growth and cellular multiplicationare synthesizing (alanine and proline)

or not syn-thesizing (lysine) prodigiosin. Concentrations of hav eceased(15). Our experiment ssuggestamino acids and conditions of incubation werethat, although the pigment can be synthesizedidentical to those described in the legend of Fig. 1. by non-proliferating cells, other metabolic ac-Symbols: 0, proline; U, alanine; 0, lysine. tivities must occur before synthesis of prodigio-

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TABLE 1. Utilization ofamino acids as sources ofcarbon or nitrogen forgrowth andprodigiosin biosynthesis"

Growth (optical density at 600 Amt of prodigiosin synthe- Synthesis of pro-nm) sized by the growing culture digiosin by sus-

Amino acid addedb (gg/mg of protein) pensions of non-

Carbon Nitrogen proliferating bac-Carbon source Nitrogen source terinac

L-Alanine 3.23 3.58 334 323 +L-Arginine 0.12 0.19 0 0DL-Aspartate 2.52 3.30 212 341 +L-Cysteine 0.09 0.17 0 0L-Glutamate 1.70 2.42 125 211 +Glycine 0.12 1.90 6 76L-Histidine 2.77 3.53 326 350 +DL-Isoleucine 0.12 0.12 0 0 -

L-Leucine 0.13 0.18 0 0 -

L-Lysine 0.09 0.28 0 0 -

L-Methionine 0.10 0.13 0 0 -

DL-Ornithine 0.32 3.06 0 208 +DL-Phenylalanine 0.14 0.13 0 0L-Proline 3.26 3.57 348 351 +DL-Serine 1.15 0.87 326 102 +DL-Threonine 0.16 0.82 0 116 -

DL-Tryptophan 0.07 0.16 0 0 -

L-Tyrosine 0.11 0.11 0 0 -

DL-Valine 0.14 0.21 0 0 -

Controld 0.13 0.17 0 0 -

a All cultures were incubated at 27 C for 48 h on a rotary shaker before growth or prodigiosin wasmeasured.bAmino acids (1%, wt/vol) were added to media lacking other sources of carbon or nitrogen. See text for

composition of the media.c Data from reference 10.d Control lacked either a source of carbon or of nitrogen.

sin. Utilization for production of energy and formaintenance of cellular metabolism of the car-

bon and nitrogen of the amino acids that causebiosynthesis of prodigiosin in non-proliferatingcells may involve primary metabolic pathwaysthat must precede synthesis of secondarymetabolites. De novo synthesis of secondarymetabolites may not be possible without an-

tecedent primary metabolism because regula-tion of the former phenomenon may dependupon some aspect of the latter.Amino acids that effected biosynthesis of

prodigiosin in non-proliferating cells may serve

a dual role in biosynthesis of prodigiosin. Theycan be sources of carbon and nitrogen forcellular metabolism, and they can be directprecursors of the pigment. Proline seems toserve a dual role. Other investigators (13, 14)have reported that the ring of proline enteredintact into the bipyrrole part of the prodigiosinmolecule. Our data demonstrated that theamino acid also was utilized as a source of car-bon and nitrogen for metabolism of non-pro-

liferating bacteria. Whether other amino acidsthat cause biosynthesis of prodigiosin in non-

proliferating cells have dual functions is notyet known.

ACKNOWLEDGMENTSThese studies were supported by grant Q-359 from the

Robert A. Welch Foundation, Houston, Tex. Randolph H.Scott was a predoctoral fellow, and Daniel V. Lim andS. M. Hussain Qadri were postdoctoral fellows, supportedby the Foundation.

LITERATURE CITED

1. Bunting, M. E. 1940. A description of some color vari-ants produced by Serratia marcescens strain 274. J.Bacteriol. 40:57-68.

2. Burton, K. 1955. A study of the conditions and mech-anism of the diphenylamine reaction for the colori-metric estimation of deoxyribonucleic acid. Biochem.J. 62:315-323.

3. Glaser, M., W. H. Bayer, R. M. Bell, and P. R. Vagelos.1973. Regulation of macromolecular biosynthesis in amutant of Escherichia coli defective in membranephospholipid biosynthesis. Proc. Natl. Acad. Sci.U.S.A. 70:385-389.

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

5. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

6. Maaloe, O., and N. 0. Kjeldgaard. 1966. Control of

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MACROMOLECULAR SYNTHESES AND PRODIGIOSIN 77

macromolecular synthesis. W. A. Benjamin Inc.,New York.

7. Mukherjee, P. P., M. E. Goldschmidt, and R. P.Williams. 1967. Enzymic formation of prodigiosinanalog by a cell-free preparation from Serratiamarcescens. Biochim. Biophys. Acta 136:182-184.

8. Neidhardt, F. C., and B. Magasanik. 1960. Studies onthe role of ribonucleic acid in the growth of bacteria.Biochim. Biophys. Acta 42:99-116.

9. Qadri, S. M. H., and R. P. Williams. 1972. Biosyn-thesis of the tripyrrole bacterial pigment, prodigio-sin, by nonproliferating cells of Serratia marcescens.Tex. Rep. Biol. Med. 30:73-83.

10. Qadri, S. M. H., and R. P. Williams. 1972. Inductionof prodigiosin biosynthesis after shift-down in tem-perature of nonproliferating cells of Serratia mar-cescens. Appl. Microbiol. 23:704-709.

11. Qadri, S. M. H., and R. P. Williams. 1973. Role ofmethionine in biosynthesis of prodigiosin by Serratiamarcescens. J. Bacteriol. 116:1191-1198.

12. Schneider, W. C. 1957. Determination of nucleic acidsin tissues by pentose analyses, p. 680-684. In S. P.Colowick and N. 0. Kaplan (ed.), Methods in enzy-mology, vol. 3. Academic Press Inc., New York.

13. Tanaka, W. K., L. B. deMedina, and W. R. Hearn.1972. Labeling patterns in prodigiosin biosynthesis.Biochem. Biophys. Res. Commun. 46:731-737.

14. Wasserman, H. H., R. J. Sykes, P. P. Peverada, C. K.Shaw, R. J. Cushley, and S. R. Lipsky. 1973. Bio-synthesis of prodigiosin. Incorporation patterns of'3C-labeled alanine, proline, glycine, and serineelucidated by Fourier transform nuclear magneticresonance. J. Am. Chem. Soc. 95:6874-6875.

15. Williams, R. P. 1973. Biosynthesis of prodigiosin, asecondary metabolite of Serratia marcescens. Appl.Microbiol. 25:396-402.

16. Williams, R. P., M. E. Goldschmidt, and C. L. Gott.1965. Inhibition by temperature of the terminal stepin biosynthesis of prodigiosin. Biochem. Biophys. Res.Commun. 19:177-181.

17. Williams, R. P., C. L. Gott, and S. M. H. Qadri. 1971.Induction of pigmentation in nonproliferating cellsof Serratia marcescens by addition of single aminoacids. J. Bacteriol. 106:444-448.

18. Williams, R. P., C. L. Gott, S. M. H. Qadri, andR. H. Scott. 1971. Influence of temperature of incuba-tion and type of growth medium on pigmentation inSerratia marcescens. J. Bacteriol. 106:438-443.

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