TRYPTOPHAN-NIACIN RELATIONSHIP IN XANTHOMONAS PRUNI'

R. G. WILSON2 AND L. M. HENDERSONDepartment of Biochemistry, Agricultural Experiment Station, Oklahoma State University,

Stillwater, Oklahoma

Received for publication 15 August 1962

ABSTRACT

WILSON, R. G. (Oklahoma State University,Stillwater) AND L. M. HENDERSON. Trypto-phan-niacin relationship in Xanthomonas pruni.J. Bacteriol. 85:221-229. 1963.-The observa-tion that Xanthomonas pruni, a bacterial patho-gen for the peach, requires niacin for growthand can use tryptophan or 3-hydroxyanthranilicacid as a substitute was confirmed. To determinewhether niacin is synthesized via the trypto-phan-3-hydroxyanthranilic acid pathway, ex-periments using labeled metabolites were under-taken. Labeled tryptophan, 3-hydroxyanthra-nilic acid, quinolinic acid, and nicotinic acid weresupplied in the basal medium in amounts suffi-cient to insure maximal growth. Nicotinic andquinolinic acids were isolated from the cellsafter the growth period. The isotope was incor-porated from the first three labeled compoundsinto niacin with dilutions approximately thesame in all cases, ranging from 7.6 to 17.1. Thedilution of isotopic niacin was 3.1- to 5.9-fold.Only labeled quinolinic acid gave rise to labeledquinolinic acid in the cell, but this acid gaverise to niacin with 10- to 12-fold reduction inspecific activity. The results indicate that ifquinolinate participates as an obligatory inter-mediate in the synthesis of niacin from trypto-phan, its concentration within the cell is verysmall and it does not equilibrate readily withexogenous quinolinate. The results confirm theconclusion, drawn from growth studies, thatniacin is needed to permit tryptophan synthesisat a sufficient rate to promote growth. In theabsence of an external source of niacin, trypto-

1 Taken from a thesis submitted in partialfulfillment of the requirements for the Ph.D.degree.

2 Present address: Graduate Department ofBiochemistry, Brandeis University, Waltham,Mass.

phan or some of its metabolites can promotegrowth by acting as precursors of niacin.

The investigation of Starr (1946) demonstratedthat Xanthomonas pruni, a peach-tree pathogen,requires an exogenous source of nicotinic acidwhen grown on a medium containing vitamin-free, acid-hydrolyzed casein. Davis, Henderson,and Powell (1951) found that tryptophan andniacin are interchangeable for supporting thegrowth of this organism. In addition, kynurenineand 3-hydroxyanthranilic acid, established asniacin precursors in other species, can replacetryptophan or niacin, within limits. Quinolinicacid exhibited a small growth-promoting activity,leaving its role as an intermediate in this bacte-rium somewhat in doubt.With the exception of these growth studies, all

efforts to find evidence for a tryptophan-niacinrelationship in bacteria or higher plants have beenunsuccessful (Yanofsky, 1954; Grimshaw andMarion, 1958; Leete, 1957; Henderson et al.,1959). To determine whether niacin is synthe-sized via the tryptophan-3-hydroxyanthranilatepathway, experiments were undertaken withlabeled tryptophan, 3-hydroxyanthranilic acid,quinolinic acid, and nicotinic acid. The resultssuggest that this species, in contrast to otherbacteria studied, does convert tryptophan andquinolinic acid to niacin.

MATERIALS AND METHODS

Materials. The amino acids and quinolinic andnicotinic acids were obtained from NutritionalBiochemicals Corp., Cleveland, Ohio. The L-tryptophan was a product of the CaliforniaCorporation for Biochemical Research. 3-Hy-droxyanthranilate (3-OHAA) was obtained fromHoffman-La Roche, Inc., Nutley, N.J. DarcoG-60 was partially inactivated by the method ofDalgliesh (1955). Nicotinic-7-C'4 acid was ob-tained from the California Corporation for 13io-

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chemical Research, and had a specific activity of5.8 or 9.2 me per mmole. Tryptophan-5-C'4 wassynthesized by Mathur and Henderson (1960),and had a specific activity of 285 ,c per mmole.Tryptophan-7a-C14 was synthesized by Hender-son, Rao, and Nystrom (1958), and by G. P.Mathur by the same procedure, and had a specificactivity of 153 or 266 ,ue per mmole. Tritium-labeled 3-hydroxyanthranilic and quinolinic acidswere prepared by the method of Wilzbach (1957).The specific activities were 717 and 670 tic permmole, respectively. Carboxyl-labeled 3-OHAAwas prepared by A. G. Fischer and R. J. Suhadol-nik according to the method of Ciereszko andHankes (1954), and had a specific activity of 220,uc per mmole. All compounds were shown to befree of radioactive impurities by paper chromatog-raphy in two or more solvent systems.

Jlethods. A culture of X. pruni, isolated fromthe leaves of a peach tree, was maintained onEmerson agar medium and subcultured at 15-dayintervals. The culture used was shown to be apathogen to the peach tree and to require niacinfor growth. Inocula were prepared from youngcells subcultured on Emerson broth. The cellswere grown in the broth (10 ml) for 20 hr, col-lected centrifugally, washed twice, and suspendedin 30 ml of isotonic saline. One drop of thissuspension was added to each culture tube.Cells from ten broth tubes were used to inoculatethe medium of Davis et al. (1951) used for theexperiments.The culture was checked routinely to make

certain that it required niacin and utilized trypto-phan or 3-hydroxyanthranilate in lieu of thevitamin. The quantities of each compound usedin these tests were those found by Davis et al.(1951) to give maximal growth in 48 hr for 3-OHAA and niacin, and in 72 hr for tryptophan.The cultures grown in tubes were aerated byshaking. Growth was determined turbidimetri-cally, at 650 m,u, at 4-hr intervals for a 72-hrperiod.

In all experiments using isotopically labeledcompounds, cells were grown in 3 liters of basalmedium. Tryptophan or nicotinic acid was addedto the medium before autoclaving but 3-hy-droxyanthranilate and quinolinate were sterilizedby Seitz filtration, then added aseptically to theautoclaved medium.

Air was continuously passed through the me-dium at the rate of approximately 10 liters per

min. The effluent air was passed through either2 N NaOH for C'4 experiments or concentratedH2SO4 for H3 experiments.To determine uptake of radioactive compound

from the medium by the organism, 2-ml sampleswere removed at approximately 4-hr intervals andthe transmission was determined. The sampleswere then centrifuged, the supernatant was with-drawn, and the amount of radioactivity present inthe medium was determined. In these same up-take experiments, when the isotope being utilizedwas C'4, the amount of radioactivity in theevolved carbon dioxide was determined. After 72hr of growth, the medium was centrifuged, andthe cells were washed with saline and collected bycentrifugation. The cells were weighed, andruptured by sonic oscillation; the pyridine nucleo-tides were extracted with hot 80% aqueousethanol.

Isolation of nicotinic and quinolinic acids. Thedisrupted cells, plus the alcoholic solution, werecentrifuged, and the residue was collected anddried. The alcohol extract was taken to drynessin vacuo, and the resulting residue was hydrolyzedwith 10 volumes of 1 N NaOH for 1 hr at 120 Cin the autoclave. The hydrolysate was neutra-lized with HCl made to volume, and sampleswere removed for microbiological assay. Nicotinicacid was determined by the microbiologicalassay with Lactobacillus plantarum. Quinolinicacid was determined as nicotinic acid after de-carboxylation with glacial acetic acid (Hender-son and Hirsh, 1949).To the remainder of the hydrolysate were

added 100.0 mg of nicotinic acid and 100.0 mgof quinolinic acid as carrier. The solution wastaken to dryness under reduced pressure, and theresulting residue was extracted with two portions(40-ml) of hot absolute methanol. The residue,after extraction, gave a negative eyanogen bro-mide test, indicating the absence of niacin (Wais-man and Elvehjem, 1941), and also a negativeferrous sulfate test (Koenigs, 1877), showing thatquinolinic acid was completely extracted. Themethanol extract was dried in vacuo, and theresidue was dissolved in 10 ml of water. Afteradjustment to pH 2, the solution was passedthrough a Dowex 50 W-X8 (200-400 mesh)column (10 X 100 mm) in the hydrogen phase.The quinolinic acid was not held on the resin,and was found in the first few fractions. Thecolumn was washed with 100 ml of distilled

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water and 100 ml of 0.3 N HCl. The nicotinicacid was then eluted from the column with 1 NHCl. The quinolinic acid was recovered byevaporation to dryness, and was crystallizedtwice from 2 ml of 580% ethanol. Niacin-con-taining fractions were pooled, evaporated to dry-ness, and the nicotinic acid was sublimed at 105C at 20 to 30 mm Hg.

Isolation of tryptophan. The dried residue of thecells, remaining after extraction with ethanol,was autoclaved in 10 volumes of 5.0 N NaOH for18 hr at 121 C. The hydrolysate was neutralizedwith HCl and centrifuged to remove unhydro-lyzed material. The resulting supernatant solutionwas adjusted to a convenient volume and sampleswere removed for microbio-assay for tryptophanusing L. plantarum. To the remainder of the solu-tion were added 100.0 mg of tryptophae ascarrier. Acetic acid was added to a final concen-tration of 57%, and the solution was passedthrough a partially deactivated Darco G-60charcoal column. The resulting filtrate wasnegative to the Hopkins-Cole test (Hopkins andCole, 1902), indicating that the tryptophan wasadsorbed. The column was washed with 50 ml of2% pyridine-5% acetic acid to remove phenylala-nine and tyrosine. The tryptophan was theneluted with 150 ml of 10%o phenol-2 %O acetic acidsolution.The tryptophan fractions were extracted with

diethyl ether to remove phenol and most of theacetic acid. The aqueous solution containing thetryptophan was then taken to dryness in vacuo.Glacial acetic acid (2 ml) was added to the resi-due, and the mixture was heated to extract thetryptophan. This solution was filtered through asintered-glass filter, and an equal volume of drybenzene was added to precipitate the tryptophanacetate. The mixture was placed in a Deepfreezeovernight and then centrifuged. The supernatantwas withdrawn; the tryptophan acetate waswashed with benzene, then ether, and dried over-night in a vacuum desiccator. The tryptophanacetate was dissolved in 1 to 2 ml of warm water.Absolute ethanol (2 volumes) was added, and thetryptophan was crystallized overnight at -10 C.The amino acid was recrystallized twice from65% ethanol, then washed with cold ethanol andwater and dried in vacuo.

Isotope determination. Analyses were made ina Packard Tri-Carb liquid scintillation spectrom-eter. Portions (0.2 ml) of dissolved samples

IL)z

0gCm

HOURS

FIG. 1. Growth of Xanthomonas pruni in re-sponse to niacin and its precursors. 0, nicotinicacid (0.05 igl/ml); A, 3-hydroxyanthranilic acid(0.4 jig/ml); *, DL-tryptophan (6.12 ,ug/mi); ,no addition.

were added to 9.8 ml of scintillation fluid (Belland Hayes, 1959), and the samples were countedfor 10 min. The C'402 collected in alkali was re-leased with perchloric acid (Van Slyke, Steele,and Plazin, 1951) collected in an ionizationchamber for counting with a vibrating reedelectrometer.

RESULTS

Incorporation of isotope from tryptophan-C"4into cellular nicotinic and quinolinic acid. Figure1 shows the growth response observed when thebasal medium was supplemented with DL-trypto-phan, 3-hydroxyanthranilic acid, or nicotinicacid. In the absence of these compounds, verylittle growth was observed. The amount oftryptophan necessary to yield maximal growthwas about five times that for 3-hydroxyanthra-nilate and 40 times that for niacin. In the presenceof tryptophan, the organism displayed a longerlag period and a slower rate of growth than wheneither of the other compounds was present.Maximal growth was obtained after 72 hr whentryptophan was being utilized, and after 48 hrfor either 3-hydroxyanthranilate or niacin.There was a close correlation between growth

and uptake of tryptophan-7a-C'4 from the me-

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224

H

H

H

zH

IUzHnU2H

WILSON AND HENDERSON

u

vzm0Hn

HOUTRS

FIG. 2. Uptake of DL-tryptophan-7a-C'4 andgrowth of Xanthomonas pruni. 0, growth; A\,zuptake.

1. 0

1.4 20

H~~~~~~OR

F1. 3 40 Uz~~~~~~~~~~~~~~

and grwho atooa rn.o rwh;

2.2 60 ~

u~~~~~~~~~~~~~

j 2. 6 80

3. 0 10012 24 36 48 60 72

HOURS

FIG. 3. Uptake of 3-hydroxyanthranilate-C'400Hand growth of Xanthomonas pruni. 0, growth; A,uptake.

dium (Fig. 2). The uptake was 42% based on theDL-tryptophan, but 84% based on the L-form.Likewise, the microbio-assay, which is specific forthe L isomer, indicated that 84% of the L-trypto-phan had been removed from the medium after72 hr of growth. These results suggested that thecells neither take up nor use the D isomer.The results of growth and uptake studies for

3-hydroxyanthranilate-C'400H are shown inFig. 3. As in the case of tryptophan, there was aclose correlation between uptake of the compoundfrom the medium and growth of the organism.Maximal growth was obtained after 48 hr, butthe maximal uptake of 3-OHAA (38%) wasreached after approximately 43 hr. From 43 hrto the end of the experiment (72 hr), radioactivityin the medium increased, indicating that the iso-tope was being released from the cells. Approxi-

J. BACTERIOL.

6 0 4040H

z~~~~~~~~~~~~~~~80

1224 36 48 60 72HOURS

FIG. 4. Uptake of nicotinic-7_-C4 acid and growthof Xanthomonas pruni. 0, growth; A, uptake.

mately half of the isotope taken up went back intothe medium. This release may be accounted forin part by lysis of cells, since the maximal growthoccurred after 48 hr.The results of growth and uptake studies with

X. pruni using nicotinic-7-C14 acid are shown inFig. 4. Growth of the organism paralleled uptakeof nicotinic acid-C14 from 0 to 36 hr. After 36 hr,84% of the C'4 had been removed from the me-dium, and growth was 90% of maximum. From36 to 48 hr, when the last 10% of the growth wasoccurring, 36% of the isotope taken up was re-leased. Thus, only 37% of the isotope taken upremained in the cells at the end of the 72-hrexperiment. The extent to which cell lysis ac-counted for the release of C14 was not determined.For all three compounds, the amount of radio-

isotope present in the carbon dioxide given offby this organism during the 72-hr period wassmall. Of the isotope taken up by the cells, lessthan 0.1 % was released as C1402 from trypto-phan-C'4, 0.7% from 3-OHAA-C14, and only0.03% from the niacin-C'4.

Table 1 shows the incorporation of radioisotopefrom tryptophan-C14 into nicotinic acid and tryp-tophan of the cells. In experiments I and II, thedilutions of C14 were 11.3- and 7.6-fold respec-tively in forming niacin, and the percentages ofincorporation were 0.096 and 0.10 respectively.The specific activity of the tryptophan laid downand the niacin formed were about the same, thusindicating that tryptophan is converted to nico-tinic acid in this organism.When tryptophan was added to the medium in

a sufficient quantity to insure maximal growth,it might be expected that the synthesis of trypto-phan by the organism would be repressed. Under

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TABLE 1. Incorporation of C14 from tryptophan into tryptophan and nicotinic acid of the cells ofXanthomonas pruni

Expt Ia Expt IIb ExptConditions

Niacin Tryptophan Niacin Tryptophan Niacin

Amount present (Ag)d ........... .................. 53 13,400 46 10,300 74Dry cells (jg per mg)d ............ ................. 0.010 2.48 0.009 2.01 0.012C'4 present (m4c) ........................... 3.3 769 5.3 1,181 0.72C14 incorporated (% of isotope added) ............. 0.096 21.8e 0.10 22.7e 0.012Specific activity before dilution (Mc/mmole)d....... 25.1 35.1 34.8 37.4 2.4Dilution of isotoped ............. .................. 11.3 7.4 7.6 5.6 64.2

a DL-Tryptophan-5-Cl4, 15.0 mg; specific activity, 95 Mc per mmole.DL-Tryptophan-7a-C14, 21.0 mg; specific activity, 89,uc per mmole.

cDL-Tryptophan-7a-C14, 15.0 mg; specific activity, 76,,c per mmole; plus 0.15 mg of unlabeled nico-tinic acid.

d Based on microbio-assay.e Based on L isomer.

these circumstances, the specific activity of theniacin synthesized would approximate that of thetryptophan administered. The data in Table 1(experiments I and II) show that the isotopepresent in the niacin isolated had been dilutedonly slightly compared to tryptophan, suggestingthat there was no source of niacin other thantryptophan. That the tryptophan isolated hadundergone dilution establishes the fact tbat thisorganism synthesizes tryptophan.The results shown in Table 1 (experiment III)

indicate that the presence of niacin in the mediumincreased the dilution of isotope in the niacinapproximately six- to eightfold. This demon-strates that even when sufficient nicotinic acid isgiven to promote maximal growth, some nicotinicacid is formed from tryptophan. It further sug-gests a turnover of nicotinate present in the cellas was also indicated in the tryptophan-C'4experiments in the absence of externally addednicotinic acid.

Microbio-assay of the L-tryptophan found in thecells indicated that there was, in general, more

tryptophan present than was added to the me-

dium, assuming utilization of only the L isomer(Table 1). This excess in the cells could not ac-count for all the dilution, suggesting considerableturnover of this amino acid during growth.

If the tryptophan were being metabolized, a

large part of the isotope might appear as carbondioxide. However, very little carbon dioxide-C'4was evolved. Approximately 85% of the L isomerof tryptophan was taken up, but only 27% of the

C'4 was found in tryptophan of protein, in nico-tinic acid, and in carbon dioxide. If the organismwere degrading and resynthesizing tryptophan, itwould seem that more of the isotope would appearas carbon dioxide-C'4. All of the data suggest sucha breakdown and resynthesis, and the absence ofisotope in carbon dioxide indicates that the path-way of degradation is not to aliphatic intermedi-ates which lead to CO2. However, the tryptophanwhich was isolated represented that which was inthe cell residue after extraction with hot 80%ethanol. Any "free" tryptophan would have beenin this alcoholic extract from which only nicotinicand quinolinic acids were isolated. No attemptswere made to determine the fate of the isotopenot present in tryptophan, niacin, and carbondioxide. No endogenous quinolinate was found inthe cells, and the amount of isotope present in theisolated quinolinic acid was negligible.

Incorporation of isotope from 3-hydroxyanthra-nilate-C'4 or -H3 into nicotinic acid. Table 2 showsthe results of the experiments in which X. pruniwas grown with 3-hydroxyanthranilate-H3 as asource of niacin. In two experiments, isotopedilutions in the niacin isolated were 12.3 and 17.1.This suggested that there was considerable syn-thesis of 3-OHAA during the growth period,presumably from tryptophan. No endogenousquinolinic acid was detected in these experiments,and the amount of isotope present in the isolatedquinolinate, after the addition of carrier; wasnegligible.To determine the amount of the hydroxyan-

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TABLE 2. Incorporation of H3 from 3-hydroxy-anthranilate-H3 into nicotinic acid of

the cells of Xanthomonas pruni

Conditions Expt I a Expt iIr

Niacin present (Ag)b ............ 40 52Niacin, Ag per mg (dry wt) of

cells'....................... 0.008 0.009H3 present (m,uc).17.1 15.9H3 incorporated (% of isotopeadded).0.20 0.18

Specific activity before dilution(,Acpermmole)b............... 58.4 41.7

Dilution of isotopeb.. 12.3 17.1

a Amount of 3-hydroxyanthranilate-H3, 1.9mg; specific activity, 717 ,uc per mmole.

b Based on the microbio-assay.

thranilate taken up by the organism, and alsothe amount of compound metabolized to carbondioxide, C'4-carboxyl-labeled 3-hydroxyanthra-nilic acid was employed. Approximately 40% ofthe isotope was removed from the medium by theX. pruni cells. About half of the maximal amounttaken up was left in the cells at the end of theexperiment; the other half went from the cellsback into the medium. The fact that less thanhalf of the hydroxyanthranilate added to themedium was taken up indicates that the organ-ism encounters some difficulty in taking thiscompound into the cell, or that the organismremoves from the medium only the amount it canutilize effectively.

Approximately 0.7% of the isotope taken upwas evolved as carbon dioxide-C14 during the72-hr period. As in the tryptophan-C14 experi-ments, this is a small portion in view of the ap-parent extensive degradation which occurred.

Incorporation of isotope from quinolinic acid-H3into cellular nicotinic and quinolinic acids. Thedata in Table 3 demonstrate that niacin can beformed from quinolinic acid by this organism.The dilution of the isotope in this conversion wasapproximately the same as that observed in thetryptophan and 3-hydroxyanthranilate experi-ments. That the quinolinate isolated had ap-proximately the same specific activity as thatadded to the medium indicates that there hasbeen no dilution with quinolinate arising in thecell. This finding is consistent with the observa-tions in the other experiments, in which thequinolinate isolated from the cells, after additionof unlabeled carrier, contained little or no isotope.

Incorporation of niacin-C14 into the X. prunicells. Experiments were performed using nicotinicacid-Cl4 as a source of the growth factor for theX. pruni. The dilution of isotope in the niacinisolated from the cells is presented in Table 4. Aswhen niacin precursors were labeled, considerabledilution of the isotope occurred. This indicatesagain that there was some synthesis of niacinduring the growth period. The dilutions were, ingeneral, smaller than those for other compounds.Values of 3.1 in experiment I and 5.2 and 5.9 inexperiment II were found. That nicotinic acidwas synthesized during the growth period wasfurther indicated by an experiment (results notshown) in which labeled niacin and unlabeledDL-tryptophan were added to the medium. Theextent of dilution of isotope in the isolated nico-tinic acid was increased from 6 to 12 by thepresence of the tryptophan.

In Table 4 (experiment II) are presented twosets of data for cells grown in medium containingniacin-C14. After 72 hr, the cells were harvestedand divided into two equal portions. One portion(no. 1) was extracted with ethanol, and the niacinwas isolated from the extract after hydrolysis.The other portion (no. 2) was hydrolyzed withalkali, without extraction, and nicotinic acid

TABLE 3. Incorporation of H' from quinolinic acidinto nicotinic and quinolinic acids of

the cells of Xanthomonas pruni

Expt Ia Expt IlbConditions

NA QA NA QA

Total amount pres-ent (,ug)c......... 50 31.1 92.4 48.1

Dry cells (pg permg)c............. o.018 0.007 0.016 0.009

H3 present (mnAc) ... 13.8 40.6 12.5 39.4H3 incorporated (%

of isotope added) . 0. 10 0.29 0.062 0.20Specific activity be-

fore dilution (,cpermmole)c ...... 67.8 675 55.5 593

Dilution of isotopec. 9.8 0.99 12.1 1.13

a Quinolinic-H3 acid, 6.8 mg; specific activity,335 Mc per mmole.

b Quinolinic-H3 acid, 15 mg; specific activity,223 Mc per mmole.

c Based on microbio-assay.

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TABLE 4. Incorporation of C14 from nicotinic acid into the cellular nicotinic acid ofXanthomonas pruni

Expt IIbConditions Expt Ia

1 2

Niacin present (total ,g)c............................... 53.3 36 36Dry cells (jsg per mg)c.................................. 0.014 0.011 0.011C14 present (m,uc) ....................................... 775 404 469C'4 incorporated (% of isotope added) ................... 11.2 3.7 4.2Specific activity before dilution (,c per mmole)c. 1,907 1,534 1,780Dilution of isotopec..................................... 3.1 5.9 5.2

a Amount of niacin-C14, 0.15 mg; specific activity, 5,880 jAc per mmole.b Amount of niacin-C'4, 0.15 mg; specific activity, 9,200,c per mmole. Different isolation procedures

were used for 1 and 2; see text.c Based on microbio-assay.

isolated. This was done to be sure that there wasno difference in isotopic dilution noted for theniacin isolated by the two methods, and thatalcoholic extraction was a valid method of re-moving the niacin from the cell. The data indi-cate that there was no significant difference in theresults obtained by the two methods. Of the iso-tope taken up, 37% remained in the cells at theend of the experiment. Of this, 38% could be ac-counted for as nicotinic acid-C'4. The amount ofisotope present in carbon dioxide-C'4 was small,and no attempt was made to isolate any otherproduct.

DIscussIoNThe results obtained with labeled quinolinate

(Table 3) could be interpreted as an indicationthat quinolinate is not an intermediate in theformation of niacin. However, the failure of thequinolinate to become diluted by quinolinatecoming from 3-OHAA may simply reflect a smallpool of quinolinate and its failure to becomeequilibrated with the acid added to the mediumand subsequently isolated from the cells. Theconcentration of quinolinic acid present in thecells in both experiments was small, approxi-mately half that of niacin on a molar basis.Whether quinolinic acid is an intermediate in

niacin synthesis or is a product of a side reactionhas long been debated. Some workers (Bonnerand Yanofsky, 1949; Krehl, Bonner, and Yanof-sky, 1950) suggested that the pathway of niacinsynthesis involves the decarboxylation of theunstable intermediate, 2- amino-3-carboxymu-conic semialdehyde, with subsequent ring closure.This route would not involve quinolinate as an

obligate intermediate. Some quinolinate mightarise from spontaneous ring closure without de-carboxylation. Niacin could then arise from thisquinolinate by decarboxylation, but this wouldrepresent only a small part of the niacin synthe-sized. That decarboxylation does occur in vivowas established by the work of Hankes and Segel(1958) and by the work with the chick embryo(Wilson and Henderson, 1960), and now for X.pruni. However, no in vitro system has beenfound that will form niacin from quinolinate.Likewise, no in vitro system has been found thatwill convert OHAA or tryptophan to niacin, andthe true status of quinolinate must await thedemonstration of an in vitro synthesis of niacin.

In the first quinolinic acid feeding experiment(Table 3), approximately 7 mg of quinolinic acidwere used. However, the yield of cells was onlyabout half that which would be expected on thebasis of the other experiments. In the secondexperiment, the amount of this compound addedto the medium was doubled; this resulted in thesame growth as previously obtained in the L-tryptophan experiments. Thus, quinolinate wasless efficient than tryptophan as a substitute forniacin, an observation in agreement with Neuro-spora growth studies (Bonner and Yanofsky,1949; Henderson, 1949).

It is possible that more quinolinic acid is re-quired because of the pH of the medium. Thebacterium would probably take up the compoundas the free acid (Bonner and Yanofsky, 1949).At the pH of the medium (6.8), most of the quino-linic acid exists as the salt rather than as the freeacid. Therefore, the organism would have somedifficulty in removing it from the medium. By

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increasing the concentration, the uptake ofquinolinic acid by the organism might be in-creased.The known mammalian, fungal, and bacterial

routes of degradation of tryptophan proceed viacommon intermediates as far as kynurenine. Herethey diverge and, in the case of mammals andfungi, the principal pathway is through 3-hy-droxykynurenine, 3-hydroxyanthranilic acid,then to pyridine carboxylic acids or to aliphaticintermediates, and thence to carbon dioxide(Gholson, Hankes, and Henderson, 1960; Hen-derson and Gholson, 1959). In the case of bacteria,the known pathways diverge at kynurenine; someorganisms convert this compound to anthranilateand thence to catechol, and others form kynurenicacid by transamination of the kynurenine fol-lowed by ring closure (Hayaishi and Stanier,1951).X. pruni appears to be unique among the

bacteria in that it possesses a tryptophan-niacinrelationship, as do animals and Neurospora. Thedata presented indicate that this organism iscapable of forming niacin from tryptophan, andof synthesizing ample tryptophan when niacinor one of its precursors is provided. A substantialpart of the C14 from tryptophan, or productsarising from tryptophan, was not accounted foras the metabolites isolated or as carbon dioxide.The isotopic carbon was apparently used in cer-tain nonspecific synthetic processes.

ACKNOWLEDGMENTS

This work was supported by a grant (RG 5766)from the Division of General Medical Sciences,National Institutes of Health, U.S. PublicHealth Service.The authors are indebted to Lloyd A. Brinker-

hoff of the Department of Botany and PlantPathology for assistance in isolating and identi-fying the culture.

LITERATURE CITED

BELL, G. G., AND F. N. HAYES. 1959. Liquidscintillation counting. Pergamon Press, Inc.,New York.

BONNER, D. M., AND C. YANOFSKY. 1949.Quinolinic acid accumulation in the conver-sion of 3-hydroxyanthranilic acid to niacinin Neurospora. Proc. Natl. Acad. Sci. U.S.35:576581.

CIERESZKO, L., AND L. V. HANKES. 1954. Inter-mediates in the synthesis of carboxyl-C14-

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