MECHANISM OF COMPETITIVE INHIBITION OF p-AMINOBENZOICACID OXIDATION BY p-AMINOSALICYLIC ACID'

NORMAN N. DURHAM AND JERRY S. HUBBARDDepartment of Bacteriology, Oklahoma State University, Stillwater, Oklahoma

Received for publication December 18, 1959

The proposed mechanism(s) associated withthe inhibition of essential metabolic reactions byvarious compounds has received a great deal ofattention in recent years, since antimetaboliteshave proved useful in several chemotherapeuticareas and in studying various metabolic reac-tions. The significance of p-aminobenzoic acidin microbial nutrition was first emphasized whenWoods (1940) and Woods and Fildes (1940)reported that this metabolite protected yeastagainst the growth inhibiting action of sulfanil-amide. Youmans, Raleigh, and Youmans (1947)reported that p-aminosalicylic acid inhibitedgrowth of virulent strains of the tubercle bacilliand this antagonism was due to anti-p-amino-benzoic acid activity. More recently, Hedgecock(1958) demonstrated that p-aminobenzoic acidcompetitively antagonized the inhibitory effectof p-aminosalicylic acid on the growth of Myco-bacterium tuberculosis.

Nutritional studies have indicated that p-aminobenzoic acid may fulfill a dual role inmicrobial metabolism. Durham (1956) reportedthat p-aminobenzoic acid was capable of servingas an oxidizable substrate for certain micro-organisms in addition to functioning as a growthfactor. Results obtained from additional investi-gations indicated that the utilization of p-amino-benzoic acid as a sole source of energy for aerobicgrowth is also competitively inhibited by p-aminosalicylic acid (Durham, 1957). Durhamand Hubbard (1959) suggested that p-amino-salicylic acid influenced the assimilation ofp-aminobenzoic acid by competing with thesubstrate for the specific transport mechanism(s)in the cell membrane thereby controlling oxida-

1 This investigation was supported in part bya research grant (E-2081) from the NationalInstitute of Allergy and Infectious Diseases,Public Health Service with the Oklahoma Agricul-tural Experiment Station Project no. 976, and inpart by a research grant (G-8935) from the Na-tional Science Foundation.

tion by regulating the intracellular accumulationof the substrate.

This paper is concerned with clarification of theconditions associated with the antimetabolicactivity of p-aminosalicylic acid toward p-amino-benzoic acid utilization and elucidation of themechanism(s) involved in this antagonism.

MATERIALS AND METHODS

A bacterium capable of growing on a chemicallydefined medium containing p-aminobenzoic acidas the sole source of carbon was isolated fromthe soil by an enrichment technique. The or-ganism was cultured and tentatively identifiedas a member of the genus Flavobacterium. Astock culture of the organism was maintainedon a mineral salts medium containing 0.1 percent p-aminobenzoic acid. The defined mediumused throughout the course of the experimenta-tion was composed of NaCl, 0.1 g; NH4C1, 0.1 g;KH2PO4, 0.324 g; K2HPO4, 0.424 g; and agar,2.0 g in 100 ml of distilled water. The basalmedium was supplemented with a mineral saltssolution (Durham, 1956) and the pH adjusted to7.0.

Cell suspensions for incubation and respi-rometer studies were prepared by harvesting thegrowth from plate cultures 22 hr old, washingtwice, and resuspending in 0.01 M phosphatebuffer of pH 7.0. The cultures were then adjustedto a standard density equivalent to a wet cellweight of approximately 1.4 mg per ml. p-Aminobenzoic acid-grown cells were usedthroughout this investigation.

Cell-free extracts were prepared using a FrenchPressure Cell under conditions which called fora pressure of 20,000 lb/in2. The crude extractwas centrifuged at 18,000 X g for 30 min. Thesupernatant collected after this treatment wasused in experiments involving cell extracts.

All respirometer experiments were performedin the Warburg apparatus (Umbreit, Burris, andStauffer, 1957) at a temperature of 37 C with

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air as the gas phase. AW'arburg vessels with doubleside arms were employed in this study. The cellsuspension was pipetted into the main chamber,0.2 ml of 20 per cent KOH into the center well,and varying concentrations of substrate andinhibitor in the side arms. Incubation experimentswere conducted under similar conditions in whichmixtures of the substrate and inhibitor wereadded to the cell suspension. The containers wereshaken in a water bath at 37 C for the desiredtime intervals. Samples were withdrawn fromthe vessel, the cells removed immediately byfiltration through a Millipore filter, and thefiltrate collected and assayed for the substrateor inhibitor. All compounds used as substratesand inhibitors for respirometer and incubationexperiments were dissolved in 0.01 M phosphatebuffer and adjusted to a pH of 7.0.

p-Aminobenzoic acid was determined aspreviously described (Durham and Hubbard,1959). p-Aminosalicylic acid was also determinedcolorimetrically by modification of a previouslydescribed procedure (Snell and Snell, 1953). Thecoupling reaction was performed as follows. Toa sample of filtrate add 3.0 ml of 20 per centp-toluenesulfonic acid in 1:50 hydrochloric acid,shake, and allow to stand 5 minutes; add 1 mlof a buffer consisting of 15.76 per cent citric acidin a 1.4 per cent sodium hydroxide solution; add2 ml of 2 per cent p-dimethylaminobenzaldehydein ethanol; mix and read in a spectrophotometeragainst a reagent blank at 450 mu. Values werecalculated from a standard curve developedconcurrently. Controls indicated that p-amino-benzoic acid interfered with the determination ofp-aminosalicylic acid. In studies in which bothp-aminobenzoic acid and p-aminosalicylic acidwere added to the reaction vessel it was necessaryto permit the p-aminobenzoic acid to be depletedfrom the medium by the cell suspension beforean accurate determination of the p-aminosali-cylic acid concentration could be made. Allinhibitor to substrate ratios are calculated on amolar basis.

RESULTS

Influence of p-aminosalicylic acid on the oxida-tion of various substrates. Investigations haverevealed that p-aminobenzoic acid induced cellsuspensions are capable of oxidizing p-amino-benzoic acid quite rapidly. The addition ofp-aminosalicylic acid to the Warburg flasksresults in a marked decrease in the rate of oxida-

tion. This inhibition becomes more pronouncedas the ratio of inhibitor to substrate is increased.Figure 1 illustrates the typical results obtainedfrom respirometer experiments when the sub-strate concentration was maintained at a constantlevel and the concentration of the inhibitor wasvaried. The control containing p-aminosalicylicacid (20 ,moles) without added substrate showsa slight but consistent increase over the endoge-nous respiration. The nature of this oxi(lative re-action has not yet been determined.

Additional studies were conducted with thep-aminobenzoic acid grown cells to determine ifp-aminosalicylic acid influenced the oxidation ofother metabolites. Results obtained from thisinvestigation indicated that the oxidation ofprotocatechuic acid was also inhibited. In con-trast to this finding was the observation that theoxidation of,-ketoadipic acid and succinic acidwere not significantly affected by the inhibitor.Figure 2 shows the influence of p-aminosalicylicacid on the oxidation of various substrates in aninhibitor to substrate ratio of 5:1. The inability

200PAS

Concentration

180-(o)

160

140

120

100 /

oa0 /;0/A

-(20)

60

QP 40-PAS control (2 0)

20

O_g Endogenous

0 10 20 30 40 50 60Time In Minutes

Figure 1. Oxidation of p-aminobenzoic acid(2 ,Amoles) in the presence of varying concentra-tions of p-aminosalicylic acid (PAS). Thefinal ratios of inhibitor to substrate are 0:1, 3:1,5:1, and 10:1.

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INHIBITION OF p-AMINOBENZOIC ACID OXIDATION

rSUC t PASAoPROTO

,0/ 6UCuc

X/D,'

#I,

227

0 30 60 90 120 0 30 60 90 120

Time In MinutesFigure 2. The influence of p-aminosalicylic acid (PAS) on the oxidation of protocatechuic

acid (PROTO), succinic acid (SUC), p-aminobenzoic acid (PAB), and B3-ketoadipic acid (KA). All sub-strates were employed in a concentration of 2 MAmoles and p-aminosalicylic acid in a concentration of10 ,umoles resulting in an inhibitor to substrate ratio of 5: 1.

of p-aminosalicylic acid to influence the oxidationof straight chain intermediates to a significantextent eliminates the possibility that the inhibitormay be killing the cells or that the oxidativeability of the cells is being otherwise impaired.

Reversal of the competitive inhibition of p-amino-benzoic acid oxidation. Results presented in figure1 indicate that the inhibition of substrate oxida-tion is proportional to the concentration of theinhibitor. The reversible nature of this com-petitive inhibition can be demonstrated by theaddition of excess substrate. Respirometer experi-ments were employed in which the competitiveinhibition of p-aminobenzoic acid oxidation byp-aminosalicylic acid was followed for 60 min ininhibitor to substrate ratios of 0:1, 3:1, 5:1,and 10:1. Figure 3 shows these results and indi-cates that within 15 min after the introduction ofadditional substrate (8 Amoles at 60 min) theoxidation proceeded at an uninhibited rate in thelower ratios and at an increased rate in thehigher ratios where the inhibition was not com-pletely reversed. Failure to reverse completelythe inhibition in the higher ratios was due to theaddition of insufficient quantities of substrate.

Influence of prior exposure to inhibitor and timeof addition on substrate oxidation. In furtherinvestigations concerning the nature of the com-petitive antagonism, manometric experimentswere performed to determine the effect of pro-longed exosure of the cells to p-aminosalicylic

litiolPAS

Concentration(rM)

j240 /

a 200 0 (20)Add 6 (0PAO

cm 160 (-/iM) X

120-

so40 /

0 LI -j I

0 30 60 90 120Time In Minutes

Figure S. Reversal of the competitive antago-nism of p-aminobenzoic acid (PAB) oxidationby p-aminosalicylic acid (PAS) by addition ofexcess substrate. The initial p-aminobenzoic acidconcentration (0 min) was 2,umoles and the inhibi-tor to substrate ratio was decreased with theaddition of 8 jAmoles of p-aminobenzoic acid at60 min.

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acid prior to addition of the substrate and theeffectiveness of the inhibitor when added to cellsactively metabolizing p-aminobenzoic acid.Figure 4 shows the results obtained with in-hibitor to substrate ratios of 5:1 and 10:1 with(a) the inhibitor added at the time of substrateaddition and (b) the inhibitor added 20 min afterthe addition of the substrate. Inhibition ofsubstrate oxidation was observed immediately inthe flasks in which the inhibitor was added at 0min. Within a few seconds following addition ofthe antimetabolite to the cells that were activelymetabolizing the substrate, a normal pattern ofcompetitive inhibition was observed. Thesefindings suggest that the sensitive "site" must bereadily accessible to the inhibitor as indicated bythe immediate susceptibility of substrate oxida-tion. Speculation then indicates that one of thefirst available sites could be the specific transportmechanism in the cell membrane. The inhibitioncould also occur at the internal oxidation site.However, if the intracellular oxidative mechanismis involved, the inhibitor must readily penetrate

0 10 20 30 40 50 60 70

Time In Minutes

Figure 4. Effectiveness of p-aminosalicylic acid(PAS) as an inhibitor when added to cells activelymetabolizing p-aminobenzoic acid (PAB). p-Aminobenzoic concentration was 2 ,umoles andthe p-aminosalicylic concentration and time ofaddition are as indicated.

the cell membrane in order to reach the sensitive"oxidative site" and exert its effect.

In related experiments, p-aminobenzoic acidgrown cells were exposed to 10 ,umoles and 20,umoles of p-aminosalicylic acid for 60 min inWarburg vessels prior to the addition of 2 ,umolesof substrate. The oxidation of p-aminobenzoicacid by these cells was followed and comparedwith the rate of oxidation when the substrate andinhibitor were added simultaneously. The resultsindicated that the degree of inhibition observedin cell suspensions previously exposed to theinhibitor appeared to be similar to the antagonismobserved when the inhibitor and substrate wereadded simultaneously.

Inhibition of the uptake of p-aminobenzoic acidby cell suspensions in the presence of p-amino-salicylic acid. Studies employing the same con-centrations of substrate and inhibitor as used inthe respirometer experiments were conducted inwhich the uptake of p-aminobenzoic acid bycell suspensions was followed colorimetrically.The data presented in table 1, experiment A, arerepresentative of the findings obtained in thisinvestigation. The results show that in theabsence of the inhibitor the substrate is depletedfrom the medium within 45 min. In the presenceof p-aminosalicylic acid the substrate is taken upat a much slower rate. The data show that theamount of p-aminobenzoic acid depleted fromn themedium decreases with increasing inhibitor

TABLE 1Cellular uptake of p-aminobenzoic acid (PAB) in

the presence and absence of varying ratios ofp-aminosalicylic acid (PAS)

Conc (jig) of PAB Remainingper ml of Filtrate

Expt. Time Ratio, PAS:PAB

0:1 3:1 5:1 10:1

A 0 min 116.0 116.0 114.6 116.015 min 80.0 87.2 97.8 103.230 min 17.3 64.0 86.8 99.045 min 0.0 46.6 76.6 97.0

B 0 sec 3.10 3.10 3.10 3.1030 sec 0.15 0.43 0.77 1.65

The initial concentration of PAB in experimentA was 116,ug per ml and 3.1 ,ug per ml in experimentB.

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concentrations. A decline in the rate of disappear-ance during the subsequent intervals was es-pecially noticeable in the 10:1 ratio.

Additional studies were conducted in which theuptake of p-aminobenzoic acid in the presence ofp-aminosalicylic acid was followed in lower con-centrations. The disappearance of substrate fromthe medium was determined in inhibitor tosubstrate ratios of 0:1, 3:1, 5:1, and 10:1 witha p-aminobenzoic acid concentration of 2.28X 10-5M (3.1 ug per ml). Table 1, experiment B,illustrates the results obtained from this experi-mentation in which the disappearance of p-aminobenzoic acid was measured after a 30-secincubation period. The data indicate that theamount of substrate remaining in the medium isdirectly proportional to the concentration of theinhibitor.

These findings suggest that the inability of thesubstrate to permeate the cell in the presence ofthe inhibitor may be responsible for the decreasedrate of oxidation. If one assumes that the in-hibition is located at the site of internal substrateoxidation, then it is unlikely that the passage ofsmall quantities of substrate into the cell shouldbe limiting. Studies following depletion of thesubstrate both at high and extremely low levelsindicated that the ratio of inhibitor to substratepresent in the system regulates the uptake ofsubstrate of the cell suspension. These findingswould then support the postulation that theinhibition is associated with the specific transportmechanism rather than the internal oxidativeenzymes.

Uptake of p-aminosalicylic acid by cell sus-pension. Colorimetric analysis of the filtratesobtained when p-aminobenzoic acid grown cellsuspensions were incubated with p-aminobenzoicacid and p-aminosalicylic acid indicated thatsome of the inhibitor was removed from themedium. Since p-aminobenzoic acid interferedwith p-aminosalicylic acid determinations, it wasnecessary that the substrate be depleted fromthe medium before inhibitor concentrations weredetermined. The assumption was made that if theuptake of p-aminosalicylic acid was proportionalto its concentration, this proportionality shouldalso be evident at lower concentrations. Table 2illustrates typical results obtained when cellsuspensions were incubated with three differentconcentrations of p-aminosalicylic acid. In therange tested, the results indicated that the rate oramount of uptake did not vary significantly with

TABLE 2Cellular uptake of p-aminosalicylic acid (PAS) by

p-aminobenzoic acid-grown Flavobacterium

Time in Min Conc (pg) PAS Remaining per ml of Filtrate

0 10.44 20.88 41.7620 6.00 15.00 39.4040 3.36 10.90 33.6060 1.04 8.36 29.4080 27.60100 25.80120 24.00

the different concentrations. The rate of dis-appearance of the inhibitor in a starting con-centration of 41.76 ,ug per ml did not exceed thedisappearance when a concentration of 10.44 ugper ml was employed. Similar rates of inhibitoruptake in the different concentrations make itdifficult to visualize a competitive inhibition atthe site of the internal oxidative enzyme sys-tem(s) of the cell since a competitive antagonismwould be dependent on the presence of varyingconcentrations of the inhibitor.

Additional experiments were performed todetermine if the antagonism could be more closelycorrelated with the external concentration ofp-aminosalicylic acid. A cell suspension waspermitted to deplete approximately 20.88 ,ug ofp-aminosalicylic acid from the medium, followingwhich 6.2 lAg of p-aminobenzoic acid were addedand the disappearance of this substrate was fol-lowed. The depletion of substrate was comparedwith the uptake observed in control cells in which6.2 ,ug of p-aminobenzoic acid and 20.88 ug ofthe inhibitor were added simultaneously. After ashort incubation period, it was observed that theamount of substrate taken up by the cells whichhad assimilated the inhibitor prior to the additionof p-aminobenzoic acid was approximately twicethe amount of substrate taken up by the cellswhen the substrate and p-aminosalicylic acid wereadded simultaneously. These findings indicatethat only the presence of the inhibitor in theexternal environment influences the utilization ofp-aminobenzoic acid since cells permitted to takeup p-aminosalicylic acid intracellularly prior toaddition of the substrate did not demonstrate adecrease in the uptake of p-aminobenzoic acid.

Additional investigations were conducted todetermine if the temperature of incubation in-fluenced the rate of uptake of p-aminosalicylic

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acid by cell suspensions. Results from thesestudies indicated that approximately one-fourthas much inhibitor was taken up at 5 C as wasdepleted from the medium when the cells wereincubated for similar periods of time at 37 C.

Since the findings in this investigation indicatedthat some of the p-aminosalicylic acid wasdepleted from the medium attempts were made torecover the inhibitor from the culture. Cellssuspensions which had been exposed to p-ammo-salicylic acid for 2 hr were washed extensivelywith phosphate buffer and the washing assayedfor the inhibitor. Cell samples were also rupturedby grinding with glass beads and the extracttested for the presence of the inhibitor. Resultsobtained from these studies indicated that neithertreatment permitted the recovery of a sufficientquantity of inhibitor to permit detection by thecolorimetric test.

Influence of extraneous carbon sources andmetabolic inhibitors on the uptake of p-amino-salicylic acid by cell suspensions. Since the pres-ence of large quantities of p-aminobenzoic acidinterfere with the colorimetric determination forp-aminosalicylic acid, other metabolites werealso employed to determine if the presence of anoxidizable substrate influenced the uptake of theinhibitor. Studies were conducted in which theuptake of p-aminosalicylic acid was determinedin cells permitted to oxidize p-aminobenzoic acidto completion. Results indicated that uptake ofthe inhibitor in these cells did not differ from theuptake observed in resting cell suspensions whichhad not been permitted to metabolize p-amino-benzoic acid.

Uptake of the inhibitor was also followed inthe presence and absence of protocatechuic acidand succinic acid. Preliminary tests indicated thatneither substrate interfered with the colorimetrictest for p-aminosalicylic acid. In addition to theregular inhibitor to substrate ratios employed,investigations were conducted with an excess ofsubstrate in an inhibitor to substrate ratio of1:3.3. Results from these experiments indicatedthat the uptake of p-aminosalicylic acid was notinfluenced by permitting the concomitant oxida-tion of extraneous carbon sources by the cellsuspensions.

Investigations were undertaken to determineif the uptake of p-aminosalicylic acid by thep-aminobenzoic acid grown cells was influencedby incubation with known metabolic inhibitors.

Compounds included in this study were sodiumazide, sodium fluoride, 2,4-dinitrophenol, po-tassium cyanide, and iodoacetate. All metabolicinhibitors were employed in a final concentrationof 1 X 10-4 M. Studies were conducted in thepresence and absence of the metabolic inhibitorsand results indicated that these compounds didnot influence the uptake of p-aminosalicylic acid.Similar studies indicated that incubation withchloramphenicol (120 ,ug per ml) and strepto-mycin (37.9 jig per ml) were also without effect.Attempts to obtain activity for p-aminobenzoic

acid in cell-free extracts to determine if the in-ternal oxidative enzyme system was sensitive top-aminosalicylic acid have been unsuccessful.

DISCUSSION

The ability of p-aminosalicylic acid to com-petitively inhibit the oxidation of p-aminobenzoicacid has been investigated in an attempt toelucidate the mechanism(s) involved in thisantagonism. Results obtained in experimentsemploying structurally diverse metabolites suchas succinic acid and ,B-ketoadipic acid confirmthe supposition that p-aminosalicylic acid isacting as an antimetabolite and is not killing orotherwise impairing the oxidative ability of thecells. Studies also demonstrated that the antag-onism can be reversed by the addition of excessquantities of the substrate. The inhibition ofp-aminobenzoic acid oxidation can be studied bymanometric or colorimetric procedures followingdepletion of the substrate from the medium.

Results obtained in this investigation indicatethat inhibition of substrate utilization becomesapparent immediately following addition of theantimetabolite. The addition of p-aminosalicylicacid simultaneously with the substrate or to cellsactively metabolizing the aromatic compoundproduced demonstrable effects within a shorttime. The inhibition could also be detected inexperiments using extremely low substrate levels.

Experimental results indicated that cellspreviously exposed to large quantities of theinhibitor for 60 min demonstrated approximatelythe same degree of inhibition as cells to which theinhibitor and substrate were added simultane-ously. Since the extent of inhibition was approxi-mately the same under both experimentalconditions, the results indicate that the inhibitorconcentrations must be similar. This could bepossible if the cell was impermeable or very

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slightly permeable to the inhibitor in which casethe antimetabolite would still be present in themedium in sufficient quantities (in the exposureexperiments) to approximate the quantities ofinhibitor added simultaneously with the sub-strate. Alternatively, p-aminosalicylic acid mayreadily permeate the cell barrier, accumulate inthe cell, and compete for the internal oxidativeenzyme system(s).

Results obtained in the investigations followingthe uptake of p-aminosalicylic acid by cell sus-pensions support the former explanation. Thedata show that uptake of the inhibitor by thecells is small and the rate of uptake is independ-ent of the initial concentration of the antime-tabolite in the medium. A competitive inhibitionat the internal enzyme system would be depend-ent on the presence of varying concentrations ofthe inhibitor and the constant uptake observedin this study would eliminate the possibility thatp-aminosalicylic acid acts inside the cell.

Related experiments in which known quantitiesof p-aminosalicylic acid were depleted from themedium by the cell suspensions prior to addingthe substrate demonstrated that the internalaccumulation of inhibitor by the cells did notinfluence p-aminobenzoic acid oxidation. At-tempts to recover p-aminosalicylic acid from thesecell suspensions were unsuccessful. These resultswould indicate that the antimetabolite is boundor metabolized immediately upon permeatingthe membrane and is not available in the "freeform" to compete for the oxidative enzyme sys-tem.

SUMMARY

The oxidation of p-aminobenzoic acid by aFlavobacterium is inhibited by p-aminosalicylicacid. The antagonism is competitive in nature andmay be followed by manometric techniques or byemploying a colorimetric procedure for studyingthe depletion of substrate from the medium.Results obtained in the investigation suggestthat p-aminosalicylic acid inhibits the oxidationof p-aminobenzoic acid by competing with the

substrate for the specific transport "sites" in thecell membrane thus regulating cellular oxidationby controlling the intracellular accumulation ofthe substrate. The data indicate that the an-tagonism does not appear to be due to an internalaccumulation of the antimetabolite or productsof its degradation.The uptake of p-aminosalicylic acid by cells

was not influenced by extraneous carbon sourcesor other metabolic inhibitors but was temperaturedependent. However, additional investigationswill be necessary to clarify the fate of this anti-metabolite in the cell.

REFERENCESDURHAM, N. N. 1956 Bacterial oxidation of p-

aminobenzoic acid by Pseudomonas fluorescens.J. Bacteriol., 72, 333-336.

DURHAM, N. N. 1957 Effect of structurallyrelated compounds on the oxidation of p-aminobenzoic acid by Pseudomonas fluo-rescens. J. Bacteriol., 73, 612-615.

DURHAM, N. N. AND HUBBARD, J. S. 1959 An-tagonism of the oxidative dissimilation of p-aminobenzoic acid by p-aminosalicylic acid.Nature, 184, 1398.

HEDGECOCK, L. W. 1958 Mechanisms involvedin the resistance of Mycobacterium tuberculosisto para-aminosalicylic acid. J. Bacteriol.,75, 345-350.

SNELL, F. D. AND SNELL, C. T. 1953 Colori-metric methods of analysis, p. 418. D. VanNostrand Co., New York.

UMBREIT, W. W., BURRIs, R. H., AND STAUFFER,J. F. 1957 Manometric techniques, reviseded. Burgess Publishing Co., Minneapolis.

WOODS, D. D. 1940 The relation of p-amino-benzoic acid to the mechanism of the actionof sulphanilamide. Brit. J. Exptl. Pathol.,21, 74-90.

WOODS, D. D. AND FILDES, P. 1940 The anti-sulfanilamide activity (in vitro) of p-amino-benzoic acid and related compounds. Chem.& Ind. (London), 59, 133-134.

YOUMANS, G. P., RALEIGH, G. W., AND YOUMANS,A. S. 1947 The tuberculostatic action ofpara-aminosalicylic acid. J. Bacteriol., 54,409-416.

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