MUTANT STRAINS OF ESCHERICHIA COLI UNABLE TO SYNTHESIZE p-AMINOBENZOIC ACID

BY J. 0. LAMPEN,* M. J. JONES, AND R. R. ROEPKE

(From the Chemotherapy Division, Stamford Research Laboratories, American Cyanam.id Compan,y, Stamford, Connecticut)

(Received for publication, March 28, 1949)

Among the growth factor-requiring strains of Escherichia coli obtained in this Laboratory by x-ray treatment or prolonged serial transfer (1, 2) were three strains which grew when p-aminobenzoic acid (PABA) was added to the basal medium. The importance of PABA in the metabolism of microorganisms was first indicated when Woods (3) reported that the bacteriostatic action of the sulfonamides is competitively antagonized by the addition of PABA. Rubbo et al. (4) soon found Clostridium aceto- butylicum requires the addition of PABA for growth, and a number of other organisms were subsequently reported to require PABA (see Peterson and Peterson (5) for references). Mutant strains which require PABA were obtained after irradiation with x-rays of the fungi Neurospora (6) and Ophiostoma (7). In addition, PABA is a part of the pteroylglutamic acid (folic acid) molecule (8), and the synthesis of pteroylglutamic acid from PABA by certain enterococci and lactobacilli has been reported (9, 10) to be inhibited by sulfonamides. It was therefore of interest to study the growth requirements of these mutants and especially to investigate the activity of various compounds related to PABA.

Most of the experiments reported in this communication were performed with mutant 273-384. Some data obtained with mutants 1861-460 and 45-462 are presented as well. A preliminary report of a portion of this work has previously been published (11).

Procedures

The mutant strains were produced and isolated by the general methods previously employed (1, 2). Mutant 273384 was isolated from a single colony culture of E. coli irradiated at 40 kv. and 20 ma. by use of a copper target for 4 hours during growth in Difco A. C. broth at 3OO.l Mutants 1861-460 and 45-462 were isolated from resting cell suspensions irradiated at 40 kv. and 20 ma. for 2 hours at 12-15”.

*Present address, Department of Microbiology, School of Medicine, Western Reserve University, Cleveland, Ohio.

1 Mutant 273-384 can be obtained from the American Type Culture Collection as No. 9723a, and the parent strain of E. coli as No. 9728.

423

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424 MUTAKT STRAIN OF E. 'COLI

The general bacteriological techniques employed have been described previously (12). The basal, synthetic medium was that of MacLeod (13) with the following composition (per liter): NaCl 5.0 gm., (NH&SO4 4.72 gm., KH2P04 2.72 gm., glucose 2.0 gm., asparagine 2.0 gm., and 1.7 mg. each of FeC12, MgC12, and Cat%. The medium was adjusted to pH 7.0 to 7.1 with,NaOH.. Unless otherwise stated all supplements were sterilized by autoclaving with the basal medium.

The amino acid mixture used for supplementation of the basal medium was essentially that of Hat, Snell, and Williams (14). However, the DL forms of leucine, lysine, and aspartic acid were used instead of the natural materials. A stock solution was prepared containing, per ml., 8 mg. of nn-aspartic acid, 2 mg. each of the other DL acids, 10 mg. of L-glutamic acid, and 1 mg. each of the other L acids. This solution, as prepared from commercial amino acids, contained traces of PABA. This was removed by adsorption at pH 3.5 with 1 gm. of Darco G-60 per 100 ml. The puri- fied material is subsequently termed amino acid supplement. The purine supplement consists of 5 y each of adenine, guanine, and xanthine per ml. of final medium.

Results

Response of Mutant i?73-384 to PABA under Various Conditions-The addition of PABA to the synthetic basal medium is sufficient to support good growth of the mutant strain. .In the presence of excess PABA (0.1 y per ml.) the turbidity attained by the mutant at 16 hours of incubation equals that attained by the parent strain of E. coli. However, the response of mutant 273-384 to limiting concentrations of. PABA is increased very markedly by the. addition of a mixture of amino acids and purines to the basal medium (Table I). In the absence of the amino acids the addition of the individual purines delayed growth, although with either guanine or xanthine the final turbidity was greater. In the presence of the amino acids any one of the purines stimulated growth. Uracil and cytosine were inactive. The stimulation observed with the entire amino acid supple- ment could often be duplicated by the addition of DL-methionine alone. This compound also prevented. the growth inhibition by the purines., However, the growth of the mutant has generally been better when the entire mixture was present and this has customarily been used. Also 5 y per ml. each of adenine, guanine, and xanthine were generally added in- stead of a single purine. The concentration of PABA at which half maxi- mal growth occurred at 16 hours was 0.0012 y per ml. on the unsupple- mented medium. This requirement was reduced to 0.0005 y per ml. by the. addition of the amino acid supplement and to 0.0001 y by the fur- ther addition of the purine .supplement. The rate of .growth. and final

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J. 0. LAMPEN, M. J. JONES, AND R. R. ROEPKE 425

turbidity reached by the mutant in the presence of the amino acid and purine supplements and excess PABA (0.1 y per ml.) were approximately the same as those obtained under identical conditions with the parent strain of E. coli.

Wyss, Lilly, and Leonian (15) reported that the concentration of added PABA required for the growth of an aminobenxoicless mutant of Neurospora crassa decreased as the pH of the medium decreased. They concluded that this organism was permeable only to the un-ionized form of the acid. We

TABLE I Effect of Amino Acids and Purines on Response of Mutant 178384 to p-Aminobenzoic

Acid

Compound

None........................ Adenine (A). ................ Guanine (G). ................ Xanthine (X). ............... Hypoxanthine ............... Uracil ....................... Cytosine .................... A + G+ X.. .............. nn-Methionine. .............. Amino acid supplement. .....

I‘ ‘I

+A+G+X.‘:. ..... ::I::

-

--

-

Additions per ml.

10 Y 10 “ 10 “ 10 “ 10 “ 10 “ 5 “ (Each)

10 ‘( 0.02 ml. 0.02 “ 5 y (Each)

-

1 --

Additions to basal medium

None Amino acid

supplement* + O.OtWO~rOm~ABA

16 hrs. -

1 _ -

.6 hrs. -

t -

--.I- - 54 hrs.

0 21 26 0 0 19 0 11 37 0 12 37 0 0 22 0 22 26 0 24 23 0 10 32 0 38 51 4 39 43

6 74 72

Turbidityresdingst (average of duplicate tub 3)

I -

-

- .6 hrs. . -

5 23 15 27 18 5 4

28 5

-

64 hrs.

5 33 23 39 28

5 6

35 6

* 0.02 ml. per ml. t 1 unit is equivalent to 13.4 million cells of E. coli per ml. (cf. (12)).

tested the effect of the acidity of the medium on tne response of mutant 273-384 to PABA. In experiments with the unsupplemented basal me- dium the concentration of PABA required did not change markedly over the range of pH 5.2 to 7.2; however, growth was slower in the more acid media. The results obtained in the presence of the amino acid and purine supplements are illustrated in Fig. 1. Citrate buffers were used in the experiments which are shown here. The response curve at pH 6.4 was essentially unchanged when ~/30 phosphate buffer was substituted for the citrate. The results with phosphate buffers at pH 6.9 and 7.2 (pH 7.0 and 7.5 before autoclaving) were similar to those at pH 6.4, although the tur-

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426 MUTAKT STRAINS OF IS. COLI

bidity reached with excess PABA was somewhat greater in the more alka- line media. It can be seen from Fig. 1 that the sigmoid curve obtained at pH 6.4 and above was replaced by a more nearly linear curve at pH 5.7 and 6.1. However, the concentration of PABA required for half maximal growth remained approximately the same (about 0.0001 y per ml.). At pH 5.2 about 4 times as much PABA were required. These data offer no

y PABA PER mi.x103 FIG. 1. The effect of the pH of the medium on the response of mutant 283-384

to p-aminobeneoic acid. The amino acid supplement (0.02 ml. per ml.), purine sup- plement, and ~/30 sodium citrate were present in all series. Aliquots of the medium were adjusted to pH 6.0, 5.5, 6.0, and 6.6 before autoclaving. In uninoculated con- trol tubes of the four series the pH was 5.2,5.7,6.1, and 6.4, respectively, after auto- claving.

positive support for the idea that the mutant is permeable only to the un-ionized form of PABA.

We have also attempted to obtain a more linear response curve at pH 7.0 by the addition to the medium of a trace element mixture (16), 10 y of xanthopterin per ml., or 0.1 r of pteroylglutamic acid per ml., or by doubling the concentration of all of the constituents of the medium. None of these additions altered the shape of the response curve.

Activity of Compounds Related to PABA-The ability of mutant 273384

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J. 0. LAJIPES, 31. J. JOSES, AND R. R. ROEPKE 427

to utilize compounds related to or containing PABA is indicated by the data of Table II. Amino acids and purines were added to the medium to increase the sensitivity of the test. Inactive compounds included benzoic

TABLE II Activity of Compounds Related to pdminobenzoic A&P .-

Compoundt Concentration required for half maximal groath$

p-Aminobenzoic acid (PABA). . 0.00012 Benzoic acid§........................................... > 1000 Aniline~................................................ > 1000 o-Aminobenaoic acids..............,.................... > 1000 m-Aminobenzoic “ 8.. . . . _ 700 p-Hydroxybenzoic acid&. . . , . . . . . . . > 1000 p..Aminophenylacetic acids.. . . . . , . . . . . . . . . . 50 p-Aminohippuric acids.. . . . . . . . . . , . . . . . . 25 2-Amino-5carboxypyrimidines. . . . . . > 1000 2-Amino-5-carboxythiaroleg . . . . . . . . . . . . . . . . . . . >lOOO Pimelic acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . >lOOO p-Aminobenzoylglutamic acid. . . . . . . . . . . . 0.08 Glutamic acid polypeptide of PABA. . 10 Pteroic acid............................................. 0.0015 Pteroylglutamic acid.............. ..___......_..._..... 2.0 Pteroyltriglutamic acid. . . . 1.0 Thymine................................................ 40 037.)

* The amino acid supplement (0.02 ml. per ml.) and the purine supplement were present in all tubes. The medium was initially adjusted to pH 7.0. When neces- sary, solutions of the materials tested were neutralized with NaOH before addition to the medium.

t The glutamic acid polypeptide of PABA was furnished by Dr. S. Ratner of New York University and the pteroyl compounds by Dr. B. L. Hutchings and Dr. E. L. R. Stokstad of the Lederle Laboratories Division, American Cyanamid Company. The other materials were in general prepared in the Chemical Laboratories of the Chemotherapy Division, Stamford Research Laboratories, American Cyanamid Company. We wish to express our gratitude to these people.

$ The criterion of growth was turbidity readings at 24 hours. t These compounds were test,ed over a range of 0.001 to 1000 y per ml. both in the

presence and absence of 0.0001 y of PABA per ml. Only benaoic acid was inhibitory within this range. Growth was delayed for 16 hours in the presence of 1060 y of benzoic acid per ml. but the final growth obtained (40 hours) was not affected.

acid, aniline, o-aminobenzoic acid, p-hydroxybenzoic acid, and the pyrimi- dine and thiazole analogues of PABA. Tatum and Beadle (6) observed that an aminobenxoicless mutant of Neurospora crassa grew slowly in the presence of pimelic acid. Our mutant strain of E. coli did not attain vis- ible turbidity with added pimelic acid. The slight activity observed with

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428 MUTANT STRAINS OF E. COLI

m-aminobenzoic acid, p-aminophenylacetic acid, and p-aminohippuric acid could well be due to contamination with traces of PABA. This probably holds as well for p-aminobenzoylglutamic acid, which has only 0.14 per cent of the activity of PABA. The sample of pteroic acid contained, as an impurity, 6 per cent of free arylsmine (calculated as PABA) and pos- sessed about 7 per cent of the activity of PABA. Pteroylglutamic acid was less than 0.01 per cent as active as PABA. This sample contained 0.2 per cent of free arylamine (as p-aminobenzoylglutamic acid). There

TABLE III

Response of Mutant 87338.4 to p-Aminobenzoic Acid and to Thymine

p-Amino- benzoic acid

Thymine

-

I-

-y per mE.

0.0005 0.01

0.1 1.0 3.0

10 30

100 300

1000 3000

Additions to basal medium*

NOW Purim Amino acid Amino acid + supplement suppkmentt SUPgLS

.6 hrs.

15 21 56 74

0 0 0 0 0 0 2 1 1 5 0 2 2 8 0 1 0 0

i4 hrs. -

-

Turbidity readings

16 hrs.

4 68

0 0

0

0 0 0

54 hrs. 6 hrs. -

, - 54 hrs

25 32 34 76 69 74

2 3 3 4 2 5 4 8 6 10

11 15 7 11 2 4 4 5 .-

- , 1 --

-

- .6 hrs. / 64 hrs.

50 53 81 78

1 1 1

22 44 53 63 27

8 -

7 11 24 31 45 56 67 37 15

* Initially adjusted to pH 7.0. t 0.02 ml. per ml.

is, therefore, no proof that pteroic acid or pteroylglutamic acid is utilized by the mutant strain.

The response of the mutant to thymine was investigated in more detail (Table III). Thymine is practically inactive alone or in the presence of purines. Light growth occurred in the presence of the amino acids and thymine. The organism was carried through five transfers on this medium. A combination of the amino acids, purines, and thymine effectively re- placed PABA. During ten transfers with this combination (30 y of thy- mine per ml.) the turbidity obtained increased about 50 per cent. In the experiment shown in Table III the combination supported almost the same turbidity as did excess PABA; however, in some experiments the turbidity

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J. 0. LAMPEN, M. J. JONES, AND R. R. ROEPKE 429

obtained with -these supplements has not exceeded half maximum. Con- centrations of thymine above 300 y per ml; appear ‘t’obe~ toxic.

We then determined Which purinds and amino acids l@ere required for growth in the presence.of thymine. When -thymine and the amino acid supplement were present, the addition of any one’,of, the four Ijurihes per- mitted growth (Table IV). Uracil, cytosine, and xanthopterin were unable to replace the purines. Methionine X&S an essential component of the amino acid mixture (Table V);’ however, the addition of methionine alone produced only delayed, submaximal growth. In the presence of methio- nine, the addition singly of glycine, serine, threonine, lysine, histidine, or tyrosine was stimulatory. Addition of the other amino acids of the mix-

TABLE IV

Response of Mu,tant i?Y3-384 to Pura’nes and Related Compounds in Presence of Amino Acids and Thymine , i,

Medium adjusted to pH:7.0. 30 y of thymine per ml. and amino acid supplement (0.02 nil. per ml.) in all tubes. ,, ’

, Additions _.._ ._.____ ------- -.-._----.----_

None.~..................... ._, ., ._ ._...,__ Adenine (A) .,, . . : Guanine (G). . : : Xanthine (X). . . . : Hypoxanthine. : . Uracil (U). . . : Cytosine.......:........................... Xanthopterin.. . . . . . . . . _:_ .‘. A+G +X +U .._......................

__.____-

Y per ml.

10 10 10 10 10 10 10 10 5 (Each)

--

Turbidity readings;* 16 hrs.

7 43 48 47 46

5 8 8

51

* Readings essentially unchanged at 64 hours

ture singly did not increase the response to methionine. In subsequent tests the growth with a combination of the seven amino acids listed in Table V has equaled that obtained with the entire mixture.

It was observed that the mutant could utilize homocystine in place of methionine in the presence of the other amino acids, thymine, and the pu- rines; however, the final turbidity reached was less than one-half of that with methionine. The relative activity of homocystine and methionine is similar to that previously observed with certain methionine-requiring mutants of E. ,wZi (12, 17).

Sensitivity of Mutant .%%%!I84 to Suyonamides-The growth of mutant 273-884 with,E’ABA, either in the unsupplemented.ba,sal medium or in the presence.of amino acids and, purines,, ivas inhibited by the addition of sub fonamides (Table-VI). This inhibition was. antagonized by increasing the

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430 MUTANT STRAINS OF E. COLI

concentration of PABA. Pteroylglutamic acid at a concentration of 100 y per ml. did not possess any demonstrable antisulfonamide activity on either medium when tested alone or in the presence of sufficient PABA to yield partial antagonism. When the combination of amino acids, purines, and thymine was present, the organism attained one-half to two-thirds maximal

TABLE V Response of Mutant %V-384 to Amino Acids in Presence of Purines and Thymine Medium adjusted to pH 7.0. 30 y of thymine per ml. and purine supplement in

all tubes.

Erper merit NO.

I

II

-i- i-

-_

-

Additions per ml.

None Amino acid supplement

‘I “ “ * nL-Methionine

nL-Methionine + glycine + DL-serine f nL-threonine -I- nL-lysine + L-histidine + L-tyrosine

Amino acid supplement* I‘ “

per ml. “ + 10 y DL-methionine

Amino acid supplement* + 50 y DL-methionine per ml.

Amino acidsupplement* + 50 y DL-homocystinet per ml.

Amino acid supplement* f 100 y x-homocys- tine per ml.

0.02 ml. 0.02 “

10 Y 40 ”

100 ‘I 10 “ 20 (‘ 40 “ 40 “ 40 I‘ 20 ‘I 20 “

0.02 ml.

- I 1

-

-

Turbidity readings

54 brs. -

6 brs. f - 0

50 2 6 3 6

0 49 6

25 16 19

2 39 23 32 25 39 25 28 18 25 23 27 2 4

20 42

21 41

8 18

8 17

- * Methionine omitted. t General Biochemicals, Inc.; recrystallized once from water.

turbidity within 24 hours, even in the presence of 1280 y of sulfanilamide or sulfadiazine per ml. Growth was delayed, however, and the final tur- bidity in the presence of the higher sulfonamide concentrations was sub- optimal.

Nutritional Variation of Mutant RTS-S84-This mutant occasionally grew on the 2nd or 3rd day of incubation in media lacking one or more of the essential supplements (thymine, purines, and amino acids). Certain of

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J. 0. IAMF’EN, M. J. JONES, .&ND R. R. ROEPRE 431

these cultures were purified by plating on A. C. agar (Difco); the single colony cultures thus obtained were transferred ten times under the original conditions and single colony cultures again isoIated. The growth require- ments of these variants will be summarized briefly. (The concentrations used were PABA 0.02 y per ml., purines and pyrimidines 10 y of each per ml., and the amino acids as in the report of Roepke et al. (l).)

TABLE VI Concentrations of Sulfonamides Necessary to Inhibit Growth of Mutant %%88.$ in

Additions to basal medium

PAB* Ami; SUppI.?- mentt

-- 7 per ml.

‘0.002 -

[0.0002 + +

0.0002 -I-

0.02 + 1.0 + 0.02 +

+

PWhe upple- ment

+

f

+

z

l-

+

Presence of Various Supplements*

-t per ml.

Thymine, 100

Pteroylglutamic acid, 100

Thymine, 100

Sulfonamide

Sulfanil- amide

“ ‘8

Sulfa- diazine

“ “ ‘,

“ -

Wdmal effective concentration of sulfonamidet

__--

16 lus. 24h. 64bm.

~-__ 7 +er ml. y jw ml. .yper ml.

2.5 10 20

6 20 40 l-12809 1280 1280 0.04 0.08 0.3

3 3 10 loo

3 3 10

160-1280~ 1280 11280

* Medium initially adjusted to pH 7.0. t 0.02 ml. per ml. $ Concentration required for half maximal inhibition; i.e., for reduction of the

turbidity at a given time to one-half of that obtained in control tubes. $ Approximately hs,lf maximal inhibition was observed over the indicated sul-

fonamide range.

Strain T grew only in the presence of either PABA or thymine. Strain LD-3 grew either with PABA or with a combination of amino acids and purines. The purine could be xanthine or, less effectively, adenine. In the presence of xanthine, delayed growth occurred with methionine, good growth with a combination of threonine, methionine, cystine, tryptophan, and arginine. Strain B-3 required either PABA or a combination of lysine, methionine, and adenine or xanthine. Strain C-2 was unable to grow in the presence of PABA but grew well when guanine or xanthine was added.

The exact phenomena involved in these variations and their genetic basis are obscure at present. From x-rayed cell suspensions of E. coZi we have obtained mutants which require the presence of purines or purines and

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amino acids. for growth but are unable to grow with PABA.2 These m,u- tants generally can use any of the four purines, although with varying effi- ciency, and usually require several amino acids. These mutants resemble in their growth requirements the varian,t st.rains derived from mutant 273-384.

Studies with ,jifutqnL./t+@Z a.n$<?fjl -&W-The nutrit.ional requirements of mutant 45-462 were studied in detail and appear to be the same as those of mutant 273-384. The ‘combination of thymine, a purine, and amino acids was again able to replace PABA. These mutants are cer- tainly very similar, if not identical.

Mutant 1861-460 grew rapidlyiin the presence of PABA but was also able to groiv slowly with methionine as the sole supplement. ’ Turbidity was observed somewhat earlierivhen both methionine and the purines. or the complete amino’ acid supplement, was present; however, the growth with such combinations never equaled: that observed with PABA. When the mutant was plated on A. C. agar and single colonies picked, all of the resulting cultures showed the same characteristics. Thus the strain does not appear to consist of a mixture of two types of cells but rather to be analogous to the variant strains of mutant 273-384described earlier.

‘, DIh7SSION

The experiments .reported here provide additional evidence that PABA (or some derived form), functions in the synthesis of the purines and methio- nine and indicate a role in the formation of thymine and of amino acids other than methionine. The fact that all three supplements are required with the unadapted mutant strain is good evidence that the activity of these materials is not due to contamination with PABA. It should be emphasized that the mutant, while able to grow in prolonged subculture with a combination of the three supplements, still grows at a suboptimal rate and generally does not attain the same maximal turbidity as in the presence of PABA. This phenomenon appears analogous to the half max- imal growth obtained with certain organisms if thymine and a purine are present in place of folic acid (18j. ,’ : ,

Previousstudies of mutant strains of E. coli (17, 19) have indicated that the conversion of cysteine’S’ to the methionine S &curs by the same path- way as in Neurospora (20); i.e.,

Cysteine -+ cystathionine -+ homoeysteine -+ methioninc

Mutant 273-384 can utilize homocystine, although poorly, for growth in the absence of PABA but cannot utilize’cystine (in the amino acid supple- ment). This would suggest that PAl3A, in some form, is involved in the

2 Roepke. R. R., and Mercer, F. E., unpublished data.

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J. 0. LAMPEN, Xl. J. JONES, AND R. R. ROEPKE 433

cysteine -+ cystathionine -+ homocysteine series of reactions. This ques- tion has been studied with mutants of E. coti which require the addition of either homocystine or methionine for growth (12). While growth with added homocystine is ‘slower than+ with ‘,added methionine, the concentra- tion of a sulfonamide required to produce half maximal inhibition of growth is approximately the same3 Thus the main action of PABA does not appear to be in the methylation .of homocysteine but in some prior re- action.4

The inactivity of pteroylglutamic acid is surprising, since this compound appears to function in the synthesis of purines and thymine by other or- ganisms. Our parent strain of E. coti and mutant 273-384 (growing in the presence of 0.01 y of PABA per ml.) both synthesize some material which is able to support maximal growth of Stieptococcus faecatis R on a medium free of pteroylglutamic acid. Miller (22)‘has demonstrated that sulfanila- mide inhibits the synthesis of folic acid by E. coti; also Winkler and de <Haan (21) have observed that pteroylglutamic acid can act as a sulfonamide antagonist for their strain of E. coti. Thus a compound similar to pteroyl- glutamic acid, but not necessarily identical with it, occurs in E. coti and may still be concerned in the syntheses discussed here.

SUMMARY

By irradiation of Escherichia cdli with x-rays three mutant strains were obtained which grew when p-aminobenzoic’acid (PABA) was added to the medium. The ability of a series of compounds related.to PABA to support growth of mutant 273-384 is reported. The addition of a mixture of pu- rines and amino acids reduces the concentration of PABA required for half maximal growth. PABA can be replaced’by a combination of amino acids, a purine, and thymine, although growth is not optimal. The purine re- quirement, can be supplied as adenine, guanine, xanthine, or hypoxanthine. Methionine is an essential component of the amino acid supplement but cannot replace the entire mixture. The data provide additional evidence that PABA functions in E. coti in the synthesis of purines and methionine

3 Lampen, J. O., and aones, M. J., unpublished data. 4 Winkler and de Haan (21) have recently studied the ability of many of the com-

pounds used here t.o antagonize the inhibition of E. coli by high concentrations of sulfonamides. They report that homocystcine with or without choline did not replace met.hionine as an antagonist. One explanation of this difference from our results might be that PAPA functions in methionine synthesis at two points; i.e., some- where in the cysteine + homocysteine series of reactions and in the methylation of homocysteine, the first reaction being inhibited by much lower concentrations of sulfonamide than is the second. We have also observed that the growth with homo- cysteine is slow and thus may have been missed with the short incubation period used by Winkler and de Haan.

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434 MUTANT STRAINS OF E. COLI

and indicate a role in the formation of thymine and of amino acids other than methionine. The point of action of PABA in the synthesis of methio- nine is discussed.

Both mutant 273384 and the parent strain of E. coli are highly resistant to sulfonamides when growing in the presence of the purines, thymine, and a mixture of amino acids.

Pteroylglutamic acid does not replace PABA for the growth of mutant 273384.

Mutant 273-384 can give rise to variants which can grow with only two (or even one) of the three supplements it originally requires in the absence of PABA. Certain of these cultures eventually lose their ability to grow in the presence of PABA.

Mutant 45-462 appears to be very similar to strain 273-384. Mutant 1861-460 grows slowly in the presence of methionine alone and appears similar to the variant strains obtained from mutant 273-384.

BIBLIOGRAPHY

1. Roepke, R. R., Libby, R. L., and Small, M. H., J. Sact., 48,401 (1944). 2. Roepke, R. R., and Mercer, F. E., J. Bact., 64,731 (1947). 3. Woods, D. D., &it. J. Exp. Path., 21, 74 (1949). 4. Rubbo, S. D., Maxwell, M., Fairbridge, R. A., and Gillespie, J. &I., Australian

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J. O. Lampen, M. J. Jones and R. R. Roepke-AMINOBENZOIC ACID

pCOLI UNABLE TO SYNTHESIZE MUTANT STRAINS OF ESCHERICHIA

1949, 180:423-434.J. Biol. Chem. 

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