The enhancement of selenite toxicity by methionine in Escherichia coli

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

99,

The Enhancement Methionine JAMES From the Department

SCALA’

363-368

of Selenite

Toxicity

in Escherichia

co/i

HAROLD

AND

of Biochemistry, Received

(1962)

Cornell August

by

H. WILLIAMS

University,

Ithaca,

New York

20, 1962

The toxicity of Se03- - to wild type Escherichia coli is enhanced by the presence of L-methionine in the growth medium. Data are presented which show t,hat the same degree of toxicity is exhibited by the methionine-requiring mutant of E. coli, A.T.C.C. 9663. n-Methionine is shown to be ineffective. Exogenous methionine suppresses the biosynthesis of selenomethionine as well as methionine. The suppression of selenomethionine biosynthesis provides a basis for the enhanced toxicity of SeOl- -. It is also possible that the biosynthesis of selenocyst(e)ine may account for part of the enhanced toxicity. Interaction between Se0 3 -- and methionine as a basis for enhanced toxicity is unlikely from the data obtained on t’he methionine-requiring mutant, A.T.C.C. 9663. INTRODUCTION

Leifson reported in 1937 that selenite is effective in inhibiting the growth of Escherichia coli during the isolation of typhoid bacilli from various natural sources (1). He also reported that sulfite enhanced the inhibitory effect of selenite (2). More complete studies by Gohar (3) and by Opienska-Blauth (4) show that selenium as selenite, is bacteriostatic to E. coli above a concentration of 23 p.p.m. Fels and Cheldelin (5) reported that selenate also suppresses the growth of E. coli and that cysteine and glutathione, but not methionine, reverse the suppression. More recently, Tuve and Williams (6, 7) and Cohen and Cowie (8,9) have done complete radioselenite uptake studies on E. coli. Tuve and Williams demonstrated that selenite is incorporated into bacterial protein as selenomethionine. Both groups have demonstrated the probable formation and incorporation into protein of selenocyst(e)ine. Cohen and Cowie reported that the methi[mine-requiring mutant ML-304-d could grow at a reduced, but exponential rate with * Predoct.oral Fellow, Division of General cal Sciences, U. S. Public Health Service.

Medi363

selenomethionine replacing methionine. The same organism, however, could not be cultured on selenomethionine agar. By isotopic competition studies, Cohen and Cowie demonstrated the suppression of radiosulfate uptake by radioselenite. On the basis of these isotopic competition studies, they reported that selenium could spare the sulfur requirement. This sparing effect is in disagreement with the growth studies of Tuve and Williams. The earlier studies (l-5) demonstrate, and later studies (7-9) show conclusively, that selenate and selenite are incorporated and reduced in the sulfate reduction pathway of E. coli. Since the oxidative properties of selenate and selenite account for their toxicity [see (7)], their reduction and incorporation into selenomethionine could represent a process of detoxification. The data of Leifson (2) and of Fels and Cheldelin (5) support this possibility. Roberts et al. (10) have shown by isotopic competition that E. coli incorporates sulfur in the most reduced form which is presented to the organism. Therefore, if sulfite is available, the reduction pathway is incorporating at this level. In an experiment such as Leifson’s (a), due to the similarity of the ions,

364

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selenite would be incorporated in large amounts and thus would be more toxic. Also, cystine and glutathione would be incorporated preferentially to sulfate or sulfite. Thus, cystine and glutathione would relieve the toxic effects of selenite or selenate by suppressing their incorporation. The studies of Roberts et al. also demonstrate that exogenous methionine completely suppresses the incorporation of radiosulfate into methionine sulfur. They also show that methionine biosynthesis is rate limiting since exogenous methionine increases the growth rate. The more recent work of Buchanan et al. (11) shows that methionine methylation is energy requiring and, therefore, rate limiting. Thus, the effect of methionine on selenate and selenite should be opposite to cystine and glutathione. The total growth studies of Fels and Cheldelin (5) demonstrated only that methionine would not reverse the selenate or selenite toxicity. Our hypothesis is that methionine would enhance the toxicity of these ions by suppressing biosynthesis of selenomethionine. This is based on the isotopic competition studies of Roberts et al. and on the more recent observations (7-9) that selenomethionine is not toxic to E. coli. We have studied growth rate and total growth of three wild-type strains and one methionine-requiring mutant under various conditions. In every case, selenite toxicity is enhanced by methionine, and selenite is toxic in low amounts to the methioninerequiring mutant. MATERIALS

AND

METHODS

The wild-type strains ML-30, K-12, and B and the methionine-requiring mutant A.T.C.C. 9663 were used in these experiments. The “S” medium containing glucose of Roberts et al. (10) was used exclusively, and sulfur was added as sodium sulfate to meet the needs of individual experiments. Selenite was added as its sodium salt. Aeration was accomplished by rotary shaking. Growth of bacteria was followed as optical density at 650 rnp in a Beckman model DU spectrophotometer and related to micrograms dry weight of bacteria/ ml. on a standard curve.

TOTAL GROWTH STUDIES Total growth was studied culture to grow overnight

by allowing a starter on a complete “C”

WILLIAMS

medium [see (lo)]. Enough cells were then aseptically transferred from this starter culture to the medium under study to bring the bacterial concentration to 0.0021 mg. dry weight, of cells/ml. Invariably, the amount of medium transferred was less than 0.75 ml.; since the volume used for total growth studies was always 125 ml., this small volume had no effect on the concentration of the “S” components. The media for total growth studies contained 0.3 flumoles sulfur/ml. when methionine was present. In the absence of methionine, the sulfate concentration was 0.6 rmole/ml. The data for total growth is reported as micrograms of growth/ml. medium in 24 hr. Optical density was taken at appropriate intervals, hut, invariably, total growth was achieved before 24 hr. GROWTH RATE STUDIES Cells for growth rate studies were taken from an exponentially growing starter culture by centrifugation at 7000 X 9 for 3 min. in a Servall refrigerated centrifuge. The cells were then resuspended and washed three times in 0.85y0 NaCl. After the third sedimentation, the cells were resuspended into a sample of t,he medium to be studied, and an aliquot of the suspension was used for inoculation. The size of the aliquot was chosen so as to bring the bacterial concentration to at least 0.015 mg. dry weight/ml. The methionine-containing media for growth rate studies contained 0.3 @mole sulfate/ml. When methionine was absent, the sulfate concentration was 0.6 pmole/ml.

GROWTH IN SULFUR-DEFICIENT

MEDIA

In order to observe growth up to and past the sulfur depletion point, the cells were prepared in the same way as for growth rate studies. The growth media, however, were different. Media devoid of usable sulfur were prepared by first growing cells on an “S” medium to sulfur depletion. This growth removed all sulfur contamination derived from the reagents. The cells were then removed by centrifugation, and the medium was sterilized by filtration. Sulfate was then added to the desired concentration. The sulfur concentrations used in these studies were approximately 0.2 pmolelml., and the inocula were sufficient to bring the bacterial concentration to at least 0.15 mg. dry weight/ml. RESULTS

Table I summarizes the results of the total growth studies. The data demonstrate the relatively high amounts of SeOs --

METHIONINE

TOTAL

GROWTH

OF

MEDIUM

AND

SELENITE

TABLE

I

E. con ML 30

AND

CONTAINING

A.T.C.C.

METHIONINE

365

TOXICITY

9663 IN 24 HR. IN

AND

SEO~ -

See text for procedure used in total growth experiments. Molar Concentration Strain

of SeOa-

Molar coqcn, of =L-methlonlne

0.00

1 :‘,z

/ i”,-F

1 ‘f?$

1 ;;g

/ Z-T

pg. dry weight/ml.

/ $7

1 “,“,-T / y,“-,x

/ ‘$s”

/ ‘f,“-F

medium

ML-30

0

590

590

595

590

595

585

571

550

530

522

195

MI,-30

10-a

605

345

130

51

37

23

11

9

9

6

2

10-a

600

325

150

48

33

27

13

8

7

4

2

A.T.C.C.

9663

TABLE TOTAL 48 X

GROWTH

IO-5 M

ML

OF E. COLI AND VARIED

II

30 IN MEDIUM

AMOUNTS

OF D- AND

Experimental Methionine

isomer

CONTAINING SEO~-L-METHIONINE

with 48 X 10-b $f SeOa- -

Methionine concentration, 40

x 10-s

0.0

4 x

8 x 10-s

10-s

pg.

dry

M

12 x 10-s

24

x 10-s

32

X 10-s

40

x 10-e

weight of cells/ml. medium

u-Methionine

480

460

475

478

480

482

482

485

r,-Methionine

620

460

42

38

33

33

33

33

necessary for toxicity to total growth in the absence of methionine. In the presence of nn-methionine, Se03 - is toxic at low levels. Similar results are obtained with the wildtype strains B and K 12. The growth of the methionine-requiring mutant A.T.C.C. 9663 is inhibited by comparable low levels of SeOa- -. Table II shows the total growth obtained when D- and r,-methionine are added separately to cultures containing Se03 -. n-Methionine is not utilized by the organism and exerts no effect on growth in the presence of selenite, while L-methionine affects total growth similar to that described in Table I. L-Methionine enhances growth in the absence of selenite and inhibits growth in the presence of selenite. Figure 1 shows how the growth rate, mg, dry weight vs. time, of wild-type E. coli is affected by the combination of methionine and SeO, -. The growth rate is unaffected by SeOl- - concentrations below 12 X 1O-4

M in the absence of nL-methionine. However, the same medium in the presence of nL-methionine reduces the growth below 40% of the control cultures. Comparable results are obtained with wild-type strains K 12 and B. Figure 2 demonstrates that even low concentrations of SeO, - reduce the growth rate of the methionine-requiring mutant A.T.C.C. 9663. Figure 3 shows the data obtained when wild-type strains are grown in the presence of Se03 -, methionine, and limiting sulfur. The sulfur depletion point appears sooner in the presence of selenite, both with and without methionine. Similar results are obtained for the wildtype strains K 12 and B. Figure 4 shows the data obtained when the methionine-requiring mutant is grown in Se03 - and limiting sulfur. The data show that the sulfur depletion point appears sooner in the presence of Se03-.

366

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WILLIAMS

In every experiment, with the exception of the total growth experiments in which growth was greatly suppressed, the culture

30

0

I 0

I

I

30

60

I

I

1

90 120 150 Time in Minutes

I

I

160

210

FIG. 1. Growth rate of E. coli ML 30 in medium containing adequate sulfate with the following additions: 1. nn-Methionine 10-Z M; 2. Se&none to 8 X 1OP M and n-methionine 5 X lo+ M; 3. SeO, - 12 X IOP M; 4. SeO,-12 X 10-5 M and on-methionine 10-a M; 5. SeOl- - 4 X 1OP to 8 X 1OP M and nn-methionine 1OP M; 6. Se03-12 X 1OP M and nL-methionine 1O-3 M. .2 ‘/

.07 -06 -

60

90

120 150 160 Time in Minutes

210

240

FIG. 3. Growth rate of E. coli ML 30 up to and after the sulfur depletion point in medium containing limiting sulfate (23 X 1OP M), and the following additions: 1. on-Methionine 10-a M; 2. nn-Methionine 10-S M and SeOr - 25 X 10-e M; 3. nn-Methionine 10-S M and SeO,-50 X 1O-6; 4. nn-Methionine 1OP M and SeO,-12 X 10m5M; 5. No additions; 6. SeOl- - 12 X 1O-5 M.

flasks were observed to turn red. This coloration usually starts just before sulfur depletion and increases thereafter until the culture medium is blood red. Washing of the cells by repeated centrifugation and microscopic examination shows the coloration to be intracellular deposits of Se8 . The color is easily duplicated by introducing some cysteine into an “S” medium which contains some selenite. DISCUSSION

All the data presented here illustrate the way in which selenite toxicity is enhanced by L-methionine. Schematically, sulfate incorporation in E. co&i is simplified as follows [see (10) for a more complete consideration] : 0

30

60

FIG. 2. Growth (methionine-requiring taining adequate and the following Se03-- 6 X 1O-5 Se03-- 4 X 1OP Se0312 X 10-a

90 120 150 Time in Minutes

rate

160

210

of E. eoli A.T.C.C. 9663 mutant) in medium consulfate, 10-J M nn-methionine additions: 1. No additions; 2. M; 3. SeOa-- 12 X 10e6 M; 4. M; 5. SeOs- - 8 X lo+ M; 6. M.

so4--

--f

SOa--

+

homoserine \ L Sz03 + cyst(e)ine --+ methionine serine

I

A comparable pathway

protein

i(

for selenite must

METHIONINE

AND

also exist with the major part of the Se going into selenomethionine. In a medium containing exogenous methionine, the organism incorporates methionine directly into protein, and biosynthesis of methionine ceases. This direct incorporation has been reported by Roberts et al. (lo), and is shown by the increased growth rate observed in curve 1 of F’ig. 1. With selenite in the medium, the presence of methionine must also prevent the biosynthesis of selenomethionine. A greatly reduced rate of selenomethionine biosynthesis would make SeOs- - available to oxidize certain functional groups of proteins [SeO, - as an oxidant is discussed by Tuve (7)]. The oxidation of functional groups, e.g., SH groups of enzymes, may render certain protein biologically inactive resulting in less total growth and a reduced growth rate, thus accounting for the results shown in Fig. l-4 and Tables I and II. Tuve (6, 7) showed that as the Se03 concentration increased, uptake increased proportionally. Likewise, the enhancement of toxicity should increase with increasing SeO, - in the presence of methionine, as our data indicate. It is also possible, that selenocyst(e)ine renders protein partially or completely inactive. The formation of selenocyst(e)ine and its incorporation is shown by the data of Cowie and Cohen (9). If selenocyst(e)ine is formed under the conditions of these experiments, it could result in biologically inactive protein. This could partially account for the reduced total growth and growth rates which were observed. The significance of this possibility would necessitate the isolation, purification, and study of enzymes formed under the conditions which have been presented. It is unlikely that a reaction between selenite and methionine is responsible for the enhancement of toxicity. Such a reaction would necessarily affect the methionine biosynthetic pathway or the mechanism of methionine incorporation. Since methionine was always in excess of Se03 -, it is doubtful that the mechanism of incorporation could be affected to an extent sufficient to produce tht observed toxicity. The data on the methionine-requiring mutant, which cannot

SELENITE

I 0

367

TOXICITY

I 30

, 60

I I I 90 120 150 Time in Minutes

, 180

I 210

I 240

FIG. 4. Growth rate of E. coli A.T.C.C. 9663 (methionine-requiring mutant) up to and after the sulfur-depletion point in medium containing limiting sulfate (23 X 10e5 M), DL-methionine 1OF M and the following additions: 1. Xo additions; 2. SeOr-- 25 X 1O-6 M; 3. SeO:,- _ 50 X lo-’ M; 4. Se(Y)10-e ill.

91 X 1OF M; 5. SeOa- - 135 X

synthesize methionine, rule out the possibility that such a reaction is affecting the biosynthetic pathway. Since the growth studies in Figs. 3 and 4 show that there is less total growth to sulfur depletion, one can conclude that selenite does not promote growth in excess of that permitted by the available sulfur. However, this does not rule out the possibility that, at the proper levels, selenite may spare the sulfur requirement. Indeed, the data of Cohen and Cowie (8, 9) indicate such an effect. The data presented in Table I suggest that a medium for the isolation of bacteria from natural products would be more efficient in preventing growth of E. coli if it contained methionine in addition to selenite. A medium containing lop3 molar each of methionine and selenite would virtually prevent all growth of E. coli, and in fact would be a very effective bacteriostatic agent for this organism.

368

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ACKNOWLEDGMENT We are grateful to Dr. Stanley A. Zahler of the Department of Bacteriology for supplying us with slants of E. coli B, K 12, and ML 30. REFERENCES 1. LEIFSON, E., J. Bacterial. 31, 26 (1937). 2. LEIFSON, E., Am. J. Hyg. 24,423 (1936). 3. GOHAR, M. A., J. Trap. Med. Hyg. 46, 29 (1943). 4. OPIENSKA-BLAUTH, J., AND IWANOWSKI, H., Acta Microbial. Polan. 1, 273 (1952) (English summary). 5. FELS, I. G., AND CHELDELIN, V. H., Arch. Biochem. 22, 323 (1949).

WILLIAMS 6. TUVE, T., AND WILLIAMS, H. H., J. Riol. Chem. 236, 597 (1961). 7. TUVE, T. W., Ph.D. Thesis, Cornell Univ., Ithaca, N. Y., 1958. 8. COHEN, G. N., AND COWIE, D. B., Comnt. rend.‘244, 686 (1957). . 9. COWIE, D. B., AND COHEN, G. N., Biochim. et Biophys. Acta 26, 252 (1957). 10. ROBERTS, R. B., ABELSON, P. H., COWIE, D. B., BOLTON, E. T., AND BRITTEN, R. J., Carnegie Inst. Wash. Publ. No. 607, pps. 318-84 (1955). 11. HATCH, F. T., LARRABEE, A. R., RENATA, E.. CATHOU, E., AND BUCHANAN, J. M., J. Biol. Chem. 236, 1095 (1961).