Interaction of thiamine and pyridoxine in Neurospora. I. Studies of the pyridoxineless mutants

Interaction of Thiamine and Pyridoxine in Neurospora. I. Studies of the Pyridoxineless Mutants’ Daniel L. Harris From the William G. Kerckhofl Laboratories of the Biological Sciewes, California Institute of Technology, Pasadena, California; and the Department of Physiology, University of Chicago, Chicago, Zllinois Received

April

14, 1952

INTRODUCTION Neurospora sitophila 299, a mut,ant st,rain frequently used for the bioassay of the pyridoximers, grows better at limiting concentrations of vitamin Bc if thiamine is also added to the culture medium (1,2). It will be shown.below that thiamine inhibits the destruction of Ba, probably by competing for the one or more enzymes which’destroy Bc, and that Bg in turn inhibits thiamine biosynthesis. In the economy of the organism, the competitive inhibition of B6 destruction leads to an improvement in growth at low Be concentrations. The inhibition of thiamine biosynthesis has, however, no detectable effect on the dry weight..

METHODS Seven pyridoxineless mutants of Neurospora erassa and one of N. silophila have been investigated. Most of these strains appear to be allelic (3); the evidence that all are is not conclusive (4). In any case, all show qualitatively identical responses to thiamine; and most of the work has, therefore, been done with one strain, 378038, which is quantitatively more favorable since it shows no growth in the absence of Be, even at elevated pH, as do many of the pyridoxineless mutants (1, 3, 4). Growth conditions and methods of assay are those commonly used in studies of Neurospora (5). In all the experiments reported here, B, was added in the form of pyridoxine. However, thiamine has the same effect if either pyridoxal or pyridoxamine is used as a source of Bg. ___..~ ’ This investigation was initiated when the author was Eli Lilly Research Fellow at the California Institute of Technology. It has been supported in part by the Wallace C. and Clara M. Abbott Memorial fund and by a research grant (No. 116) from the National Institutes of Health, U. S. Public Health Service. 294

B,j-B1 INTERACTION.

295

I

EXPERIMENTAL

The most direct evidence that thiamine inhibits the destruction of BP,is given in Table I. In this representative experiment, strain 37803A was grown at a limiting pyridoxine concentration with or without excess exogenous thiamine. Extracts of the mycelia were t,hen assayed for B, wit.h the same strain. Several points will be noted in these data. (a) Virtually all of the pyridoxine has disappeared from t,he culture media. and has presumably been taken up by the mycelia. (b) Only a small part of the exogenous Be was recovered in the mycelia. It is thus apparent that much has been destroyed. In view of the difficulty in extracting all of the Be, the absolute values shown may he somewhat low and the author is not convinced that t.here is substantial TABLE I oj Bs Destruclion by Thiamine Strain 37803A grown 7 days at 25” in 500 ml. medium supplemented with pyridoxine and thiamine as noted. Mycelia were washed and then autoclaved in 10 vol. distilled water, 30 min., 15 lb. Aliquots (0.2-10 ml.) of the extracts wereassayed for B( with 37803A in presence of excess thiamine. Necessary control and recovery experiment.8 were included in the bioassay. Inhibition

13, added, m&moles Bs added, mrmoles Dry wt.., g. B, found in medium, mpmoles Be found in mycelium, mwmoles hIillimicromoles &/!I.

0

100 0.565 3 20.7 36.7

1250 100 1.046 4 38.6 36.8

destruction of Be by the mycelium grown in the presence of excess exogenous thiamine. Strauss (4) has also noted a destruct.ion of exogenous Be by strains 37803 and 44602. In his experiments the strains were grown in the absence of exogenous thiamine. (c) Approximately half as much Be is found in the mycelium grown in the absence of exogenous vitamin B1 as in that grown in the presence of B1. As will be noted below, an identical ratio can be derived from other data. We may conclude that exogenous B1 inhibits t.he destruction by the growing mycelium of the exogenous Be. (d) The concentration of Be is the same in both mycelial mats, or, to put it the other way, the dry weight is directly proportional to the amount of Be within the mycelium. The above data provide critical evidence that. HI it1hibit.s 116 de-

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DANIEL

L.

HARRIS

struction. Taken together with the experiments cited below, all of which are consistent with this interpretation, they are conclusive. The reactions which one can infer from these observations may be stated as follows : (Cj -a-.+

13a ,

(‘)

+

enzymes

3

dry weight

/ (E)

3

(1)

/

‘dF) B ‘1 1

CD) \I s

According to this scheme, the gene mutation blocks the synthesis (A) of endogenous Be and thus interrupts growth. Exogenous Bs may, however, be substituted and will be converted (B) as the coenzymes pyridoxal and pyridoxamine phosphate to enzymes which catalyze reactions (C) ultimately resulting in protoplasmic syntheses as measured by dry weight. Obviously, numerous other reactions (D) are essential to the syntheses, and, if excess Bc is present, they will limit the growth. Exogenous Be has, however, two fates. Part is converted (B) to a useful form, part is converted (F) to some inactive form (X). B1, apparently, can inhibit this reaction. The biosynthesis (E) of B1 is essentially intact. It will be shown below that at high Bg concentrations it is somewhat inhibited. It is logical to suppose that the destruction (F) of Be is enzymatically catalyzed. Indeed, any other assumption would require special justification. If this is the case, B1 conceivably prevents the destruction by competing with Be for the destructive enzyme(s). The data presented below are consistent with this hypothesis and will be examined in terms of the reactions written. Analysis of the effect of thiamine on the growth curves of the Be-less mutants yields results in qualitative and quantitative agreement with the data of Table I. Figure 1 show that thiamine has no effect on either the maximum dry weight or the shape of the growth curve of strain 37803. Indeed, if we assumethat one-half of the exogenous pyridoxine is destroyed, and shift the lower curve of Fig. 1 to the left accordingly, the two curves can be superimposed. With minor changes in intercepts with ordinate or abscissa, but none in shape, identical curves can be

BI~--B~ INTERACTION.

297

I

drawn for each of the pyridoxineless mutants. The fraction of exogenous Bg destroyed differs with strain, varying from 40 to SO?$,,but is constant for a given strain from experiment to experiment and is within broad limits independent of Be concentration. The data are thus consistent with the view that B1 competitively inhibits Bs destruction and suggest that the destructive enzyme is not readily saturated by Be even at the highest concentrations tested. Somewhat more direct evidence that B1 competitively inhibits Ba destruction is obtained from a study of the amount of thiamine required 70-

40-

s-

0.25

I P YRIOOXIME

4 Imbt&f/2Orl.)

lb

t

64

FIG. 1. Effect of pyridoxine and thiamine on dry weight of the pyridoxineless mutant, 378038. Lower curve, no B1 added to culture medium; upper curve, excessB1 (64 X 10-S)mole added to medium. Incubation conditions: 25”C., 72 hr., 125-ml. Erlenmeyer flasks. Curves are empirical, but were drawn with the same template.

by the mold at various concentrations of Be. We are interested in the amount of B1 required to produce the maximum growth possible at any given concentration of Bs, i.e., the amount required to pass from the lower to the upper curve of Fig. 1. That this amount is dependent upon Ba concentration is implicit in Fig. 1, but is shown directly in Fig. 2a, in which the upper curve of Fig. 1 is plotted as 100% maximum growth. The amount of thiamine required to saturate the mold is difficult to ascertain with precision, but the best estimate appears to be that shown by the dotted line on the surface of the three-dimensional solid, and reproduced as the uppermost curve of Fig. 2b. In the latter graph addi-

298

DANIEL

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HARRIS

tional curves for other degreesof saturation with thiamine corresponding to 99,95,90,80,70, and 60% maximum growth are also given, and these all form a family of curves characterized by a slow rise to a maximum followed by a rapid fall to zero. Apart from the absolute magnitude of the values shown, the general shape of the curves seems characteristic

4 Es tmpM/.?O

nlJ

Q (mpM/ZOm/.l

Fro. 2. Concentration of thiamine required to produce optimal growth. discussion, see text. Incubation conditions: 25”C., 72 hr., 125-ml. flasks.

For

and relatively certain. Comparable results have been obtained in two other experiments. In terms of the reactions written above [Eq. (l)], these curves may be interpreted as follows. If Bi competitively inhibits Be destruction, at least two factors will be of importance: (a) At strictly limiting concentrations of Bs, i.e., less than 4 mpmoles Be/flask, one may suppose that as Bs is increased,

Be-B1

INTERACTION.

299

I

the absolute amount of thiamine required to compete successfully for the destructive enzyme would also increase. (b) However, to the degree that substances other than Be become limiting, the loss of Be becomes inconsequential, and the thiamine requirement therefore drops rapidly to zero. The interaction of these two factors is more clearly shown in Fig. 2c which demonstrates that the competitive inhibition of Be destruction (as measured by the ratio B1/ Bs, which is properly regarded as an inhibition index) becomes progressively less important as Bg is increased, i.e., as factors other than Bg become limiting to growth. The data thus far presented have given a static picture of the destruction of pyridoxine. Table IT demonstrates that the thiamine effect does The Eifecl

TABLE II of B1 and Time on Dry Weight

Strain 378038; BF, = 1 mpmolej20 ml.; 25°C. Days growth No B,

2 14.4

3 16.8

4 18.8

5 22.6

6 21.2

7 22.8

8 24.2

10 24.2

24 24

2 mpmoles Ratio

20.6 1.43

26.6 1.59

29.6 1.57

31.6 1.39

32.2 1.52

35.0 1.53

34.4 1.42

37.3 1.55

37 1.55

B,

TABLE

III

The Effect of Dry Weight of Delayed Strain

Addition

of B1

378038; Bg = 2 mpmoles/l5 ml.; B, = 50 mpmoles; 7 days, 25°C.

Days Dry wt.

Per cent Bc available

0 62

loo

1 50 80.5

2 44 71

3 33 53

Incubation: 4 33 53

5 31 50

7 30 48.4

not disappear or even diminish with time. Table III shows that if thiamine is added after growth is initiated it has progressively less effect. These dat,a suggest t,hat,pyridoxine is destroyed during the initial stages of growt,h, i.e., before the coenzymes pyridoxal and pyridoxamine phosphate become bound to the corresponding apoenzymes, and that this destruction is largely if not entirely irreversible. It is possible to calculate directly from the observed dry weights the percentage of pyridoxine not destroyed, i.e., available to the mold. (This calculation is justified at, IOH’concentrat,ions of Be by the data of Table I and Fig. 1.) It will he noted (Table III) that the available pyridoxine drops rapidly at first and then approaches, perhaps asymptotically, the limiting value of ahout 18%:,. This suggests t,hat the rat.e of dest~ruct~ion of BF, is propor-

300

DANIEL

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tional to its concentration. More extensive data are, however, obviously needed. Stokes et al. (1) showed that strain 299 could grovv and synthesize its own Bs if ammonium salts were present and the pH of the medium was above 5.6; this observation has since been confirmed and extended to other strains (3, 4). As Table IV shows, thiamine will increase the dry weight of strain 44602a under these conditions by a factor of 3 or 4, a figure in good agreement with data obtained for this strain using exogenous Bs. We may conclude that exogenous thiamine inhibits the destruction of endogenous as well as of exogenous BE. The above data show conclusively that exogenous B1 inhibits the destruction of both exogenous and endogenous Bs. They suggest strongly that this inhibition is competitive. It is not immediately apparent, however, why exogenous thiamine should be required. A priori, one TABLE IV Effect of Thiamine on Dry Weight of &60.2a PH

Supplement 0 B,, 50 m@moles Ratio

5.7 0.5 mg. 2 mg. 4.0

6.5 9 mg. 36 mg. 4.0

7.2 14 mg. 39 mg. 2.s

would expect the B,j-lessmutants to be capable of synthesizing thiamine in amounts sufficient to meet their metabolic needs. The amount of thiamine in the mycelium of the Bo-less mutant, 378038, grown at various concentrations of Bs was assayed with the aid of 9185, a mutant strain of Neurospora which requires intact thiamine for growth (2). As a control, analyses were also made of the mycelium of the wild type (1A) grown in the presence of various concentrations of biotin. The results (Table V) suggest that the concentration of B1in the mycelium of the wild type is essentially independent of biotin. In the mutant grown at very low levels of Ba, the concentration of B1 in the mycelium approaches that found in the wild, type; but as Be is increased, the concentration of B1 drops markedly. Pyridoxine thus seems to interfere with thiamine biosynthesis. Further data in support of this finding will be presented in later publications (6). Table V also shows the minimum amount of exogenous thiamine required to produce optimal growth at the stated Be concentrations. These figures were calculated from the data of Fig. 2c. It will be noted

BgBl

INTERACTION.

301

I

that at, limiting Be concentrations, 12 times as much exogenous as endogenous thiamine is needed, even though the concentration of thiamine within the mycelium is virtually normal. It thus would seem that neither the mutant nor the wild type is able to synthesize the massive doses of thiamine required to prevent the destruction of Bs. To the mutant this has serious consequences; to the wild type it is of no moment, for the wild type synthesizes more Bs than it requires for growth. Thus Strauss (4) found that wild type 4A contains after 4 days growth approximately 200 mpmoles BJg. mycelium, while the culture medium TABLE V E$ixt of Biotin and Pyridoxine on the Synthesis of Thiamine Strains 1A and 378038 were grown for 7 days at 25°C. in 500 ml. of media. Mycelium was washed and thiamine extracted by autoclaving mycelia in 200 ml. of 0.1 N HCl for 30 min. at 15 lb. Aliquots (0.2-5 ml.) were assayed for B, with 9185. Necessary control and recovery experiments were included. The amounts of exogenous B, required for optimal growth of 378038 were calculated from Fig. 2c. IA Biotin, mpmoles Dry weight, mg. B1 found, mpmoles BI concn., mpmoleslg.

0.008 0.07 125 336 50 loo 330 294

Pyridoxine, mpmoles Dry weight, mg. B, found, mpmoles B1 concn., mpmoles/g. Exog. B1 required, mpmoles

4 48 12.5 277 104

0.6 719 150 210 378038

12.5 190 21.5 113 250

50 489 50 102 600

200 922 60 66 800

800 1115 75 67 0

contains 100 mpmoles. Preliminary experiments with wild type 1A indicate that after 7 days the culture medium alone may contain as much as 1000 mpmoles. The massive doses of thiamine required provide further evidence that in preventing Bs destruction, thiamine acts as a substrate rather than as a catalyst, i.e., competitively. It, will be recalled from Fig. 1 that exogenous thiamine has a negligible effect on the dry weight of the mutant grown at, high Be concentrations, even though the endogenous B1 is then at a minimum. In fact, as Be is increased, both the concentration of endogenous thiamine (Table V) and the relative requirement

302

DANIEL

L.

HARRIS

for thiamine (Fig. 2c) fall steadily. One must, therefore, conclude that this minimum concentration of B1, i.e., 60-70 mHmoles BJg. dry weight, must be enough to meet the minimum demands of the organism for BI as a catalyst, at least so far as dry weight is concerned. That it is adequate is suggested by the data of Tatum and Bell (2) from which one can calculate that the thiamineless mutants 9185 and 18558 require approximately 10-20 mFmoles B1 to produce 1 g. of dry weight. Other strains, 1090 and 17084, require somewhat more, perhaps 60-100 rnhmoles/g. Even some of this exogenous B1 may be destroyed, since these authors recovered in extracts of the mycelium only half of the exogenous thiamine. DISCUSSION The above data lead to several significant conclusions: (a) A relatively constant fraction of the endogenous or exogenous BE is destroyed by the B&ess mutants of Neurospora in the absence of exogenous thiamine. The fraction destroyed varies from 40 to 80% depending on the strain. (b) This destruction can be prevented by the addition of relatively large quantities of exogenous B1, the amount required depending upon the amount of and need for Bb. In doing this, thiamine appears to act in a noncatalytic manner. The data strongly suggest that thiamine acts as a substrate which competitively inhibits Be destruction. (c) Exogenous Bs inhibits thiamine biosynthesis. There is no convincing evidence, as yet, that endogenous B, does so as well. It will be shown in a later paper (6) that Bg and BJike compounds inhibit the growth of several thiamineless mutants when they are grown in media supplemented with the pyrimidine and thiazole moieties of thiamine. But little if any inhibition is seen if thiamine itself is the supplement. This inhibition is relieved competitively and efficiently by pyrimidine, but apparently only indirectly by thiazole. Although other interpretations are not excluded, the data suggest strongly that Be competes with pyrimidine for the site on some enzyme, quite possibly the enzyme which couples pyrimidine and thiazole to form thiamine and that B, may, in fact, be coupled with thiazole to form an inactive or inhibitory compound. In the Be-less mutants this would lead to a loss of both BC and B1. If this view is correct, the thiamine effect in the Be-less mutants would be due to a saturation of this enzyme with thiamine, particularly

Be-B1 INTERACTION.

I

303

during the early stages of growth before pyridoxal and pyridoxamine phosphate arising from endogenous or exogenous Bg become bound to the corresponding apoenzymes. Such saturation would competitively prevent the loss of Ba and would explain the relatively large quantities of thiamine required, quantities which neither the mutant nor the wild type seem able to synthesize. As noted above, the loss of Bs in the wild type is of no moment since the wild type synthesizes more Bs than it requires. . A somewhat similar situation may obtain in the yeast Saccharmyces carlsbergensis. According to Rabinowitz and Snell (7) this yeast requires pyridoxine for growth only if thiamine is also present in the culture medium or in the inoculum. Although they did not explain the requirement for pyridoxine, they did suggest that thiamine was toxic and succeeded in getting growth by the addition of an antimetabolite of thiamine, neopyrithiamine. It seems altogether possible that pyridoxine may inhibit the biosynthesis of thiamine in this yeast and thus reduce the endogenous thiamine to nontoxic levels. For those interested in using Neurospora for bioassay of the pyridoximers it may be noted that it is not only convenient but in fact advisable to add thiamine to the culture medium when performing bioassays, particularly when analyzing natural products. Addition of thiamine increases not only the precision, but also the sensitivity and specificity of the assay. It may also be noted that the strain used in this investigat.ion, 37803, is, because of its insensitivity to pH and absolute requirement for Be, more satisfactory for bioassay than is strain 299. The loglog plot of Fig. 1 may also prove useful for this purpose. SUMMARY

All. pyridoxineless mutants of Neurospora crassa and Neurospora sitophila grow better if thiamine is also added to the culture medium. On the basis of the evidence which includes (a) an analysis of the growth curves; (b) the requirement for thiamine at various concentrations of pyridoxine; (c) the interrelation of vitamins Be, B1, and time; and (d) assay of the mycelial content of Be and B1; it is concluded that BI competitively inhibits the destruction of BG. Be in turn interferes with thiamine biosynthesis, but sufficient endogenous B1 is present to satisfy normal metabolic requirements, at least so far as growth is concerned. A specific mechanism is suggested which will be further developed in later publications.

304

DANIEL L. HARRIS REFERENCES

1. STOKES, J. L., FOSTER, J. W., AND WOODWARD, C. R., JR., Arch. B&hem. 2, 235 (1943). 2. TATUM, E. L., AND BELL, T. T., Am. J. Botany 33, 15 (1946). 3. HOULAHAN, M. B., BEADLE, G. W., AND CALHOUN, H. G., Genetics 34, 493 (1949). 4. STRILUSS, B. S., Arch. Biochem. 30, 292 (1951). 5. HOROWITZ, N. H., J. Biol. Chem. 171, 255 (1947). 6. HARRIS, D. L., in preparation. 7. RABINOWITZ, J. C., AND SNELL, E. E., Arch. Biochem. Biophys. 33, 472 (1951).