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The induced synthesis of catalase in Rhodopseudomonas spheroides
BIOCHIMICA ET BIOPHYSICA ACTA
503
T H E INDUCED SYNTHESIS OF CATALASE IN
RHODOPSEUDOMONAS S P H E R O I D E S RODERICK K. CLAYTON* Department o/ Biochemistry, The Technical University o/Norway, Trondheim (Norway) and Biology Division, Oak Ridge National Laboratory**, Oak Ridge, Tenn. (U.S.A.)
(Received April ioth, 1959)
SUMMARY Synthesis of catalase is induced in Rps. spheroides on contact with air. In cultures grown anaerobically in the light the catalase content is 0.002 % of the dry wt. In aerobic cultures grown in darkness the catalase content is about o.15 %. The rate of induced synthesis of catalase is low in a culture growing exponentially and high in a culture whose growth is limited through depletion of carbon sources. The slow induced synthesis in the former case is not accelerated by adding casein hydrolyzate. Rapid induced synthesis is maintained in a growing culture if glutamate and acetate are omitted from the growth medium. Conditions of gratuity prevail for this induced synthesis of catalase if the culture is aerated gently in the light; rates of growth and induced synthesis are then independent of prior aeration and of catalase content. The rate of aerobic growth in darkness is also independent of catalase content. The kinetics of induction are similar for a culture aerated gently in the light (photosynthetic growth) and a culture aerated vigorously in darkness (aerobic growth). Synthesis of catalase is not related, directly or inversely, to synthesis of bacteriochlorophyll. The induced synthesis of catalase requires sources of carbon, nitrogen, and energy. These requirement and the effects of inhibitors suggest that the protein of catalase is synthesized de novo. Although aeration causes this induced synthesis, energy for the process can be derived either from air or light.
INTRODUCTION Our understanding of the biosynthesis of proteins has been advanced by the study of inducible enzymes 1. An inducible enzyme that possesses a prosthetic group (e.g., a hemoprotein) could be useful in studying the mechanism by which prosthetic group and protein are joined. In cases where the physiological function of an enzyme is uncertain, the function might be elucidated in an organism that can be made to contain either a high or a low concentration of the enzyme. The phenomenon of excitability, traditionally studied by sensory physiologists, is exhibited wheu an organism responds to a change in its environment by making an enzyme. Induced enzyme synthesis is * Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. ** Operated by Union Carbide Corporation for the U.S. Atomic Energy Commission. Biochim. Biophys. Acta, 37 (196o) 5o3-512
504
R. K. CLAYTON
also a special example of cellular differenti,,tion. Thus a variety of problems can be broached through the study of induced enzyme synthesis. With these prospects in mind, an induced synthesis of catalase in the facultative photoheterotroph Rhodopseudomonas st~heroides is being investigated. This organism can grow either aerobically in the dark or anaerobically by photosynthesis" oxygen is not liberated in its photosynthetic metabolism. The induced synthesis of catalase occurs on the introduction of oxygen. This paper describes some aspects of the kinetics, energetics, and chemistry of this induced synthesis. In the study of induced enzyme synthesis, and especially in the interpretation of the kinetics of induction, a considerable clarification is achieved if "conditions of gratuity", as defined by Mol~oD A~D COHI~~, are made to prevailL Such conditions are said to prevail if neither the presence of the enzyme nor its inducer influences the general cellular metabolism. In studying the induced synthesis of/~-galactosidase in Escheriehia coli, MOSOD AND COHN2 achieved gratuity by using methyl-fl-D-thiogalactoside as the inducer. This substance is not a substrate for metabolism, and under the conditions of growth, fl-galactosidase was not involved in the general metabolism of the cells. Conditions of gratuity were attained by CHANTRENNE AND COURTOIS3, in the study of induced catalase synthesis in yeast, by using a mutant that lacks respiratory enzymes (here the inducer is air). For the induced synthesis of catalase in Rps. spheroides the attainment of gratuity under illumination with gentle aeration will be demonstrated. METHODS
Rps. spheroidas, strain 2.4.1, was cultivated in a synthetic medium containing minerals, chelating agents, growth factors, malate, glutamate, and acetate, as described by COI-IEN-BAZlRE at al.4. Photosynthetic cultures were grown anaerobically under illumination at 3°o in glass-stoppered bottles; illumination was provided by three 4o-W tungsten lamps at a distance of about 12 in. Aerobic cultures were grown in darkness at 25 ° in agitated vessels. Catalase was assayed by the iodometric method of HERBERT5. Intact cells wern exposed to toluene for 4 min immediately before assay to render them freely permeable to H20 v Samples that could not be assayed at once were treated with 0.2 vg/ml of tetracycline and placed in darkness; this treatment prevented further changes in their catalase content. Nonenzymic and peroxidatic decomposition of H~O 2 in the assay were ruled out through observation of the reaction kinetics, oxygen evolution, and the effects of azide and heating. The catalase of Rp~. spharoidas has been purified and characterized6. A knowledge of its specific activity permits the expression of catalase content as percentage of the dry wt. From its molecular weight, the number of catalase molecules per bacterium can be estimated. Growth was measured turbidimetrically, at 68o m/~, by the method of COHENBAZIRE at al. 4. Calalasa content and growth cycle In a mature culture of Rps. spharoidas grown anaerobically in the light (henceforth abbreviated Lt. Anaer.), the catalase content is about o.oo2 % of the dry wt. If this culture is used as an inoculum for a culture grown aerobically in darkness, growth Biochim. Biophys. Acta, 37 (196o) 553-512
INDUCED SYNTHESIS OF CATALASE
505
and catalase synthesis proceed as shown in Fig. i where the data from 4 experiments are superimposed. In one experiment covering the range o to 5 h the inoculum was IO %; here the data for cell mass and catalase content were divided by IOO to correspond to a o.i % inoculum. In the other 3 experiments the inoeulum was o.z %. 7-
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Fig. I. G r o w t h a n d c a t a l a s e s y n t h e s i s in Rps. spheroides growing aerobically in d a r k n e s s a t 25 0.
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Fig. I shows that, in a culture growing aerobically in darkness from an inoculum of I t . Anaer. culture, an initial outburst of catalase synthesis is followed b y a slackening, after which the synthesis of catalase keeps pace with growth. As growth becomes limited*, the synthesis of catalase continues for several hours. The inverse experiment is shown in Figs. 2 A and B. Fig. 2A represents a culture growing exponentially in the dark with aeration, which was transferred to anaerobic
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Fig. 2. A. G r o w t h a n d c a t a l a s e s y n t h e s i s in Rps. spheroides t r a n s ferred a t t = 1. 5 h f r o m aerobic conditions (dark, 25 ° ) to anaerobic c o n d i t i o n s (light, 30°). B. G r o w t h a n d c a t a l a s e s y n t h e s i s in Rps. spheroides g r o w i n g anaerobically in t h e l i g h t a t 3 °0 . I n o c u l u m : o.I % (v/v) of a m a t u r e c u l t u r e g r o w n aerobically in d a r k n e s s .
* I n all of t h e e x p e r i m e n t s to be described, g r o w t h in t h e c o m p l e t e m e d i u m b e c a m e l i m i t e d t h r o u g h a n e x h a u s t i o n of sources ot carbon. T h i s w a s a s c e r t a i n e d b y m e a s u r i n g final cell yield as a f u n c t i o n of t h e initial c o n c e n t r a t i o n s of m a l a t e , acetate, a n d g l u t a m a t e .
Biochim. Biophys. Acta, 37 (196o) 5o3-512
R.K. CLAYTON
5o6
conditions in the light (in replicate glass-stoppered bottles) and allowed to grow to completion. Fig 2B portrays the development of a Lt. Anaer. culture from a o.I % inoculum of a culture grown aerobically in darkness. Fig. 2A reveals that, when Rps. spheroides is transferred from aerobic conditions in darkness to anaerobic conditions in the light, growth and the synthesis of catalase cease. Growth is resumed after several hours (during this lag the photosynthetic pigments are being synthesized4), b u t the total catalase in the culture remains constant. Eventually, when the catalase is diluted to a level of about o.ooi %, the synthesis of this enzyme is resumed (Fig. 2B). As growth becomes limited, the catalase content rises to about 0.002 %.
Is new protein synthesized? F u n d a m e n t a l to the understanding of a n y example of induced enzyme synthesis is a knowledge of whether the enzyme-protein is synthesized de hove. A conclusive affirmative answer to this question has been obtained for the induced synthesis of fl-galactosidase in E. coli through studies of radioisotope incorporationT, 8. In our case this type of experiment has not been performed, but considerable circumstantial evidence supports the idea that a de hove synthesis of protein occurs. This evidence is found in the requirements for carbon, nitrogen, and energy and in the action of inhibitors. Figs. 3 and 4 illustrate experiments showing t h a t nonproteillaceous sources ot carbon and nitrogen are required for the induced synthesis of catalase in Rps. spheroides. I n the experiment of Fig. 3 Lt. Anaer, cells were washed and resuspended, in 04
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Fig. 3. Growth and induced catalase synthesis in Rps. spheroides. Cells grown anaerobically in light, washed, and resuspended in basal medium (see text) containing o.o2 M ammonium malate (curves A) or o.02 M sodium malate (curves B). Suspensions aerated in darkness from t = o.
Fig. 4. Growth and induced catalase synthesis in Rps. spheroides. Cells grown anaerobically in light, resuspended in basal medium (see text), incubated anaerobically in light for 48 h, then washed and resuspended in o.o2 M potassium phosphate (pH 6.8) plus additives. Suspensions aerated in darkness from t = o. Additives: curve A, none; curve B, (NH4)zSO4; curve C, Na malate; curve D, NH 4 malate; curve E, Na acetate + Na-H glutamate; and curve F, NH 4 malate + Na acetate + N a - H glutamate. All additives were present initially at a concentration of o.o2 M.
Biochlm. Biophys. Acta, 37 (196o) 5o3-512
INDUCED SYNTHESIS OF CATALASE
507
2 aliquots, in "basal medium" plus 0.02 M ammonium malate (aliquot A) or 0.02 M sodium malate (aliquot B). The basal medium was the same as the complete growth medium except that it lacked ammonium ion, nitrate, malate, acetate, and glutamate. The resuspended aliquots were aerated in darkness. Growth and catalase content were measured at intervals. In the presence of ammonium malate, a vigorous synthesis of catalase proceeded for more than 6 h. In the presence of sodium malate, this synthesis ceased after 4 h, probably because endogenous reserves of fixed nitrogen were exhausted. In the experiment illustrated in Fig. 4 Lt. Anaer. cells were resuspended in basal medium and incubated anaerobically in the light for 48 h, to exhaust endogenous reserves of carbon and nitrogen. The culture was then washed and resuspended in 0.02 M potassium phosphate, pH 6.8. Six portions, containing various additives (see Fig. 4), were aerated in darkness and growth and catalase content were assayed at intervals. No catalase was synthesized by cells suspended in phosphate buffer (curve A), nor by cells suspended in phosphate buffer plus ammonium sulfate or sodium malate (curves B and C). Synthesis of catalase proceeded in the presence of phosphate plus ammonium malate (curve D), or phosphate plus sodium acetate plus monosodium glutamate (curve E). Rates of induced catalase synthesis were proportional to growth rates. Thus the induced synthesis of catalase requires sources of carbon and nitrogen other than intracellular proteins. The roles of light and air in making energy available, and of air as an inducer, were demonstrated in the following experiment. A mature Lt. Anaer. culture was diluted 2.5-fold with complete medium, shaken with air for I min, and dispensed into glass-stoppered bottles, completely filled. These were placed in darkness; at intervals one was opened, and its contents were assayed for catalase. Other bottles were placed in the light after various intervals of darkness, and after 3o-min illumination their contents were assayed. One bottle was opened after 13o min in darkness. The contents were shaken with air for I min, returned to the bottle, illuminated for 3o rain, and assayed. Two bottles, one illuminated and the other in darkness from the start of the experiment, contained o.ooi % methylene blue. The dye was bleached, signalling the exhaustion of dissolved oxygen, after IiO min (illuminated) and 30 min (in darkness) (see Fig. 5). Samples kept in darkness (curve D) showed some increase in catalase during the first 2o min. This is interpreted as an induced synthesis of catalase in which the air introduced initially served both as inducer and as a means of liberating energy through respiration. The first illuminated sample (curve L, o to 30 min) showed a much larger increase in catalase content, for 2 reasons: ('0 In the light the reduction of dissolved oxygen was slower, so that the concentration of inducer remained higher and (b) energy provided by photosynthesis was greater than that provided by respiration at a low oxygen tension. In successive illuminated samples (after longer periods of darkness) the induced synthesis of catalase was progressively smaller because the inducer, air, was being exhausted. The slight induced synthesis in a completely anaerobic sample (curve L, 135 to 165 mill) might have arisen by the interaction of light with oxidizing entities generated by the initial aeration. We shall see later that, in a culture that has had no contact with air for 24 h, an increase in illumination does not act as an inducer. Curve A, L represents the sample that was aerated again after 13o min and then illuminated. Induced synthesis of catalase in this sample was nearly as great as that in the first illuminated sample; therefore inducibility was not lost on standing in darkness Biochim. Biophys. Acta, 37 (196o) 5o3-512
508
R. K. CLAYTON
without air.These results show that the induced synthesis of catalase in Rps. spl~eroides requires energy made available by light or air, but air (not light) acts as inducer. Several inhibitors were found to exert parallel effects on growth and on the induced synthesis of catalase. These results strengthen the likelihood that the latter process involves a synthesis of new protein. Thus chloramphenicol caused a 50 % inhibition both of growth and catalase synthesis at a concentration of I/~g/ml and complete inhibition of both processes at IO/~g/ml. For tetracycline the corresponding figures are o.oi and o.I/~g/ml. 0.4
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Fig. 5. I n d u c e d c a t a l a s e s y n t h e s i s in Rps. spheroides i l l u s t r a t i n g roles of l i g h t a n d air. Cells g r o w n a n a e r o b i c a l l y in light, d i l u t e d 2.5-fold w i t h c o m p l e t e m e d i u m , a e r a t e d i m i n , a n d d i s p e n s e d i n t o g l a s s - s t o p p e r e d b o t t l e s a t t = o. C u r v e D, b o t t l e s k e p t in dark. C u r v e s L, bottles in d a r k u n t i l s t a r t of curve, t h e n in light. C u r v e A, L, b o t t l e in d a r k u n t i l s t a r t of curve, t h e n c o n t e n t s a e r a t e d I m i n , r e t u r n e d to bottle, a n d i l l u m i n a t e d . Dissolved O 8 e x h a u s t e d in d a r k e n e d b o t t l e a t t i m e t n ; in i l l u m i n a t e d b o t t l e a t t i m e tL.
After irradiation with u.v. an inhibition of growth and catalase synthesis occurred that became progressively more severe. A dose that depressed the rate of growth to 50 To of normal 4 h after irradiation reduced the rate of induced catalase synthesis to 6o % of normal. In dim light (below saturation for photosynthesis) the rates of growth and catalase synthesis were depressed to the same extent. At one intensity both rates were 3o TO of maximal, at another, 7 ° %. A plot of growth rate versus pH had the same shape as a plot of rate of induced catalase synthesis versus pH. For both cases the rate was maximal at pH 7.1.
Kinetics o/induction: gratuity," ]eedback m~chanisms The kinetics of the induced synthesis of catalase in Rps. spheroidos appear to be dominated by the physiological state of the organisms. This is illustrated in Fig. 6, where the numbers on the curves represent approximate values for molecules of new catalase/ceU/min. The data of curves A, B, and C were obtained with suspensions that were illuminated and aerated very gently (about 5 bubbles/min through 50 ml of suspension in a large test tube). Curve A shows the rapid, sustained induced synthesis of cataiase, initiated and maintained by aeration in a Lt. Anaer. culture, Biochim. Biophys. Acta, 37 (196o) 5o3-512
INDUCED SYNTHESIS OF CATALASE
509
whose growth had become limited by a depletion of carbon sources (other types of limitation have not been investigated). If such a culture is diluted with complete medium at the start of aeration (curve B), the rate of induced synthesis falls dramatically as growth is resumed. A Lt. Anaer. culture in exponential growth shows, when aerated, a slow but fairly constant rate of induced synthesis (curve C). In this case the differential rate of synthesis (new catalase/new cell mass) is nearly constant. Curve D shows the constitutive synthesis of catalase in an illuminated anaerobic culture in exponential growth. 0.5-
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Fig. 6. I n d u c e d s y n t h e s i s of c a t a l a s e in Rps. spheroides in different s t a g e s of growth. C u r v e A, c u l t u r e g r o w n a n a e r o b i c a l l y in l i g h t to m a t u r i t y (growth l i m i t e d b y e x h a u s t i o n of carbon) t h e n a e r a t e d g e n t l y in l i g h t f r o m t = o. C u r v e B, s a m e as A, b u t c u l t u r e d i l u t e d fivefold w i t h c o m p l e t e m e d i u m a t s t a r t of aeration. G r o w t h b e g a n w i t h o u t lag. C u r v e C, i l l u m i n a t e d , a n a e r o b i c c u l t u r e in e x p o n e n t i a l growth, a e r a t e d in light from t = o. C u r v e D, i l l u m i n a t e d , a n a e r o b i c c u l t u r e in e x p o n e n t i a l growth, s h o w i n g t h e c o n s t i t u t i v e s y n t h e s i s of c a t a l a s e in t h e a b s e n c e of air. T h e o r d i n a t e is n o r m a l i z e d to a n initial cell d e n s i t y (at t = o) of i m g (dry wt.)/ml. A c t u a l initial a n d final cell d e n s i t i e s in m g / m l were 2.I a n d 2.i (A), 0.42 a n d o.7i (B), o.33 a n d o.5I (C), a n d o.63 a n d 0.95 (D). T h e n u m b e r s a d j a c e n t to t h e c u r v e s are a p p r o x i m a t e v a l u e s for molecules of n e w catalase/cell]miu.
The greater inducibility ot cells in a carbon-limited culture is shown again in Fig. 7. Curves A and B show the data of Fig. 2B, which represent anaerobic growth and catalase synthesis in the light. Curve C shows the catalase content of cells withdrawn from this culture and aerated in the light for 30 min ("induced cells"). The inducibility rises sharply as growth is limited. An increased rate of induced enzyme synthesis in "stationary-phase" cells has been reported for many systems 9, 20. In some cases9 the slow induction in exponentially growing cells is augmented by adding amino acids. In our case no such augmentation could be demonstrated by adding casein hydrolyzate or yeast extract. On the contrary, the inducibility remains high in a growing culture if glutamate and acetate are omitted from the medium, which ordinarily contains glutamate, acetate, and ammonium malate. An interpretation is that a specific organic substance, or group Biochim. Biophys. Acta, 37 (196o) 5o3-5I";'
5IO
R.K. CLAYTON
of substances, represses induction in a growing culture and that this substance is destroyed as growth becomes limited through exhaustion of certain carbon sources. 5.5E
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2s 3o ~2 34 36 TIME(hr) Fig. 7. Rate of induced synthesis of catalase in Rps. spheroides versus growth phase. Curves A and B represent data on cells grown anaerobically in the light at 3°o and assayed immediately. Curve C shows the catalase content of cells withdrawn from this culture and aerated in the light for 3° rain ("induced cells"). Such a specific inhibitor has been found for the synthesis of ornithine transcarbamylase in E. coli. Arginine, a product (one step removed) of the reaction of this enzyme, represses its synthesis n. Through this feedback mechanism the syntheses of citruUine and arginine can be regulated. In the present case, repressors could be the products of peroxidatic reactions involving catalase. Their identification might yield evidence concerning the natural substrates of catalase, acting peroxidatically. The kinetics of induction might be modified further in this system by the presence or absence of conditions of gratuity. Gratuity can be defined, for our purposes, as follows. The action of oxygen as an inducer of catalase synthesis in Rps. spheroides is not modified b y other effects of aeration such as a shift from photosynthetic to oxidative metabolism, nor does the newly formed catalase influence the subsequent induction. One can hope that this definition of gratuity was satisfied under the combination of illumination and gentle aeration employed in the experiments of Fig. 6. When a suspension aerated in this way was placed in a glass-stoppered bottle with methylene blue and illuminated, the dye was not bleached for 2 to 4 h. Respiratory utilization of oxygen was therefore slight; the energy for anabolic activities was provided mainly through photosynthesis. I t is possible, however, that this degree of aeration was inhibitory to photosynthesis. I t is also possible that catalase influences its own induced synthesis through a feedback mechanism: a rise in catalase content could alter the steady-state concentrations of H20 ~ and of the catalase-H.~O 2 complex under constant aeration. CHANTRENNE described evidence for such a mechanism in yeast is. Finally, the rate of photosynthesis in the pre~ence of air could depend on the
Biochim. Biophys. Aaa, 37 (196o) 5o3-512
INDUCED
SYNTHESIS
~II
OF CATALASE
catalase content. These questions were explored in the experiment illustrated in Fig. 8. A mature Lt. Anaer. culture of Rlbs. spheroides was diluted fivefold with complete medium under strict anaerobiosis. An aliquot was aerated gently in the light and growth and catalase content were assayed at 3o-min intervals (curve A). The remainder was allowed to grow anaerobically in the light; samples were withdrawn at 3o-min intervaL. These were assayed immediately (curve B) or after 3o rain gentle aeration in the light (curves C). Curve B shows the constitutive synthesis of catalase in an anaerobic culture. The initial dilution of the culture led to a marked increase in the average illumination of the cells, owing to a decrease in shading. This change did not stimulate the synthesis of catalase, therefore light does not play the role of an inducer in this system. The increase in illumination was sufficient to cause a temporary cessation of bacteriochlorophyll synthesis4; this provides evidence against an hypothesis ~a that holds that the syntheses of catalase and chlorophyll are competitive, so that the former is accelerated when the latter is retarded. Curves C show the induced synthesis of catalase after various periods of anaerobic growth in the light, starting with a freshly diluted "stationary" culture. For each of these curves the initial catalase content was less than o.ooz5 %. The corresponding increments of curve A show the induced synthesis after the same interval of photosynthetic growth with aeration. Here the catalase content at the start of an increment was as high as o.oI8 %. Curve C' is a reconstruction of curves C for comparison with curve A. We see that each increment of curve A is equal to the corresponding C curve. Moreover, the rate of growth was not influenced by aeration (upper curve in Fig. 8). ~- 4 A 1 0 o
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Fig. 8. Effect of prior a e r a t i o n o n t h e i n d u c e d s y n t h e s i s of catalase in Rps. sphe~,oides.Cells g r o w n anaerobically in t h e l i g h t to m a t u r i t y , t h e n diluted fivefold w i t h c o m p l e t e m e d i u m , u n d e r s t r i c t anaerobiosis, a t t = o. A, a l i q u o t a e r a t e d in t h e l i g h t f r o m t = o. B, a l i q u o t i l l u m i n a t e d w i t h o u t a e r a t i o n f r o m t = o. C, s a m p l e s i l l u m i n a t e d w i t h o u t a e r a t i o n f r o m t ~ o, t h e n a e r a t e d in t h e l i g h t for 30 rain. C u r v e C' is c o n s t r u c t e d b y j o i n i n g c u r v e s C t o g e t h e r .
Biochim.'Biophys. Acga, 37
(i96o) 5o3-512
512
R. K. CLAYTON
Thus a prior histoly of aeration influences neither the rate of growth nor the rate of induced catalase synthesis, within the limits of this experiment. More specifically, rates of growth and induction are independent of catalase content for values of the latter ranging from o.0o25 to o.o18 %. In another experiment, which gave the same results, the catalase content ranged from 0.0o26 to 0.045 %. To summarize the results of this experiment: (a) Conditions of gratuity as defined in this section were satisfied. (b) The induced synthesis of catalase is not related to a cessation of bacteriochlorophyll synthesis. (c) A feedback mechanism involving catalase and H , O , does not operate under the conditions of this experiment. Conditions of gratuity provide a firm basis for the interpretation of data concerning induced enzyme synthesis1; it is for this reason that this point has been emphasized. It appears, however, that the induction of catalase synthesis in Rps. spheroides follows the same pattern whether gratuity prevails or not. Thus the kinetics of induction under vigorous aeration in darkness are similar to those under gentle aeration in light. Also, the rate of aerobic growth in darkness does not depend appreciably on the catalase content: when a Lt. Anaer. culture is transferred to aerobiosis in the dark, growth usually continues at the same rate without a lag. These facts emphasize our ignorance of the function of catalase in this organism. ACKNOWLEDGEMENTS
This work was performed during the tenure of a National Science Foundation Senior Postdoctoral Fellowship. REFERENCES 1 M. CORN, Bacteriol. Rev., 21 (1957) 14o. 2 j . MONOD AND M. COHN, Advances in Enzymol., 13 (1952) 67. 3 H. CI-IANTRENNEAND C. COURTOIS, Biochim. Biophys, Acta, 14 (1954) 3974 G. COI-IEN-BAZlRE, W. R. SISTROM AND R. Y. STANIER, J. Cellular Comp. Physiol., 49 (1957) 25. 5 D.HERBERT, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. II, Academic Press, Inc., New York, 1955, p. 784 • 6 R. K. CLAYTON, Biochim. Biophys. Acta, in the press. 7 D. S. HOGNESS, M. COHN AND J. MONOD, Biochim. Biophys. Acla, 16 (1955) 99 s B. ROTMAN AND S. SPIEGELMAN, J. Bacteriol., 68 (I954) 419. 9 y . MARUYAMA AND H. MITUI, J. Biochem., 45 (1958) 169. 10 M. J. PINSKY AND J. L. STOKES, J. Bacteriol., 64 (1952) 337n L. GORINI AND W. K. MAAS, Biochim. Biophys. Acta, 25 (1957) 2o8. 12 H. CnANTRENNE, Biochim. Biophys. Acta, 16 (1955) 4 lo. 13 D. APPLEMAN,Plant Physiol., 27 (1952) 613.
Biochim. Biophys. Acta, 37 (1960) 503-512
503
T H E INDUCED SYNTHESIS OF CATALASE IN
RHODOPSEUDOMONAS S P H E R O I D E S RODERICK K. CLAYTON* Department o/ Biochemistry, The Technical University o/Norway, Trondheim (Norway) and Biology Division, Oak Ridge National Laboratory**, Oak Ridge, Tenn. (U.S.A.)
(Received April ioth, 1959)
SUMMARY Synthesis of catalase is induced in Rps. spheroides on contact with air. In cultures grown anaerobically in the light the catalase content is 0.002 % of the dry wt. In aerobic cultures grown in darkness the catalase content is about o.15 %. The rate of induced synthesis of catalase is low in a culture growing exponentially and high in a culture whose growth is limited through depletion of carbon sources. The slow induced synthesis in the former case is not accelerated by adding casein hydrolyzate. Rapid induced synthesis is maintained in a growing culture if glutamate and acetate are omitted from the growth medium. Conditions of gratuity prevail for this induced synthesis of catalase if the culture is aerated gently in the light; rates of growth and induced synthesis are then independent of prior aeration and of catalase content. The rate of aerobic growth in darkness is also independent of catalase content. The kinetics of induction are similar for a culture aerated gently in the light (photosynthetic growth) and a culture aerated vigorously in darkness (aerobic growth). Synthesis of catalase is not related, directly or inversely, to synthesis of bacteriochlorophyll. The induced synthesis of catalase requires sources of carbon, nitrogen, and energy. These requirement and the effects of inhibitors suggest that the protein of catalase is synthesized de novo. Although aeration causes this induced synthesis, energy for the process can be derived either from air or light.
INTRODUCTION Our understanding of the biosynthesis of proteins has been advanced by the study of inducible enzymes 1. An inducible enzyme that possesses a prosthetic group (e.g., a hemoprotein) could be useful in studying the mechanism by which prosthetic group and protein are joined. In cases where the physiological function of an enzyme is uncertain, the function might be elucidated in an organism that can be made to contain either a high or a low concentration of the enzyme. The phenomenon of excitability, traditionally studied by sensory physiologists, is exhibited wheu an organism responds to a change in its environment by making an enzyme. Induced enzyme synthesis is * Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. ** Operated by Union Carbide Corporation for the U.S. Atomic Energy Commission. Biochim. Biophys. Acta, 37 (196o) 5o3-512
504
R. K. CLAYTON
also a special example of cellular differenti,,tion. Thus a variety of problems can be broached through the study of induced enzyme synthesis. With these prospects in mind, an induced synthesis of catalase in the facultative photoheterotroph Rhodopseudomonas st~heroides is being investigated. This organism can grow either aerobically in the dark or anaerobically by photosynthesis" oxygen is not liberated in its photosynthetic metabolism. The induced synthesis of catalase occurs on the introduction of oxygen. This paper describes some aspects of the kinetics, energetics, and chemistry of this induced synthesis. In the study of induced enzyme synthesis, and especially in the interpretation of the kinetics of induction, a considerable clarification is achieved if "conditions of gratuity", as defined by Mol~oD A~D COHI~~, are made to prevailL Such conditions are said to prevail if neither the presence of the enzyme nor its inducer influences the general cellular metabolism. In studying the induced synthesis of/~-galactosidase in Escheriehia coli, MOSOD AND COHN2 achieved gratuity by using methyl-fl-D-thiogalactoside as the inducer. This substance is not a substrate for metabolism, and under the conditions of growth, fl-galactosidase was not involved in the general metabolism of the cells. Conditions of gratuity were attained by CHANTRENNE AND COURTOIS3, in the study of induced catalase synthesis in yeast, by using a mutant that lacks respiratory enzymes (here the inducer is air). For the induced synthesis of catalase in Rps. spheroides the attainment of gratuity under illumination with gentle aeration will be demonstrated. METHODS
Rps. spheroidas, strain 2.4.1, was cultivated in a synthetic medium containing minerals, chelating agents, growth factors, malate, glutamate, and acetate, as described by COI-IEN-BAZlRE at al.4. Photosynthetic cultures were grown anaerobically under illumination at 3°o in glass-stoppered bottles; illumination was provided by three 4o-W tungsten lamps at a distance of about 12 in. Aerobic cultures were grown in darkness at 25 ° in agitated vessels. Catalase was assayed by the iodometric method of HERBERT5. Intact cells wern exposed to toluene for 4 min immediately before assay to render them freely permeable to H20 v Samples that could not be assayed at once were treated with 0.2 vg/ml of tetracycline and placed in darkness; this treatment prevented further changes in their catalase content. Nonenzymic and peroxidatic decomposition of H~O 2 in the assay were ruled out through observation of the reaction kinetics, oxygen evolution, and the effects of azide and heating. The catalase of Rp~. spharoidas has been purified and characterized6. A knowledge of its specific activity permits the expression of catalase content as percentage of the dry wt. From its molecular weight, the number of catalase molecules per bacterium can be estimated. Growth was measured turbidimetrically, at 68o m/~, by the method of COHENBAZIRE at al. 4. Calalasa content and growth cycle In a mature culture of Rps. spharoidas grown anaerobically in the light (henceforth abbreviated Lt. Anaer.), the catalase content is about o.oo2 % of the dry wt. If this culture is used as an inoculum for a culture grown aerobically in darkness, growth Biochim. Biophys. Acta, 37 (196o) 553-512
INDUCED SYNTHESIS OF CATALASE
505
and catalase synthesis proceed as shown in Fig. i where the data from 4 experiments are superimposed. In one experiment covering the range o to 5 h the inoculum was IO %; here the data for cell mass and catalase content were divided by IOO to correspond to a o.i % inoculum. In the other 3 experiments the inoeulum was o.z %. 7-
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Fig. I shows that, in a culture growing aerobically in darkness from an inoculum of I t . Anaer. culture, an initial outburst of catalase synthesis is followed b y a slackening, after which the synthesis of catalase keeps pace with growth. As growth becomes limited*, the synthesis of catalase continues for several hours. The inverse experiment is shown in Figs. 2 A and B. Fig. 2A represents a culture growing exponentially in the dark with aeration, which was transferred to anaerobic
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Fig. 2. A. G r o w t h a n d c a t a l a s e s y n t h e s i s in Rps. spheroides t r a n s ferred a t t = 1. 5 h f r o m aerobic conditions (dark, 25 ° ) to anaerobic c o n d i t i o n s (light, 30°). B. G r o w t h a n d c a t a l a s e s y n t h e s i s in Rps. spheroides g r o w i n g anaerobically in t h e l i g h t a t 3 °0 . I n o c u l u m : o.I % (v/v) of a m a t u r e c u l t u r e g r o w n aerobically in d a r k n e s s .
* I n all of t h e e x p e r i m e n t s to be described, g r o w t h in t h e c o m p l e t e m e d i u m b e c a m e l i m i t e d t h r o u g h a n e x h a u s t i o n of sources ot carbon. T h i s w a s a s c e r t a i n e d b y m e a s u r i n g final cell yield as a f u n c t i o n of t h e initial c o n c e n t r a t i o n s of m a l a t e , acetate, a n d g l u t a m a t e .
Biochim. Biophys. Acta, 37 (196o) 5o3-512
R.K. CLAYTON
5o6
conditions in the light (in replicate glass-stoppered bottles) and allowed to grow to completion. Fig 2B portrays the development of a Lt. Anaer. culture from a o.I % inoculum of a culture grown aerobically in darkness. Fig. 2A reveals that, when Rps. spheroides is transferred from aerobic conditions in darkness to anaerobic conditions in the light, growth and the synthesis of catalase cease. Growth is resumed after several hours (during this lag the photosynthetic pigments are being synthesized4), b u t the total catalase in the culture remains constant. Eventually, when the catalase is diluted to a level of about o.ooi %, the synthesis of this enzyme is resumed (Fig. 2B). As growth becomes limited, the catalase content rises to about 0.002 %.
Is new protein synthesized? F u n d a m e n t a l to the understanding of a n y example of induced enzyme synthesis is a knowledge of whether the enzyme-protein is synthesized de hove. A conclusive affirmative answer to this question has been obtained for the induced synthesis of fl-galactosidase in E. coli through studies of radioisotope incorporationT, 8. In our case this type of experiment has not been performed, but considerable circumstantial evidence supports the idea that a de hove synthesis of protein occurs. This evidence is found in the requirements for carbon, nitrogen, and energy and in the action of inhibitors. Figs. 3 and 4 illustrate experiments showing t h a t nonproteillaceous sources ot carbon and nitrogen are required for the induced synthesis of catalase in Rps. spheroides. I n the experiment of Fig. 3 Lt. Anaer, cells were washed and resuspended, in 04
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Fig. 3. Growth and induced catalase synthesis in Rps. spheroides. Cells grown anaerobically in light, washed, and resuspended in basal medium (see text) containing o.o2 M ammonium malate (curves A) or o.02 M sodium malate (curves B). Suspensions aerated in darkness from t = o.
Fig. 4. Growth and induced catalase synthesis in Rps. spheroides. Cells grown anaerobically in light, resuspended in basal medium (see text), incubated anaerobically in light for 48 h, then washed and resuspended in o.o2 M potassium phosphate (pH 6.8) plus additives. Suspensions aerated in darkness from t = o. Additives: curve A, none; curve B, (NH4)zSO4; curve C, Na malate; curve D, NH 4 malate; curve E, Na acetate + Na-H glutamate; and curve F, NH 4 malate + Na acetate + N a - H glutamate. All additives were present initially at a concentration of o.o2 M.
Biochlm. Biophys. Acta, 37 (196o) 5o3-512
INDUCED SYNTHESIS OF CATALASE
507
2 aliquots, in "basal medium" plus 0.02 M ammonium malate (aliquot A) or 0.02 M sodium malate (aliquot B). The basal medium was the same as the complete growth medium except that it lacked ammonium ion, nitrate, malate, acetate, and glutamate. The resuspended aliquots were aerated in darkness. Growth and catalase content were measured at intervals. In the presence of ammonium malate, a vigorous synthesis of catalase proceeded for more than 6 h. In the presence of sodium malate, this synthesis ceased after 4 h, probably because endogenous reserves of fixed nitrogen were exhausted. In the experiment illustrated in Fig. 4 Lt. Anaer. cells were resuspended in basal medium and incubated anaerobically in the light for 48 h, to exhaust endogenous reserves of carbon and nitrogen. The culture was then washed and resuspended in 0.02 M potassium phosphate, pH 6.8. Six portions, containing various additives (see Fig. 4), were aerated in darkness and growth and catalase content were assayed at intervals. No catalase was synthesized by cells suspended in phosphate buffer (curve A), nor by cells suspended in phosphate buffer plus ammonium sulfate or sodium malate (curves B and C). Synthesis of catalase proceeded in the presence of phosphate plus ammonium malate (curve D), or phosphate plus sodium acetate plus monosodium glutamate (curve E). Rates of induced catalase synthesis were proportional to growth rates. Thus the induced synthesis of catalase requires sources of carbon and nitrogen other than intracellular proteins. The roles of light and air in making energy available, and of air as an inducer, were demonstrated in the following experiment. A mature Lt. Anaer. culture was diluted 2.5-fold with complete medium, shaken with air for I min, and dispensed into glass-stoppered bottles, completely filled. These were placed in darkness; at intervals one was opened, and its contents were assayed for catalase. Other bottles were placed in the light after various intervals of darkness, and after 3o-min illumination their contents were assayed. One bottle was opened after 13o min in darkness. The contents were shaken with air for I min, returned to the bottle, illuminated for 3o rain, and assayed. Two bottles, one illuminated and the other in darkness from the start of the experiment, contained o.ooi % methylene blue. The dye was bleached, signalling the exhaustion of dissolved oxygen, after IiO min (illuminated) and 30 min (in darkness) (see Fig. 5). Samples kept in darkness (curve D) showed some increase in catalase during the first 2o min. This is interpreted as an induced synthesis of catalase in which the air introduced initially served both as inducer and as a means of liberating energy through respiration. The first illuminated sample (curve L, o to 30 min) showed a much larger increase in catalase content, for 2 reasons: ('0 In the light the reduction of dissolved oxygen was slower, so that the concentration of inducer remained higher and (b) energy provided by photosynthesis was greater than that provided by respiration at a low oxygen tension. In successive illuminated samples (after longer periods of darkness) the induced synthesis of catalase was progressively smaller because the inducer, air, was being exhausted. The slight induced synthesis in a completely anaerobic sample (curve L, 135 to 165 mill) might have arisen by the interaction of light with oxidizing entities generated by the initial aeration. We shall see later that, in a culture that has had no contact with air for 24 h, an increase in illumination does not act as an inducer. Curve A, L represents the sample that was aerated again after 13o min and then illuminated. Induced synthesis of catalase in this sample was nearly as great as that in the first illuminated sample; therefore inducibility was not lost on standing in darkness Biochim. Biophys. Acta, 37 (196o) 5o3-512
508
R. K. CLAYTON
without air.These results show that the induced synthesis of catalase in Rps. spl~eroides requires energy made available by light or air, but air (not light) acts as inducer. Several inhibitors were found to exert parallel effects on growth and on the induced synthesis of catalase. These results strengthen the likelihood that the latter process involves a synthesis of new protein. Thus chloramphenicol caused a 50 % inhibition both of growth and catalase synthesis at a concentration of I/~g/ml and complete inhibition of both processes at IO/~g/ml. For tetracycline the corresponding figures are o.oi and o.I/~g/ml. 0.4
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Fig. 5. I n d u c e d c a t a l a s e s y n t h e s i s in Rps. spheroides i l l u s t r a t i n g roles of l i g h t a n d air. Cells g r o w n a n a e r o b i c a l l y in light, d i l u t e d 2.5-fold w i t h c o m p l e t e m e d i u m , a e r a t e d i m i n , a n d d i s p e n s e d i n t o g l a s s - s t o p p e r e d b o t t l e s a t t = o. C u r v e D, b o t t l e s k e p t in dark. C u r v e s L, bottles in d a r k u n t i l s t a r t of curve, t h e n in light. C u r v e A, L, b o t t l e in d a r k u n t i l s t a r t of curve, t h e n c o n t e n t s a e r a t e d I m i n , r e t u r n e d to bottle, a n d i l l u m i n a t e d . Dissolved O 8 e x h a u s t e d in d a r k e n e d b o t t l e a t t i m e t n ; in i l l u m i n a t e d b o t t l e a t t i m e tL.
After irradiation with u.v. an inhibition of growth and catalase synthesis occurred that became progressively more severe. A dose that depressed the rate of growth to 50 To of normal 4 h after irradiation reduced the rate of induced catalase synthesis to 6o % of normal. In dim light (below saturation for photosynthesis) the rates of growth and catalase synthesis were depressed to the same extent. At one intensity both rates were 3o TO of maximal, at another, 7 ° %. A plot of growth rate versus pH had the same shape as a plot of rate of induced catalase synthesis versus pH. For both cases the rate was maximal at pH 7.1.
Kinetics o/induction: gratuity," ]eedback m~chanisms The kinetics of the induced synthesis of catalase in Rps. spheroidos appear to be dominated by the physiological state of the organisms. This is illustrated in Fig. 6, where the numbers on the curves represent approximate values for molecules of new catalase/ceU/min. The data of curves A, B, and C were obtained with suspensions that were illuminated and aerated very gently (about 5 bubbles/min through 50 ml of suspension in a large test tube). Curve A shows the rapid, sustained induced synthesis of cataiase, initiated and maintained by aeration in a Lt. Anaer. culture, Biochim. Biophys. Acta, 37 (196o) 5o3-512
INDUCED SYNTHESIS OF CATALASE
509
whose growth had become limited by a depletion of carbon sources (other types of limitation have not been investigated). If such a culture is diluted with complete medium at the start of aeration (curve B), the rate of induced synthesis falls dramatically as growth is resumed. A Lt. Anaer. culture in exponential growth shows, when aerated, a slow but fairly constant rate of induced synthesis (curve C). In this case the differential rate of synthesis (new catalase/new cell mass) is nearly constant. Curve D shows the constitutive synthesis of catalase in an illuminated anaerobic culture in exponential growth. 0.5-
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Fig. 6. I n d u c e d s y n t h e s i s of c a t a l a s e in Rps. spheroides in different s t a g e s of growth. C u r v e A, c u l t u r e g r o w n a n a e r o b i c a l l y in l i g h t to m a t u r i t y (growth l i m i t e d b y e x h a u s t i o n of carbon) t h e n a e r a t e d g e n t l y in l i g h t f r o m t = o. C u r v e B, s a m e as A, b u t c u l t u r e d i l u t e d fivefold w i t h c o m p l e t e m e d i u m a t s t a r t of aeration. G r o w t h b e g a n w i t h o u t lag. C u r v e C, i l l u m i n a t e d , a n a e r o b i c c u l t u r e in e x p o n e n t i a l growth, a e r a t e d in light from t = o. C u r v e D, i l l u m i n a t e d , a n a e r o b i c c u l t u r e in e x p o n e n t i a l growth, s h o w i n g t h e c o n s t i t u t i v e s y n t h e s i s of c a t a l a s e in t h e a b s e n c e of air. T h e o r d i n a t e is n o r m a l i z e d to a n initial cell d e n s i t y (at t = o) of i m g (dry wt.)/ml. A c t u a l initial a n d final cell d e n s i t i e s in m g / m l were 2.I a n d 2.i (A), 0.42 a n d o.7i (B), o.33 a n d o.5I (C), a n d o.63 a n d 0.95 (D). T h e n u m b e r s a d j a c e n t to t h e c u r v e s are a p p r o x i m a t e v a l u e s for molecules of n e w catalase/cell]miu.
The greater inducibility ot cells in a carbon-limited culture is shown again in Fig. 7. Curves A and B show the data of Fig. 2B, which represent anaerobic growth and catalase synthesis in the light. Curve C shows the catalase content of cells withdrawn from this culture and aerated in the light for 30 min ("induced cells"). The inducibility rises sharply as growth is limited. An increased rate of induced enzyme synthesis in "stationary-phase" cells has been reported for many systems 9, 20. In some cases9 the slow induction in exponentially growing cells is augmented by adding amino acids. In our case no such augmentation could be demonstrated by adding casein hydrolyzate or yeast extract. On the contrary, the inducibility remains high in a growing culture if glutamate and acetate are omitted from the medium, which ordinarily contains glutamate, acetate, and ammonium malate. An interpretation is that a specific organic substance, or group Biochim. Biophys. Acta, 37 (196o) 5o3-5I";'
5IO
R.K. CLAYTON
of substances, represses induction in a growing culture and that this substance is destroyed as growth becomes limited through exhaustion of certain carbon sources. 5.5E
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2s 3o ~2 34 36 TIME(hr) Fig. 7. Rate of induced synthesis of catalase in Rps. spheroides versus growth phase. Curves A and B represent data on cells grown anaerobically in the light at 3°o and assayed immediately. Curve C shows the catalase content of cells withdrawn from this culture and aerated in the light for 3° rain ("induced cells"). Such a specific inhibitor has been found for the synthesis of ornithine transcarbamylase in E. coli. Arginine, a product (one step removed) of the reaction of this enzyme, represses its synthesis n. Through this feedback mechanism the syntheses of citruUine and arginine can be regulated. In the present case, repressors could be the products of peroxidatic reactions involving catalase. Their identification might yield evidence concerning the natural substrates of catalase, acting peroxidatically. The kinetics of induction might be modified further in this system by the presence or absence of conditions of gratuity. Gratuity can be defined, for our purposes, as follows. The action of oxygen as an inducer of catalase synthesis in Rps. spheroides is not modified b y other effects of aeration such as a shift from photosynthetic to oxidative metabolism, nor does the newly formed catalase influence the subsequent induction. One can hope that this definition of gratuity was satisfied under the combination of illumination and gentle aeration employed in the experiments of Fig. 6. When a suspension aerated in this way was placed in a glass-stoppered bottle with methylene blue and illuminated, the dye was not bleached for 2 to 4 h. Respiratory utilization of oxygen was therefore slight; the energy for anabolic activities was provided mainly through photosynthesis. I t is possible, however, that this degree of aeration was inhibitory to photosynthesis. I t is also possible that catalase influences its own induced synthesis through a feedback mechanism: a rise in catalase content could alter the steady-state concentrations of H20 ~ and of the catalase-H.~O 2 complex under constant aeration. CHANTRENNE described evidence for such a mechanism in yeast is. Finally, the rate of photosynthesis in the pre~ence of air could depend on the
Biochim. Biophys. Aaa, 37 (196o) 5o3-512
INDUCED
SYNTHESIS
~II
OF CATALASE
catalase content. These questions were explored in the experiment illustrated in Fig. 8. A mature Lt. Anaer. culture of Rlbs. spheroides was diluted fivefold with complete medium under strict anaerobiosis. An aliquot was aerated gently in the light and growth and catalase content were assayed at 3o-min intervals (curve A). The remainder was allowed to grow anaerobically in the light; samples were withdrawn at 3o-min intervaL. These were assayed immediately (curve B) or after 3o rain gentle aeration in the light (curves C). Curve B shows the constitutive synthesis of catalase in an anaerobic culture. The initial dilution of the culture led to a marked increase in the average illumination of the cells, owing to a decrease in shading. This change did not stimulate the synthesis of catalase, therefore light does not play the role of an inducer in this system. The increase in illumination was sufficient to cause a temporary cessation of bacteriochlorophyll synthesis4; this provides evidence against an hypothesis ~a that holds that the syntheses of catalase and chlorophyll are competitive, so that the former is accelerated when the latter is retarded. Curves C show the induced synthesis of catalase after various periods of anaerobic growth in the light, starting with a freshly diluted "stationary" culture. For each of these curves the initial catalase content was less than o.ooz5 %. The corresponding increments of curve A show the induced synthesis after the same interval of photosynthetic growth with aeration. Here the catalase content at the start of an increment was as high as o.oI8 %. Curve C' is a reconstruction of curves C for comparison with curve A. We see that each increment of curve A is equal to the corresponding C curve. Moreover, the rate of growth was not influenced by aeration (upper curve in Fig. 8). ~- 4 A 1 0 o
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1~o
T I M E (rnin)
Fig. 8. Effect of prior a e r a t i o n o n t h e i n d u c e d s y n t h e s i s of catalase in Rps. sphe~,oides.Cells g r o w n anaerobically in t h e l i g h t to m a t u r i t y , t h e n diluted fivefold w i t h c o m p l e t e m e d i u m , u n d e r s t r i c t anaerobiosis, a t t = o. A, a l i q u o t a e r a t e d in t h e l i g h t f r o m t = o. B, a l i q u o t i l l u m i n a t e d w i t h o u t a e r a t i o n f r o m t = o. C, s a m p l e s i l l u m i n a t e d w i t h o u t a e r a t i o n f r o m t ~ o, t h e n a e r a t e d in t h e l i g h t for 30 rain. C u r v e C' is c o n s t r u c t e d b y j o i n i n g c u r v e s C t o g e t h e r .
Biochim.'Biophys. Acga, 37
(i96o) 5o3-512
512
R. K. CLAYTON
Thus a prior histoly of aeration influences neither the rate of growth nor the rate of induced catalase synthesis, within the limits of this experiment. More specifically, rates of growth and induction are independent of catalase content for values of the latter ranging from o.0o25 to o.o18 %. In another experiment, which gave the same results, the catalase content ranged from 0.0o26 to 0.045 %. To summarize the results of this experiment: (a) Conditions of gratuity as defined in this section were satisfied. (b) The induced synthesis of catalase is not related to a cessation of bacteriochlorophyll synthesis. (c) A feedback mechanism involving catalase and H , O , does not operate under the conditions of this experiment. Conditions of gratuity provide a firm basis for the interpretation of data concerning induced enzyme synthesis1; it is for this reason that this point has been emphasized. It appears, however, that the induction of catalase synthesis in Rps. spheroides follows the same pattern whether gratuity prevails or not. Thus the kinetics of induction under vigorous aeration in darkness are similar to those under gentle aeration in light. Also, the rate of aerobic growth in darkness does not depend appreciably on the catalase content: when a Lt. Anaer. culture is transferred to aerobiosis in the dark, growth usually continues at the same rate without a lag. These facts emphasize our ignorance of the function of catalase in this organism. ACKNOWLEDGEMENTS
This work was performed during the tenure of a National Science Foundation Senior Postdoctoral Fellowship. REFERENCES 1 M. CORN, Bacteriol. Rev., 21 (1957) 14o. 2 j . MONOD AND M. COHN, Advances in Enzymol., 13 (1952) 67. 3 H. CI-IANTRENNEAND C. COURTOIS, Biochim. Biophys, Acta, 14 (1954) 3974 G. COI-IEN-BAZlRE, W. R. SISTROM AND R. Y. STANIER, J. Cellular Comp. Physiol., 49 (1957) 25. 5 D.HERBERT, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. II, Academic Press, Inc., New York, 1955, p. 784 • 6 R. K. CLAYTON, Biochim. Biophys. Acta, in the press. 7 D. S. HOGNESS, M. COHN AND J. MONOD, Biochim. Biophys. Acla, 16 (1955) 99 s B. ROTMAN AND S. SPIEGELMAN, J. Bacteriol., 68 (I954) 419. 9 y . MARUYAMA AND H. MITUI, J. Biochem., 45 (1958) 169. 10 M. J. PINSKY AND J. L. STOKES, J. Bacteriol., 64 (1952) 337n L. GORINI AND W. K. MAAS, Biochim. Biophys. Acta, 25 (1957) 2o8. 12 H. CnANTRENNE, Biochim. Biophys. Acta, 16 (1955) 4 lo. 13 D. APPLEMAN,Plant Physiol., 27 (1952) 613.
Biochim. Biophys. Acta, 37 (1960) 503-512