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STABILITY REVTERSIBILITY ADAPTATION SELENOMETHIONINE CHLORELLA

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SHRIFT ET AL-ADAPTATION TO SELENOMETHIONINE population of Escherichia coli undergoing induced ,8-galactosidase synthesis. J. Bact. 78: 613-623. 4. COHN, M. & K. HORIBATA. 1959. Physiology of the inhibition by glucose of the induced synthesis of the 8-galactoside-enzyme system of Escherichia coli. J. Bact. 78: 624-635. 5. GALE, E. F. & R. DAVIES, eds. 1953. Adaptation in micro-organisms, 3rd Symposium of the Society for General Microbiology. University Press, Cam- bridge, England. 6. HASE, E., Y. MORIMURA, & H. TAMIYA. 1957. Some data on the growth physiology of Chlorella studied by the technique of synchronous culture. Arch. Biochem. Biophys. 69: 149-165. 7. HASE, E., Y. MORIMURA, S. MIHARA, & H. TAMIYA. 1958. The role of sulfur in the cell division of Chlorella. Arch. fur Mikrobiol. 31: 87-95. 8. KELLNER, K. 1955. Die Adaptation von Ankis- trodesmus brawmii an Rubidium und Kupfer. Biol. Zentr. 74: 662-691. 9. NovicK, A. & M. WEINER. 1957. Enzyme induction as an all-or-none phenomenon. Proc. Nat. Acad. Sci., U.S. 43: 553-566. 10. PIRSON, A. & K. KELLNER. 1952. Physiologische Wirkungen des Rubidiums. Ber. deut. botan. Ges. 65: 276-286. 11. PREER, J. R., JR. 1957. Genetics of the protozoa. Ann. Rev. Microbiol. 11: 419-438. 12. PREER, J. R., JR. 1959. Nuclear & cytoplasmic dif- ferentiation in the protozoa. In: Developmental Cytology, D. Rudnick, ed. Ronald Press Co. Pp. 3-20. 13. RYAN, F. J. & L. K. SCHNEIDER. 1949. Mutations during the growth of biochemical mutants of Esch- erichia coli. Genetics 34: 72-91. 14. SHRIFT, A. 1954. Sulfur-selenium antagonism. I. Antimetabolite action of selenate on the growth of Chlorella 7Iligaris. Am. J. Botan. 41: 223-230. 15. SHRIFT, A. 1954. Sulfur-selenium antagonism. II. Antimetabolite action of seleno-methionine on the growth of Chlorella vuilgaris. Am. J. Botan. 41: 345-352. 16. SHRIFT, A. 1959. Nitrogen & sulfur changes asso- ciated with growth uncoupled from cell division in Chlorella vulgaris. Plant Physiol. 34: 505-512. 17. SHRIFT, A., JOANN NEVYAS, & SIETSKE TURNDORF. 1961. Stability & reversibility of adaptation to selenomethionine in Chlorella vulgaris. Plant Physiol. 36: 509-519. 18. TAMIYA, H., K. SHIBATA, T. SASA, T. IWAMURA, & Y. MORIMURA. 1953. Effect of diurnally inter- mittent illumination on the growth & some cellular characteristics of Chlorella. In: Algal Culture From Laboratory to Pilot Plant, John S. Burlew, ed. Carnegie Inst. Wash. Publ. 600. Pp. 76-84. STABILITY & REVTERSIBILITY OF ADAPTATION TO SELENOMETHIONINE IN CHLORELLA VULGARIS 1, 23 A. SHRIFT 4, JOANN NEVYAS, & SIETSKE TURNDORF DIVISION OF BTOLOGY, UNIVERSITY OF PENNNSYLVANIA, PHILADELPHIA Adaptation of Chlorella vulgaris populations to selenomethionine manifests itself as a resistance to the growth uncoupling effect of the antimetabolite. Whereas initial exposure to the antimetabolite inhibits division but allows cell growth to proceed, adapted populations will divide in the presence of the analogue without the extended period of this uncoupled growth. All cells in the population have been shown capable of the change from sensitivity to resistance (27). The present paper will show that the transformation was retained despite 220 generations in the complete IReceived November 11, 1960. 2 This investigation was supported in part by a Nation- al Science Foundation Grant 5968. 3 Pre,sented at the Pacific Slope Biochemical Confer- ence, University of California, Davis, September 8, 1960. 4 Present address: Kaiser Foundation Research Insti- tute, S. 14th St. and Cutting Blvd., Richmond, Cal. absence of the amino acid analogue. Reversal to sen- sitivity, however, could be achieved by means of three specific environmental treatments, performed in line with the hypothesis that the adaptive mechanism in- volves induction of a new steady state level of the enzyme pathway leading from sulfate to methionine. MATERIALS & METHODS Methods for the culture and growth measurements of Chlorella vulgaris have been described in other papers (24, 26). The general protocol of the sub- cultures described in this paper is shown in table I. Inoculum sizes were kept at 2.0 X 105 cells/ml, and growth and division in most instances were allowed to continue until the deceleration or stationary phases of the growth curve had been reached. During this interval a 1,000-fold increase in cell number normally occurred, the equivalent of about ten generations. 509 Downloaded from https://academic.oup.com/plphys/article/36/4/509/6088232 by guest on 20 September 2021
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SHRIFT ET AL-ADAPTATION TO SELENOMETHIONINE

population of Escherichia coli undergoing induced,8-galactosidase synthesis. J. Bact. 78: 613-623.

4. COHN, M. & K. HORIBATA. 1959. Physiology ofthe inhibition by glucose of the induced synthesisof the 8-galactoside-enzyme system of Escherichiacoli. J. Bact. 78: 624-635.

5. GALE, E. F. & R. DAVIES, eds. 1953. Adaptationin micro-organisms, 3rd Symposium of the Societyfor General Microbiology. University Press, Cam-bridge, England.

6. HASE, E., Y. MORIMURA, & H. TAMIYA. 1957.Some data on the growth physiology of Chlorellastudied by the technique of synchronous culture.Arch. Biochem. Biophys. 69: 149-165.

7. HASE, E., Y. MORIMURA, S. MIHARA, & H. TAMIYA.1958. The role of sulfur in the cell division ofChlorella. Arch. fur Mikrobiol. 31: 87-95.

8. KELLNER, K. 1955. Die Adaptation von Ankis-trodesmus brawmii an Rubidium und Kupfer. Biol.Zentr. 74: 662-691.

9. NovicK, A. & M. WEINER. 1957. Enzyme inductionas an all-or-none phenomenon. Proc. Nat. Acad.Sci., U.S. 43: 553-566.

10. PIRSON, A. & K. KELLNER. 1952. PhysiologischeWirkungen des Rubidiums. Ber. deut. botan. Ges.65: 276-286.

11. PREER, J. R., JR. 1957. Genetics of the protozoa.Ann. Rev. Microbiol. 11: 419-438.

12. PREER, J. R., JR. 1959. Nuclear & cytoplasmic dif-ferentiation in the protozoa. In: DevelopmentalCytology, D. Rudnick, ed. Ronald Press Co.Pp. 3-20.

13. RYAN, F. J. & L. K. SCHNEIDER. 1949. Mutationsduring the growth of biochemical mutants of Esch-erichia coli. Genetics 34: 72-91.

14. SHRIFT, A. 1954. Sulfur-selenium antagonism. I.Antimetabolite action of selenate on the growth ofChlorella 7Iligaris. Am. J. Botan. 41: 223-230.

15. SHRIFT, A. 1954. Sulfur-selenium antagonism. II.Antimetabolite action of seleno-methionine on thegrowth of Chlorella vuilgaris. Am. J. Botan. 41:345-352.

16. SHRIFT, A. 1959. Nitrogen & sulfur changes asso-ciated with growth uncoupled from cell division inChlorella vulgaris. Plant Physiol. 34: 505-512.

17. SHRIFT, A., JOANN NEVYAS, & SIETSKE TURNDORF.1961. Stability & reversibility of adaptation toselenomethionine in Chlorella vulgaris. PlantPhysiol. 36: 509-519.

18. TAMIYA, H., K. SHIBATA, T. SASA, T. IWAMURA,& Y. MORIMURA. 1953. Effect of diurnally inter-mittent illumination on the growth & some cellularcharacteristics of Chlorella. In: Algal CultureFrom Laboratory to Pilot Plant, John S. Burlew,ed. Carnegie Inst. Wash. Publ. 600. Pp. 76-84.

STABILITY & REVTERSIBILITY OF ADAPTATION TO SELENOMETHIONINEIN CHLORELLA VULGARIS 1,23

A. SHRIFT 4, JOANN NEVYAS, & SIETSKE TURNDORFDIVISION OF BTOLOGY, UNIVERSITY OF PENNNSYLVANIA, PHILADELPHIA

Adaptation of Chlorella vulgaris populations toselenomethionine manifests itself as a resistance tothe growth uncoupling effect of the antimetabolite.Whereas initial exposure to the antimetabolite inhibitsdivision but allows cell growth to proceed, adaptedpopulations will divide in the presence of the analoguewithout the extended period of this uncoupled growth.All cells in the population have been shown capableof the change from sensitivity to resistance (27).The present paper will show that the transformationwas retained despite 220 generations in the complete

IReceived November 11, 1960.2 This investigation was supported in part by a Nation-

al Science Foundation Grant 5968.3 Pre,sented at the Pacific Slope Biochemical Confer-

ence, University of California, Davis, September 8, 1960.4 Present address: Kaiser Foundation Research Insti-

tute, S. 14th St. and Cutting Blvd., Richmond, Cal.

absence of the amino acid analogue. Reversal to sen-sitivity, however, could be achieved by means of threespecific environmental treatments, performed in linewith the hypothesis that the adaptive mechanism in-volves induction of a new steady state level of theenzyme pathway leading from sulfate to methionine.

MATERIALS & METHODS

Methods for the culture and growth measurementsof Chlorella vulgaris have been described in otherpapers (24, 26). The general protocol of the sub-cultures described in this paper is shown in table I.Inoculum sizes were kept at 2.0 X 105 cells/ml, andgrowth and division in most instances were allowed tocontinue until the deceleration or stationary phasesof the growth curve had been reached. During thisinterval a 1,000-fold increase in cell number normallyoccurred, the equivalent of about ten generations.

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In sulfur deficient media cell number increased onlyabout ten times or three generations. The criteriaused to measure degree of adaptation were duration ofthe uncoupled growth phase, rate of exponential cellmultiplication (calculated from the equation k =

lnN, - lnN1/t, - t, with t in hr), and final num-bers of giant cells/ml (cells more than 10 microns indiameter). In liquid medium, cells exposed to 3.0 X10-s Ai selenomethionine for the first time character-istically show uncoupled growth that lasts approxi-mately 150 hours; exponential cell multiplication witha calculated constant, k, of about 0.040; and a finalgiant count of about 1.5 X 105 cells/ml. Fully adapt-ed cultures, on the other hand, have an uncoupledphase of about 22 hours, an average constant of 0.061,and a final giant count of about 3.0 X 104 cells/ml(27). Because of the variation in these featuresfrom one experiment to the next, suitable controlswere run during each subculture. Points along theexponential phase of multiplication were selected andk was calculated by the method of least squares. In-tercepts on the time axis of the growth curve, withthe inoculum size as base line, were calculated fromk and used as estimates of the durations of the un-coupled growth and lag phases.

RFSULTS & DISCUSSION

STABILITY OF ADAPTATION. Two serial subcul-tures of Chlorella vz'lgaris in the presence of seleno-metlbionine will eliminate the extended phase of un-coupled growth characteristic of populations exposedto the analogue for the first time (27). Inasmuchas all cells can adapt, in this respect comparable topopulations of microorganisms that undergo adaptiveenzyme formation (9, 13, 18, 29), subcultures in theabsence of selenomethionine were undertaken in orderto determine the number of generations necessary forloss of the adaptation.

In table II are included data for a population de-rived from a culture grown once with selenomethion-ine, and once without the analogue. The cells that

were then withdrawn for re-exposure to the analogueshowed growth uncoupling which lasted only 87 hours.This was shorter than the 120 hours of the control,grown simultaneously, that had been exposed to se-lenomethionine for the first time. An increase inthe frequency of divisions also occurred as seen fromthe exponential multiplication constant of 0.046 incontrast to 0.036 for the inhibited control. No changein magnitude of the final giant count was observedl.

Rather than the expected reversal, subculture forabout ten generations in the absence of selenomethion-ine had little or no effect on adapted cells that hadbeen cultured once with selenomethionine. The re-sponse was similar to that obtained with these cells,during their second direct subculture in selenometh-ionine.

Failure to deadapt is also strikingly shown by cellsdrawn from a population that had first been subjectedto two successive subcultures with selenomethionine(table III). After ten generations without the ana-logue, the descendents, on re-exposure to seleno-methionine showed values of the same order of mag-nitude as those found in cultures directly sub-culturedwith selenomethionine three times. The cells hladagain responded as if the intervening absence ofselenomethionine had been without effect.

I(lentical results were obtained with cells that hadbeen serially grown three, four, and five times withselenomethionine. Twenty generations away fromlthe analogue had no effect on cells drawn either fromcultures serially subcultured three times (table IV)or from cultures serially subculturecl four times (tableV) with the analogue. Resistance was also main-tained by cells that had undergone five consecutivepassages with the analogue, even after 30 generationswithout it (table VD1).

A more exhaustive attempt at deadaptation wasundertaken with cells that had been successively cul-ture(l six times with selenomethionine. Table VIIslhows that no deadaptation had occurred even after22 passages, the e(quivalent of about 220 generations.The duration of the uncoupled phase during the 22

TABLE I

GENERAL PROTOCOL OF SU13CULTURINGS

AD.PTED SERIES CONTROL SERIES

Se

S Se D- L-

S Se S Se S Se S Se S Se

S

S SC -S I)- L-

S Se S S S

S: 3.1 X 10- .r sulfate as oiliy sulfur source. Se: 3.0 X 10-5%r selenolliethionine; 3.1 X 10-4.%r sulfate.-S: iNo sulfur compounds added. D-: 3.1 X 10-4 M D-methioniiie as only sulfur source. I- : 3.1 X 10-4 mL-Iletllioniiie as only sulfur source.

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511SHRIFT ET AL-STABILITY & REVERSIBILITY OF ADAPTATION

TABLE IIEFFECTS OF CULTURAL CONDITIONS ON DEGREE OF ADAPTATION IN CHLORELLA VULGARIS

CULTURED ONCE WITH SELENOMETHIONINE*

ADAPTED SERIES

1 X Se

U: 104k: 0.038G: 1.0 X 105

S Se -S D- L-

U: 13 69 ** 14 17k: 0.064 0.050 0.057 0.05 1

G; 3.0 x 105

S Se S Se S Se S Se S Se

U: 15 87 9 19 21 147 13 146 1.4 147k1: 0.068 0.046 0.062 0.050 0.068 0.047 0.068 0.052 0.062 0.056G: 3.3X 105 2.4X 105 3.3x105 3.5x105 3.0x105

CONTROL SERIES

S

U: .. .k:(l.-

T:.k: (G:

S Se -s D- L-

18 107 *;c 26 280.066 0.037 0.058 0.055

3.1x 105

S Se

T: 7 120k: 0.064 0.044G: 4.4X105

I100.055

I130.072

I140.070

o U: Duration of uncoupled growth phase or of lag phase (hr). k: Exponlential growth rate conistant. G:Giant cells/ml at completion of subculture. Other symbols are the same as in table I.

**Final population of adapted series: 1.5X106cells/ml; control series: 1.OX O1"cells/ml.

passages ranged from 26 to 45 hours with a mean and95 % confidence interval of 33 + 6 hours. Thesevalues are in contrast to values for cultures which onfirst exposure to selenomethionine exhibited uncoupledphases lasting from 130 to 165 hours. The figure33 ± 6 is significantly larger than the mean valueof 13 ± 7 obtained for the lags of the untreated con-trol cultures that had been grown simultaneously. Acomparable relationship was observed between cul-tures consecutively grown with selenomethionine andtheir untreated controls (27). No significant differ-ence in exponential rates of multiplication was foundbetween the adapted series and the untreated controls.

Final numbers of giants remained unchanged.It is evident that C. vulgaris populations, once

they are transformed by culture in the presence ofselenomethionine, are able to maintain their trans-formation indefinitely after removal of the causativeagent. Even populations that have only partiallyadapted do not revert when their cells are so cultured.On the contrary, these partially adapted cells continueon their way to complete adaptation on re-exposureto selenomethionine as if the interim had been withouteffect. Under the conditions specified, therefore, theadaptation can be considered a stable one.

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REVERSIBILITY OF ADAPTATION. The fact that thetransformiation is retained (lespite 220 generationswithout the analogue, probably rules out any mech-anism whose explanation invokes an induction of anew enzyme system capable of either destroying orutilizing the selenomethionine. Such an adaptive en-zvmne wouild have been expectedl to (lisappear afterwithd(rawal of the analogue, as has been establishedfor other induced systems (9, 13, 18, 29). A testablehypothesis to explain the stable transformation canbe drawn up based on the known biochemiiical proper-ties of selenomethionine.

Protein hydrolysates of growth uncoupled cellslack methionine, as revealed by paper chromatography,wlhereas other sulfur-containing amino acids, in thechromiiatographic region of cyst(e)ine, are present(26). In view of the ability of selenomethionine toreplace methionine in several biological systems (2,5. 14, 17, 30) it is likely that alteredl proteins whichcontain the analogue are being synthesized by thegrowth uncoupled C. vldgaris cells. The reductionof sulfate to cvsteine and the incorporation of thissulfur amiiino acid into protein, however, were notaffecte(l. The question arises as to whether or notthe altered enzymes retain their activitv. Such a re-tenltion canl be inferredl from the observations thatlarge increases in dry -weight, oxygen uptake, andprotein nitrogen occur during uncouple(d growth (26).Furthermore, a methionine-requiring mutant ofEschcrichia coli has been grown with selenomethion-ime in the complete absence of methionine (2, 5) anda cellular fraction, prepare(d froml this organism, hasbeen slhovnI to activate selenomiiethionine as well if

not better than methionine itself (17). There is alsoabundant evidence from other microorganisms thatamino aci(l analogues can be incorporated into pro-teins, an(l that some altered enzymes nevertheless re-tain activity (3, 6, 8, 15, 16, 20, 34, 35).

When the growth uncoupled population resumesexponential divisions there is a return to the wveight,size, and protein level of normal cells. Most signifi-cant is the fact that methionine is once more presentin the proteins (26). One can therefore concludethat the adaptive process involves the developmentof an ability to incorporate methionine, derivedI fromsulfate, into cellular proteins in the presence of seleno-methionine.

The following assumptions have been ma(le toexplain this renewed ability:

A. The competitive biochemical block by seleno-methionine, wlhich prevents methionine entry intoproteins, also causes accumulation of methionine andof intermediates on the route from sulfate to meth-ionine. Antinmetabolites have been demonstrated tocause such an accumulation of metabolites in otherorganisms ( 15, 28).

B. One might expect that the higher levels ofmetabolites would repress the biosynthesis of certainof the enzymes responsible for the synthesis of thesemetabolites, as is the case in some microorganisms(12, 19, 31). In particular, methionine has beenshown to repress the formation of enzymes whiclh leadto the synthesis of this amino acid in E. coli and inProtecus iiiorgan ii (1, 4, 32, 33). If such a repressionis operative in Cliloreila -vuilgaris, then selenometlhioni-

TABLE IIIFAILURE OF CHLORELLA VULGARIS, SUBCLILTURED TWICE WITH SELENONIETIIIONINE,

TO DEADAPT AFTER SUBCULTURE WITHOUT THE ANALOUCE*

ADAPTED SERIES

2 X Se

U: 69k: 0.050G: 3.0x 105

S Se

U: 9 19k: 0.062 0.050G: _ 2.4X

U: ...

CONTROL SERIES

S

U: 18

Se

107k: 0.060 0.037G: 3.1X105

S Se

U: 7 120k: 0.064 0.044G: 4.4X105

S Se

105

S Se

190.0502.0X 105

U: 7k: 0.058

140****

. . .

1-4X 105

* U: Duration of uncoupled growth phase or of lag phase (hr). k: Exponential growthGiant cells/ml at completion of subculture. Other symbols are the same as in table I.

** Too fews points to calculate k; duration of uncoupled phase estimated.

rate constant. G:

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513SHRIFT ET AL-STABILITY & REVERSlBILITY OF ADAPTATION

TABLE IVEFFECTS OF CULTURAL CONDITIONS ON DEGREE OF ADAPTATION IN CHLORELLA VULGARIS

AFTER THREE SERIAL SUBCULTURES WITH SELENOMETHIONINE *

ADAPTED SERIES

3 X SeU: 19k: 0.050G: 2.4x105

S Se -S D-L-

U: 20 24 24 5k: 0.065 0.057 0.070 0.046G: 1.7 x 105

S Se S Se S Se S Se S Se

U: 17 17 8 25 7 99 4 112 0 47k: 0.061 0.051 0.055 0.061 0.054 0.058 0.055 0.060 0.052 0.057G: 55x104 3X104 8x104 1.1x105 6X104

S

U: 30<: 0.078G: I

CONTROL SERIES

S

U: 7k: 0.064G:

U:k-.G:

Se

240.0531.1X 105

Se

1200.0444.4X 105

S Se -S D- L-

7 140** *** 20 160.058 ... ** 0.058 0.050

1.4X105

S Se

U: 12 161k: 0.057 0.049G: 1.9x

100.057

1160'055

105

S90.058

S Se

U: 19 125**k: 0.072 .. . **G: 2.0X 105

*U: Duration of uncoupled growth phase or of lag phase (hr). k: Exponential growth phase constant. G:Giant cells/ml at completion of suibculture. Other symbols are the same as in table I.

** Too few points to calculate k; duration of uncoupled growth phase estimated.*** Final population of adapted series: 3.2X 106 cells/ml; control series: 2.8X 106 cells/ml.

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TABLE \TFAILURE OF CHLORELLA VUL.GARIS, SUBCULTURED 4 TENIES VITII SELFCNOMtETIIIONINE,

TO DEAA.\PT AFTEIR SUBCULTURE \\ITl[OUT TIIE ANAL.OGUE

ADAPTEI) SERIES

4 X Se

U: 24

U:k:G:

U:k :

CONT. OLIILS

0.0571.7 x105

S Se

8 250.055 0.061

3x104

S Se

29 320.077 0.067

1.2X 10)

S Se

U: 21k: 0.078G:

250.0694X 10

S

U: 7k: 0.058G:

Se

1.40X 1,..

1.4x1 W')

S Se

U: 12k: 0.057G:

1610.0491.9X 105

S Se

U: 19k: 0.072G:

S

U: 19k: 0.065G:

125**

2.0x 10

Sc

1340.0312.1X 1(5

*U: Duration of uncoul)led growth plhase or of lag plhase (lhr). k: Exi)o11clitialGianit cells/ mil at copll)letioll of sul)culturc. Otlher symlbols a-ire tlle samiie as in table 1.

*' Too few poinits to calculate k; duration of uncoupled growxthl phase estimated.

ine, or the accumulated, endogenously produced meth-ionine, or both could be expected to enhance the re-

pression during the period of uncoupled growth.This might account for the failure to detect methioninein the proteins of the uncoupled cell, but it would notadequately explain either the reappearance of methi-onine in the proteins when exponential divisions re-

sume or the maintenance of the adaptation after re-

moval of the analogue.There is evidence that certain constitutive enzymes

can be induced to higher levels if their substrates are

exogenously supplied to the cells (11, 22). In anotherinstance, a temporary increase in constitutive en-

zymes, induced by a product of an enzyme sequence,

has been noted (23). On this basis, one can reason

that the higher levels of sulfur intermediates withinC. vutlgaris will induce an increased synthesis of one

or more of the constitutive enzymes responsible forthe further metabolism of these intermediates.

C. The enzyme pathway, now operating at a new

steady state level, will lead to a more rapid rate ofmethionine synthesis, the consequent displacement ofthe selenomethionine, a reappearance of methioninein the cellular proteins, and a renewed ability of theuncoupled cell to divide at exponential rates.

grow tIl ratc cOllstalit. G:

D. As long as the adapted cells are given sulfate,even in the absence of selenomethionine, the high rateof methionine synthesis should be maintained indefi-nitely, for according to this picture the actual inducerof the new enzyme level is not selenomethionine butone or more of the endogenous sulfur intermediates.

Three types of experiments have been performed,based on these assumptions, in an effort to cause re-version of the adapted cells to their original state ofsensitivity toward selenomethionine. The rationalefor these experiments has been as follows:

A. SULFUR STARVATION: this treatment shouldlead to a depletion of endogenous sulfur intermediatesand a consequent lowering of enzyme levels duringthe limited population increase, amounting to two orthree generations, that occurs under such restingstate conditions. The starved cells, on re-exposureto selenomethionine, should once more be sensitive tothe analogue.

B. CULTURE WITH D-METHIONINE: since C.vulgaris is able to use D-methionine as its only sourceof sulfur (24, 25), the route from sulfate to methion-ine can be circumvented. Growth of adapted cellswith this isomer should therefore also lead to de-

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SHRIFT ET AL-STABILITY & REVERSIBILITY OF ADAPTATION

TABLE VIEFFECTS OF CULTURAL CONDITIONS ON DEGREE OF ADAPTATION IN CHLORELLA VULGARIS

AFTER 5 SERIAL SUBCULTURES WITH SELENONIETHIONINE*

ADAPTED SERIES

5 X Se

U: 25k: 0.061G: 3.0X104

S Se -S D- L-

U: 21 27 28 19k : 0.076 0.066 0.075 0.058G: 1.0X 105

S Se S Se S Se S Se S SeU: 22 28 23 21 23 81 20 104 21 57k: 0.081 0.075 0.084 0.066 0.069 0.054 0.079 0.067 0.083 0.066G: 5X104 5X104 5X104 6x104 6x104

S Se

U: 14 17k: 0.068 0.063G : 3X104

S Se

U: 29 32k: 0.073 0.064G : 44x104

CONTROL SERIES

S Se

U: 12 161k: 0.057 0.049G: 1.9 X 105

S Se -S D-

U: 19 125** 26 20k: 0.072 ** 0.069 0.055G: 2.0X 105

S Se S S SU: 19 134 30 23 20k: 0.065 0.031 0.076 0.074 0.079G: 2.1X105

S Se

U: 7 130**k: 0.061G: 2.3x105

S Se

U: 14 165**k: 0.064 **G: 1.3 x 105

*U: Duration of uncoupled growth phase or of lag phase (hr). k: Exponential growth rate Constant. G:Gian1t cells/nml at Completion of subculture. Other symbols are the sanme as inl table I.

** Too few points to calculate k; duration of uncoupled growth phase estimated.*** Final population of adapted series: 1.5 X 1O' cells/ml; control series: 2.3 X 106 cells/ml.

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pression of intermediates and their respective enzymes.

C. CULTURE WITH L-MATETHIoNINE: L-methion-ine also serves as an only sulfur source (24, 25); thesame argument could be expected to hold here.

Table II, discussed previously in connection withthe failure of cells to deadapt after ten generationsaway from the analogue, summarizes the effects of thereversal conditions on cells that previously had beencultured once in the presence of selenomethionine.Unlike the subculture in complete medium, with sul-fate as sulfur source, each of the three treatmentsresulted in deadapted cells. After sulfur starvation,re-exposure to selenomethionine gave an uncoupledphase that lastedl 147 hours and a growth rate constantof 0.047. The selenomethionine control, grown si-multaneously, gave comparable values of 120 hoursand 0.044. Treatment with D-methionine gave riseto cells whose uncoupled phase lasted 146 hours, butwhich divided at a somewhat faster rate, the growthrate constant being 0.052. After L-methionine treat-ment, the uncoupled phase also lasted 147 hours, butthe growth rate constant was 0.056. In all three casesthe final number of giants was of the same order ofmagnitude as in the selenomethionine control. Itshould be noted that under conditions of sulfur starva-tion in this experiment, the population multipliedfrom 0.22 X 106 cells/ml to only 1.5 X 106 cells/ml,an increase of approximately three generations. Thesubsequent growth pattern in the presence of seleno-methionine, therefore, cannot be attributed to selectionof sensitive mutants during the starvation period.In the two methionines, about nine generations de-veloped between the time of inoculation and with-drawal of inoculum cells for re-exposure to seleno-methionine.

That the three treatments in themselves had noadverse effects can be seen from the growrth char-acteristics of the so-treatedl controls anid adapted cellsthat were transferred to complete niediumii. Evensulfur-starved cells grew normally. Enlarged cells,typical of another species, CGllorclla cllipsoidca, (lur-ing sulfur starvation (10) were not observed.

A secon(l deadlaptationi trial in whiclh D-methionlneculture and sulfur starvation were app)lied to adlapte(dcells that had been grown once with the analogue gaveidentical results.

Cells grown three tinies with the analogute werealso subjected to the three conditions (table IV). Incontrast to the selenomethionine control which had anuncoupled growth phase of 161 hours and a divisionrate constant of 0.049, sulfur starvation gave rise tocells whose uncoupled growtth lastedl 99 hours andwhose exponential divisions had a constant of 0.058;D-methionine treatment caused an uncoupled periodof 112 hours and a constant of 0.060; and L-methioninesubculture gave cells with an uncoupled phase of 47hours and a constant of 0.057. None of the treat-ments caused an increase in final giant/ml count tothe level of the selenomethionine control, except per-haps the D-methionine subculture. Deadaptation interms of the criteria used therefore appears to havebeen less complete with these adapted cells than withadapted cells grown once with the analogue.

Table VI shows that incomplete deadaptation couldalso be brought about in cells that had been seriallysubcultured five times witlh selenomethionine.

Similar data were obtaine(d with cells that hadfirst heen subcultured six times with selenomlethioninefollowed by 13 passages without it (table VIIIT fig1). Whereas the 13 passages had had no effect onthe clegiee of adaptation, each of the tllree treatments

TABLE VIIFAILIURE OF CHLOREILA VULGARIS, SUBCULTUREf) 6 TIMIES WRI[H SETENOMIETIION\TNF,

TO DEADAPT AFTER 22 SUPCULTURF WITHIlOUTj TIlI AN-ALC(UE

ADAPTEID SERIES

UNCOUPLEDPHASEAFTER

TRANSFER SLOPE OFTO EXPONEN-

SELENOME- TIALTHIONINE PHASE

(hr)29452627393632

0.0690.0710.0770.0750.0530.0550.069

CONTROL SERIES

+ SELENO-METHIONTINE

GIANTSAT END OFSUBCULTURE

105 cells/ml

0.30.10.30.30.10.20.1

DURATIONOF

UNCOUPLEDPHASE

(hr)130*165*160*162150145*

SLOPE OFEXPONEN-

TIALPHAXSE

***..4.

0.0440.043

GIANTSAT END OFSUBCULTURE10) CellS./ml

2.31.32.0

1.82.41.3

DURATIONOF LAGPHASE

(hr)7

1456

222314

33 0.067+J-6** +0.009**

* Too few points to calculate k; duration of uncoupled growth phase estimated.** Mean & 95 (% confidence interval

13 0.066±7** +0.001**

No.PASSAGESWNIITHOUTSELENOME-THIONINE

121314151722

UNTREATED

SL.OPE OFEXPONEN-

TIALPTIASE

0.0610.0640.0630.0680.0730.0720.059

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SHRIFT ET AL-STABILITY & REVERSIBILITY OF ADAPTATION

TABLE VIIIEFFECTS OF CULTURAL CONDITIONS ON DEGREE OF ADAPTATION IN CHLORELLA VULGARIS AFTER 6 SERIAL

SUBCULTURES WITH, & 13 SUBCULTURES WITHOUT, SELENONIETHIONINE*

ADAPTED SERIES

6 x Se -> 13 X S

S Se -S D- L-

U: 18 26 *** 6 12k: 0.078 0.077 0.054 0.054G: 3X104

S Se S Se -S S Se D- S

U: 16 27 16 108 + ... 89 10 10k: 0.074 0.075 0.079 0.065 ... 0.064 0.056 0.071G: 3X104 5X104 3X104

S Se

U: 24 39k: 0.068 0.053G: XlX104

S Se

18 1310.058 0.037

3x104

S Se

23 1180.066 0.043

lX104

S

270.073

Se L-

59 20.073 0.0454X104

Se

910.0643x 104

CONTROL SERIES

S

S Se -SL-

U: 5 160** *** 12 15k: 0.063 ...

** 0.056 0.056

G:-

2X105 _

S Se

TU1: 6 162k: 0.068 0.044{ : 1.8X105

I\S Se

U: 22 150k: 0.073 0.043G: 2.4X105

S -S

160.065

S D-

14 130.079 0.061

S L-

6 ...

0.064 ...

* U: Duiration of uncoupled growth phase or of lag phase (hr). k: Exponential growth rate constant. G:Giant cells/ml at completion of subculture. Other symbols are the same as in table L

** Too few points to calculate k; duration of uncoupled growth phase estimated.*** Final populations of adapted series: 3.2 x 106 cells/ml; control series: 4.7 x 106 cells/ml.f Final population of adapted series: 3.0 x 106 cells/ml; control series: 2.7x 106 cells/ml.

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PLANT PHYSIOLOGY

lengthened the uncoupled growth period. Depressionof the growth rates was not as marked, however, norwere there significant increases in the final numbersof giants/ml. An additional subculture of each typecaused further increases in the uncoupled phases, andexcept for the L-methionine treatment, further de-clines in the division rates. Final numbers of giantsremained unaffected. After 220 generations withoutthe analogue, descendents of these adapted cells alsodeadapted with these treatments and to the same extentobserved after 13 subcultures without the analogue.The selenomethionine control, in this experiment, hadan uncoupled phase that lasted, unaccountably, for202 hours.

All the physiological reversal experiments haveproven successful. Populations exposed once toselenomethionine deadapted completely. Least dead-aptation occurred with cells that had been serially sub-cultured several times with selenomethionine. Herethe inability to completely revert was most apparentfrom the failure of giant cells to develop with thefrequency found in cultures exposed to selenomethion-ine for the first time. Whereas first exposure to theanalogue inhibits the division mechanism and allowscell growth to continue before exponential divisionsresume, deadaptation of fully adapted cells yieldedpopuilations whose division was inhibited, and growthas well, to judge from the low frequency of giants.One possible explanation is that the duration of thesulfur starvation treatment and the number of genera-

50 100 150 200 250 300TIME - HOURS

FIG. 1. Effects of cultural conditions on degree ofadaptation in Chlorella vutlgaris after 6 serial subcultureswith, and 13 subcultures without selenomethionine. Seetable VIII for complete data.

tions with D- or L-methionine were insufficient. Theleast effective of the deadaptation procedures wasconsistently the subculture with L-methionine. Thereasons for this undoubtedly lie in the unknown metab-olism of this isomer. The biochemical pathwayswhereby the sulfur of both isomers of methionine istransformed into all other sulfur metabolites requiredfor normal growth of the alga is completely unknown.

Despite the obscurity of the intermediate steps inthe reduction of sulfate and utilization of methioninesulfur in C. vulgaris, the successful deadaptation at-tempts in all trials lend validity to the assumptionsmade earlier in explanation of the adaptive mecha-nism. It thus appears possible to induce a populationof cells to develop a new steady state level of consti-tutive enzymes, and to have this transformation passedon to the progeny indefinitely after complete removalof the causative agent. Theoretical treatments of suchtransformations have been discussed by Delbruck (7)and by Pollock (21).

SUMMARYResistance to the growth uncoupling effect of

selenomethionine, an adaptation which develops inall cells of Chlorella zulgaris, was maintained despiteremoval of the analogue. Cells that had undergonefrom one to six consecutive subcultures with the anti-metabolite were subcultured without it and on re-exposure were as resistant as before. As many as 22passages away from selenomethionine, the equivalentof 220 generations, failed to cause deadaptation ofcells dlerived from a population that had previouslybeen cultured six times with the analogue. Completeor partial reversal could be achievedl at any time,however, if a(lapted cells were subjected to sulfurstarvation, subculture with D-methionine as only sul-fur source, or subculture with L-methionine as onlysulfur source. The reversal data fit the hypothesisthat the permanent adaptation to selenomethionine inthis alga involves induction of a higher steady statelevel of the enzymes responsible for re(luction of sul-fate to methionine.

ACKNOWLEDGMENTS

The authors wish to thank Professor John R.Preer and Mrs. Lolita Moore for many fruitful dis-cussions.

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SHRIFT ET AL-STABILITY & REVERSIBILITY OF ADAPTATION

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