Cholesterol Inhibition of Isopentenyl Pyrophosphate A3 A2-Isomerase in Mycoplasma laidlawii

Copyright0 1970 American Society for Microbiology Printed inU.S.A.

Cholesterol

Inhibition of Isopentenyl Pyrophosphate

A3 A2-Isomerase

in

Mycoplasma

laidlawii

PAUL F. SMITH A MARIE R. SMITH

Department ofMicrobiology, SchoolofMedicine, UniversityofSouthDakota,

Vermillion, South Dakota 57069

Received for publication10December 1970

Cholesterol inhibits isopentenyl A',A2-isomerase of Mycoplasma laidlawii in an

apparently competitive fashion. The conversion of mevalonic acid toisopentenyl pyrophosphateis slightlystimulated.Organismsgrownin thepresenceof

mevalonic-2-14C acid contain small amounts ofradio-label in nucleic acid and protein frac-tions. Most of the label is found in the lipids and is reduced dramatically in

or-ganisms grown with cholesterol. No significant accumulation of phosphorylated

intermediatesofpolyterpene biosynthesiswasobserved in cellsorculture superna-tantfluid. All of the radioactivity appearing in the nucleic acid fraction occurs in

theminornucleoside, isopentenyl adenosine, ofthe transfer ribonucleic acid. The necessity for synthesis by the organisms of this minor nucleoside from mevalonic acid mayexplain the site ofenzyme inhibition by cholesterol of polyterpene

bio-synthesis.

Mycoplasma laidlawii differsfromtheso-called parasitic mycoplasmasby its lack of a nutritional requirementfor a planar 3-hydroxysteroland by its capacity to synthesize hydroxylated poly-terpenes (22). The sterol requirement of two

species,

M. arthritidis strain 07 and M. gal-linarium strain J, is considered to be the result of

insufficiency

for enzymatic synthesis of

polyterpenes

(11, 12, 24). In the former species all trans

3,3'-dihydroxycarotenes

will replace thesterol requirement. It lacks all of the enzymes in the pathway to polyterpenes but apparently can

hydroxylate

suchcompounds. M.gallinarium lacks onlythree enzymes inthepathway, i.e., those

involved

in the conversion ofmevalonic acid to A'-isopentenyl pyrophosphate. Growth of this organism with

A'-isopentenyl

pyrophosphate substituting for sterol results in the formationof

hydrocarbon

and hydroxylated polyterpenes (23). Inhibitors of polyterpene biosynthesis inhibit growth of M.

laidlawii

but not of the sterol-requiring species (26).If theinhibitionoccurs at a site in the pathway before condensation of isoprenyl phosphates forms the long-chain

polyterpene,

the organism can overcome this inhibition ifcholesterolissupplied in the growth medium. It is found that cholesterol substitutes for the

carotenoids

in the unsaponifiable

lipids

ofthe organism. Cholesterol has been shown to spare the

biosynthesis

of carotenoids in M.

laidlawii (19).

These results have

led

to the

conclusion that carotenoids and sterols possess analogous functions inmycoplasmas.

This study reports attempts to define the enzymatic site of cholesterol inhibition of the

biosynthesis

of

polyterpenes

in M. laidlawii.

MATERIALS AND METHODS

M. laidlawii strain B was grown in lipid-free mediumaspreviously described (25). Theorganisms

were harvested in the mid-exponential phase of

growth,usually after 16 hr of incubation. Experiments designedtoassessthe fateof mevalonic acid when the organism was grown in the presence of cholesterol

employed culture media of thefollowing composition

per 100 ml: control medium-chloroform:methanol

(2:1, v/v)-extracted tryptose, 2 g; chloroform:

methanol-extractedPPLOserumfraction (Difco),100

mg; potassium oleate, 100 jumoles; mevalonic-2-'4C acid, as the N,N-dibenzylethylenediammonium-bis(3,5-dihydroxy-3-methyl pentoate), 1.0 jAc and

10,moles; and ethanol, 1 ml. Test medium was

identicaltothe control except for addition of2 mg

ofcholesterol in ethanol solution.

Cholesterolwaspurified through dibromide

forma-tion followed byregeneration with zinc powder and

crystallization from absolute ethanol or acetic acid

(6).DL-Mevalonic-1-'4C and -2-'4C acid lactoneswere

obtainedfromNew EnglandNuclearCorp., Boston,

Mass. Afterthe benzenewasevaporated, the residual

lactones were hydrolyzed with potassium hydroxide,

and then thepH level was adjusted to 8.0 by the

addition ofacid (2). DL-Mevalonic acidasthe

N,N-dibenzylethylenediammonium-bis(3,5-dihydroxy-3

-methylpentoate) and

A3-isopentenyl

pyrophosphate

27

on March 20, 2021 by guest

http://jb.asm.org/

as thetris(cyclohexylammonium) salt wereobtained

from Mann ResearchLaboratories,Orangeburg, N.Y.

A3-Isopentenyl-1-14C pyrophosphate was synthesized

from mevalonic-2-14C acid with enzymes isolated

from yeast (3) or withpig liver enzymes (2, 18). The

A3-isopentenyl pyrophosphate produced was purified bythedescending paperchromatographic method of

Nandiand Porter(18).

Enzymatic assays used M. laidlawii B sonically

treated in 0.2 M potassiumphosphate buffer (pH 7.5)

for 5 min in a 10-kc Raytheon sonic oscillator. Protein

was determined by the method of Lowry et al. (17).

The conversion of mevalonic acid to A3-isopentenyl

pyrophosphate, which involves the three enzymes,

adenosine triphosphate

(ATP)-mevalonate-5-phos-photransferase (E.C. 2.7.1.36),

ATP-5-phospho-mevalonate phosphotransferase (E.C. 2.7.4.2), and

ATP-5-pyrophosphomevalonate carboxylase (E.C.

4.1.1.33), wasassayed as onecomplete reaction by

trapping the 14CO2 liberated from mevalonic-1-'4C

acid as previously described (11). Isopentenylpyro-phosphate Al,A2-isomerase (E.C. 5.3.3.2) was

assayed by the method ofAgranoffet al. (1).

Cho-lesterol was added to the reactionmixturesinsolutions

of ethanolaftertheaddition ofsonicallytreated cells

but before addition of substrate. This procedure

resulted in micellar stabilization ofthe cholesterol.

Thedistributionof14Cfrom '4C-mevalonicacid in

crude chemical fractions was determined with

organisms grown both in the presence and absence

of cholesterol. Washed cell pellets from 100-ml

cultures wereextracted with 10% trichloroacetic acid

at0 Ctogive low-molecular-weight components and

at 100 Ctogivethe nucleicacid fraction, andfinally

with chloroform-methanol (2: 1, v/v) to give the

crude lipid fraction. The resultant residue contained

crudeprotein. Detection ofpossible accumulation of

precursors of phosphorylated polyterpene

inter-mediates in the cells and culture fluid both of control and cholesterol-grown organisms was accomplished by chromatography through diethylaminoethyl (DEAE) celluloseby usingammonium carbonate of

increasingionic strength (5). Fractions (20ml) were

collected,lyophilized,andassayedforradioactivity.

Isolation of transfer ribonucleic acid (RNA) from

organismsgrown with orwithout cholesterol and in

the presenceofmevalonic-2-'4C acid was performed

bythemethod describedby Zubay (27) through the

isopropyl alcohol precipitation steps. After dialysis

against deionized water, the transfer RNA was

enzymatically hydrolyzed bythe methodof Hall (7).

Afterlyophilization of the reaction supernatant fluid

and solution in water, a samplewas held for

radioi-sotope counting. The remainder was

co-chromato-graphed in two solvent systems (8),

n-butanol-water-ammonia (86:14:5) and isopropanol-concentrated

HCI-water (680:170:144), by the descending method

with authentic isopentenyl adenosine

[N6-(3-methyl-2-butenyl)-adenosine; supplied by Harry B. Wood,

Jr., National Cancer Institute]. After detection of

nucleosidesonthe paper by ultraviolet-lightscanning, allabsorbingareaswerecutoutand counted.

Radioi-sotope countingwasperformed either in a Tracerlab

proportionalcounterorin aPackardliquid

scintilla-tion counter aspreviously described (11).

RESULTS

Enzymatic synthesis of A3-isopentenyl

pyro-phosphate from mevalonic acid actually was

stimulated since the amount of 14CO2 liberated frommevalonic-1-'4Cacidwasmorethandoubled

bythepresenceof cholesterol (Fig. 1). Increased

cholesterol concentration up to a level of 0.05 mg/ml (12.5 mg of protein/ml) resulted in increasing liberation of "CO2. This result was

consistent in repetitive experiments with different lots of M. laidlawii.

The isopentenyl pyrophosphate Al,

A2-iSOm-erase of M. laidlawii shows the opposite effect

in the presence of cholesterol. Increasing con-centrations of cholesterol inhibit the formation of ofA2-isopentenyl pyrophosphate with complete inhibition occurring at a level of 0.03 mg/ml (8.4 mg ofprotein/ml; Fig. 2). When the cho-lesterol concentration was held at 0.015 mg/ml (2.4 mg ofprotein/ml) and the concentration of substrate, A3-isopentenyl pyrophosphate, was

varied, the inhibition by cholesterol was ap-parently competitive. Plotting the reciprocals of

the rates and of substrate concentrations, the

curvesforcontrol and cholesterol-inhibited

reac-tions were linear and intersected at a common point (Fig. 3). Cholesterol reduced the Km from 5.8 X 10-5to 1.5 X 10-4M. These resultsindicate that theinhibition ofpolyterpene biosynthesisby cholesteroloccurs ataspecific enzymatic site,the isopentenyl pyrophosphate A3,A2-isomerase.

A search for thebasisofinhibition atthisstep in the pathway wassoughtsince theexpectation would have beeninhibition ofthe pathwayat the level of mevalonic acid. Organisms were grown inthepresence ofmevalonic-2-'4C acid both with and without cholesterol to determine thefate of mevalonic acid as well asthe possible accumula-tion of A3-isopentenyl pyrophosphate. A small proportion of the total "4C appears in the small molecule fraction and in the crude nucleic acid and protein fractions. The predominant amount is

incorporated

into the

lipids

(Table 1). There is little change in the amount

incorporated

in all fractions except total lipids inorganisms grown with cholesterol. Mevalonicacid presumably can serve as a precursor for branched-chain amino

acids, explainingits presence inthecrude protein fraction. The labeling ofnucleic acids suggested that mevalonic acid might be a precursor of A2-isopentenyl adenosine in the transfer RNA. Co-chromatography of the enzymatically re-leased nucleosides from transfer RNA from organisms grown in mevalonic-2-14C acid with authentic isopentenyl adenosine resulted in the appearance of all thelabelintransfer RNA with

isopentenyl

adenosine (Table 2). All ofthe

14C-J. BACTERIOL.

on March 20, 2021 by guest

http://jb.asm.org/

label in the total nucleicacid wasassociatedwith transfer RNA.

Nosignificantaccumulationofphosphorylated intermediates of the biosynthetic pathway to polyterpenes was found in culture supernatant fluidsororganisms.A traceofacompoundeluting atthe point whereisopentenol phosphateappears was found in the culture supernatant of cho-lesterol-grown organisms.Thismaterial afteracid hydrolysisofthephosphate radical possessedthe characteristics ofa five-carbon alcohol by gas-liquidchromatography.

DISCUSSION

This study revealed three facts. The sparing actionof cholesterolonpolyterpene biosynthesis byM. laidlawii isaresult ofspecificinhibitionof isopentenyl A3,

A2-isomerase.

The

organism

must alsosynthesize isopentenyl adenosine. Mevalonic

acid

probably serves as a precursor for the A2-isopentenyl radical ofisopentenyl

adenosine.

The nature of cholesterol inhibition of the isomerase apparently is competitive. However, any conclusive statement must await further

10r 0 .1.1 E 0 0. a' 0~ NI.. 5 O 0 0.05 Cholesterol (mg/ml)

FIG. 1. Effect of cholesterol on conversion of

mevalonic acid-1-14C to A3-isopentenyl pyrophosphate.

Reaction mixtureconsistedofadenosine triphosphate,

20umoles; MnCl2, 30,moles; reduced glutathione,

30

;umoles;

potassiumfluoride, 15 jumoles; potassium

phosphate buffer(pH7.5), 500jAmoles;mevalonic-1-1.4C

acid, 10 jAmoles (0.5

lAcAmoles);

ethanol or ethanol

solution of cholesterol, 0.05 ml; andsonically treated

M.laidlawiiprotein,50mg;inatotal volumeof5.0ml.

After3hrof incubationat37 Cintscrew-captubes, the reaction wasstoppedby heatingto 75C. Thereaction

mixture wasacidifiedwith N sulfuric acidand

'4CO2

trappedasbarium carbonatebyaerationwithnitrogen.

Afteradditionofasmallamountofcarrier, the

precipi-tateofbarium carbonatewaswashed, dried,andcounted

inaproportionalcounter. 3r 0 a. 0. 0 0 0 10 20 30 Time (min)

FIG. 2. Effect of cholesterol on isopentenyl A3,A2_

isomerase activity of M. laidlawii B. (i) Control;

(0) cholesterol, 0.015 mg/ml; (-) cholesterol, 0.03

mg/ml, and heat-inactivated control. The reaction

mixture consisted of reduced glutathioiie, 15 i.moles;

MgCI2, 2 ,moles; potassium fluoride, 60 ,moles; A'-isopentenyl-1-14C pyrophosphate, synthesized with

yeast enzymesfrom 20

jumoles

ofmevalonic-2-14C acid

(0.5 uc/,umole); Tris-maleate buffer (pH 8.0), 50

,Amoles;sonically treatedM. laidlawii protein, 18.8 mg;

ethanol or ethanol solution of cholesterol, 0.05 ml; in

total volumeof2.2ml. Incubation was at 37 C.Samples

(0.5 ml) were removed at 0, 5, 10, and 30mitt. A

0.5-ml amount of 6% trichloroacetic acid was addedtoeach

sample, thesolution was centrifuged, and the

superna-tant fluid was extracted with 3 ml of

diethzyl

ether.

Samples (0.05 and0.10ml) of the aqueous layerwere

countedinaproportionalcounter.

studies with

compounds

ofsimilar structure.The only similar portions of the three molecules in question, cholesterol,

A3-isopentenyl

pyrophos-phate,and

A2-isopentenyl

pyrophosphate, are the hydrocarbonends. CH2 P-P-O-CH2-CH2-C CH3

A3

isopentenyl pyrophosphate CH3

/

P-P-O-CH2-CH=C CH3 A2-isopentenyl pyrophosphate

on March 20, 2021 by guest

http://jb.asm.org/

Downloaded from

TABLE 1. Distributiont of 'IC in crude chemical

fractions oJ Mycoplasma laidlawii grown with

mevalonic-2-14C acid withand

withoutcholesterol

Fraction ~No Cholesterol Fraction cholesterola growna

Whole cells ... ... 2,794 1, 377

Coldtrichloroacetic acid. 17 23

Hot trichloroaceticacid. 245 235

Chloroform-methanol... 2,046 1,127

Residue ... .... 188 144

-Values express counts

gram (dry weight) of cells.

per minute per

milli-1.5

V/s

FIG. 3. Lineweaver-Burk plots of

isopeniteniyl-A3, A2-isomerase activity in presence anid absenice of cholesterol. The reactioni mixture was the same as

described in thelegendtoFig. 2,except A3-isopentenyl-1-'4Cpyrophosphate wassynthesizedbyusinlgpigliver

enzymesanidits concentrationt variedfrom 0.8to24.0

,umoles and cholesterol concenitration was conistant

at 0.015 mg/ml. Km valuesare expressedasmolarity.

CH3 CH3

I ~~/

R-CH-CH2CH2-CH2-CH CH3 cholesterol

If this apolar end is an attachment site for the

enzyme,competitioncould beexpected.Holloway

and Popjac (13) postulate both lipophilic and

polar binding sites forprenyl pyrophosphates in the A2-isopentenyl and geranyl transferase from

pig liver. Shah, Cleland, and Porter (20) have

shown the existence of an acid-stable,

enzyme-bound component during the isomerization of

isopentenyl pyrophosphate by the pig liver

enzyme.They conclude thata covalentthioether

bond exists between a sulfhydryl group on the

enzyme and the carbonium ion formed by protonation of the methylene carbon atom of isopentenyl pyrophosphate. Our limited data allow for little or no reliable speculation. How-ever, interaction ofenzyme with the apolar end

of thesubstrate doesoccurinthepigliversystem,

suggesting a basis for the apparent competition

by cholesterolwiththe enzymeof M. laidlawii.

TABLE2. Distributioni of 'IC in ntucleosides of

tranisfer RNA from Mycoplasma laidlawii grown with mevalonic-2-'4Cacida

Solvent Ab Solvent Bc RF Counts/min RF counts/min 0. 80d 200 0.83d 194 0.25 0 0.63 0 0.13 0 0.43 0 0.05 0 0.34 0 0 0 0.27 0 0 0

Total transfer RNA hydrolysate amount was

equivalent to that chromatographed, 180 counts/

min.

bni-Butanol-water-ammonium hydroxide. 86:

14:5.

c Isopropanol-concentrated HCl-water, 680:

170:144.

dSpot containing authentic isopentenyl

adeno-sine.

The pig liver enzyme isinhibited by sulfhydryl

inhibitors. No reports exist which demonstrate

specificinhibitionof thisenzyme by intermediates

in the pathway to polyprenols. Polyprenols are

known to inhibit incorporation of acetate into unsaponifiable lipids (14). Siperstein and Guest

(21) suggested that the primary site of feedback control induced byexogenous cholesterolinliver

is the reduction of hydroxymethyl glutaryl coenzymeA(CoA)tomevalonicacid. Cholesterol

alsoinhibitsincorporation of mevalonicacid into squalene in rat liver (16) and Tetrahymena pyriformis (4) but not into ubiquinones. Thus, there areindicationsformultiplesites of feedback inhibitionbycholesterol.

Isopentenyl adenosine has not been identified specificallyasbeing among the minor nucleosides

of mycoplasmas. However, hydrolysates of

transfer RNA from M. laidlawii B and M.

I/v

J. BACTERIOL.

on March 20, 2021 by guest

http://jb.asm.org/

gailisepticum exhibit cytokinin activity (9). Isopentenyl adenosine and its methythio-derivative have cytokinin action (10). From our studies, it would appear that isopentenyl adeno-sine does occur in the transfer RNA of M. laidlawii B. Mevalonic acid

apparently

serves as precursor for the isopentenyl radical of this compound in this organism since 14C in the nucleic

acids arising

from mevalonic-2-t4C acid is

associated

solely with the

isopentenyl

adeno-sine. The

biosynthesis

of

isopentenyl

adenosine has been shown to occur in yeast and rat liver by the transfer of

A2-isopentenyl

group from A2-isopentenyl pyrophosphate to an adenosine receptor in suitable transfer RNA

(15).

This mechanism requires the isomerization of A3-isopentenyl pyrophosphate to A2-isopentenyl pyrophosphate, the reaction inhibited

by

cho-lesterol inM. laidlawii. Since the

biosynthesis

of isopentenyl

adenosine

in M. laidlawii

undoubt-edly

occurs

by

the same

pathway,

complete suppression of the isomerase

probably

does not occur in vivo.

High

concentrations

(0.1

to 0.2 mg/ml) of cholesterol inhibit growth of myco-plasmas. Otherenzymatic sites in the

pathway

to polyterpenes

beyond

the isomerase may also be inhibited;

these,.

however, have not been

ex-amined.

ACKNOWLEDGMENT

This investigation was supported by Public Health Service research grant At 04410-08 fromtheNational Institute ofAllergy

andInfectious Diseases.

LITERATURE CITED

1. Agranoff,B.W., H.Eggerer, U.Henning,andF.Lynen. 1960.

Biosynthesisof terpenes. ViI. Isopentenyl pyrophosphate

isomerase. J. Biol. Chem. 235:326-332.

2. Anderson,D.G., and J.W.Porter.1962. Thebiosynthesisof

phytoeneandothercarotenesbyenzymesofisolatedhigher

plantplastids. Arch.Biochem.Biophys.97:509-519. 3. Bloch, K., S.Chaykin,A. H.Phillips,andA.DeWaard. 1959.

Mevalonic acidpyrophosphate andisopentenyl

pyrophos-phate. J.Biol. Chem. 234:2595-2604.

4. Conner,R.L.,J.R.Landrey, C.H.Burns,andF. B.Mallory. 1968. Cholesterol inhibition of pentacyclic triterpenoid biosynthesis in Tetrahymenapyriformis. J.Protozool. 15: 600-605.

5.Dugan,R.E.,E.Rasson, and J.W. Porter.1968.Separation

of water-soluble steroid and carotenoid precursors by

DEAE-cellulosecolumnchromatography. Anal. Biochem. 22:249-259.

6. Fieser,L. F. 1955.Cholesterol, A6-cholesten-3-one, and A4-cholesten-3-one.Org. Syn.35:43-49.

7. Hall, R. H.1964. Isolation of N6 (aminoacyl)adenosinefrom yeast ribonucleic acid.Biochemistry 3:769-773.

8. Hall, R. N. 1965. Ageneral procedurefor the isolation of "minor" nucleosides from ribonucleic acid hydrolysates. Biochemistry 4:661-670.

9. Hayashi, H.,H.Fisher, and D. Soll. 1969. Transfer ribonucleic acid from Mycoplcvma. Biochemistry 8:3680-3686. 10. Hegelson, J. B. 1968. The cytokinins.Science161:974-981. 11. Henrikson, C. V., and P. F. Smith. 1965. Conversion of

mevalonic acid to -y,-y-dimethylallyl pyrophosphate by Mycoplasma. J. Bacteriol. 92:701-706.

12. Henrikson, C. V., and P. F. Smith. 1966. Growth response of Mycoplasma to carotenoid pigments and carotenoid inter-mediates. J. Gen. Microbiol. 45:73-82.

13. Holloway, P. W., and G. Popjak. 1967. The purification of 3, 3-dimethylallyl- and geranyl-transferase and of iso-pentenyl pyrophosphate isomerase from pig liver. Biochem. J. 104:57-70.

14. Isler, O., R. Ruegg, G. Saucy, J.Wursche, K. F. Gey, and A. Pletscher. 1959. Semi-, mono-, sesqui- and triterpenes as cholesterol precursors. Ciba Found. Symp. Biosyn. Terpenes Sterols, 1958, p. 135-146.

15. Kline, L. K., F. Fittler, and R. H. Hall. 1969. N6-(A2-iso-pentenyl)adenosine. Biosynthesis in transfer ribonucleic acidinvitro.Biochemistry 8:4361-4371.

16. Krishnaiah, K. N., V. C. Joshi, and T. Ramasarma. 1967. Effect of dietary cholesterol and ubiquinone on isoprene synthesis in rat liver. Arch. Biochem. Biophys. 121:147-153. 17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

18. Nandi, D. L., andJ.W. Porter. 1964. The enzymatic synthesis of geranyl geranyl pyrophosphate by enzymes of carrot root andpigliver. Arch. Biochem. Biophys. 105:7-19. 19. Rothblat, G. H., and P. F. Smith. 1961. Nonsaponifiable lipids

of representative pleuropneumonialike organisms. J. Bacteriol. 82:479-49 1.

20. Shah, D. H., W. W. Cleland, andJ. W. Porter. 1965. The

partialpurification, properties and mechanism of action of pig liver isopentenyl pyrophosphate isomerase. J. Biol. Chem.240:1946-1956.

21.Siperstein, M. D., and M. J. Guest. 1960. Studies on the site of feedback control of cholesterol biosynthesis. J. Clin. Invest. 319:642-652.

22.Smith, P. F. 1968. Lipids of Mycoplasma. Adv. Lipid Res. 6:69-103.

23. Smith, P. F. 1968. Nature of unsaponifiable lipids of Myco-plasma sp., strain J, grown with isopentenyl pyrophosphate as asubstitute for sterol. J. Bacteriol. 95:1718-1720. 24. Smith, P. F., and C. V. Henrikson. 1965. Comparative

bio-synthesis of mevalonic acid by Mycoplasma. J. Bacteriol. 89:146-153.

25. Smith, P. F., and C. V. Henrikson. 1965. Glucose containing phospholipids in Mycoplasma laidlawii, strain B. J.Lipid

Res.6:106-111.

26. Smith, P. F., and C. V. Henrikson. 1966. Growth inhibition of Mycoplasma byinhibitors of polyterpenebiosynthesis and its reversal by cholesterol. J. Bacteriol. 91:1854-1858. 27. Zubay, G. 1966. Isolation of transfer RNA, p. 455-460. In

G. L. Cantoni and D. R. Davies (ed.), Procedures in nucleic acidresearch.Harper and Row,New York.

VOL.

103,

1970 31

on March 20, 2021 by guest

http://jb.asm.org/