JOURNAL OF BACTERIOLOGY, Aug. 1972, p. 547-556 Copyright 0 1972 American Society for Microbiology Vol. 111, No. 2 Printed in U.S.A. Regulation of Homocysteine Biosynthesis in Salmonella typhimurium1 MICHAEL A. SAVIN, MARTIN FLAVIN, AND CLARENCE SLAUGHTER Laboratory of Biochemistry, National Heart and Lung Institute, Bethesda, Maryland 20014 Received for publication 10 April 1972 The regulation of the homocysteine branch of the methionine biosynthetic pathway in Salmonella typhimurium has been reexamined with the aid of a new assay for the first enzyme. The activity of this enzyme is subject to syner- gistic feedback inhibition by methionine plus S-adenosylmethionine. The syn- thesis of all three enzymes of the pathway is regulated by noncoordinate re- pression. The enzymes are derepressed in metJ and metK regulatory mutants, suggesting the existence of regulatory elements common to all three. Experi- ments with a methionine/vitamin B,2 auxotroph (metE) grown in a chemostat on methionine or vitamin B12 suggested that the first enzyme is more sensitive to repression by methionine derived from exogenous than from endogenous sources. metB and metC mutants grown on methionine in the chemostat did not show hypersensitivity to repression by exogenous methionine. Therefore, it appears that the metE chemostat findings are peculiar to the phenotype of this mutant; such evidence suggests a possible role for a functional methyltetra- hydrofolate-homocysteine transmethylase in regulating the synthesis of the first enzyme. Thus there appear to be regulatory elements which are common to the repression of all three enzymes, as well as some that are unique to the first enzyme. The nature of the corepressor is not known, but it may be a deriv- ative of S-adenosylmethionine. metJ and metK mutants of Salmonella have a normal capacity for S-adenosylmethionine synthesis but may be blocked in synthesis or utilization of a corepressor derived from it. Methionine is synthesized in Salmonella typhimurium by a highly branched pathway, the two terminal branches converging in the methylation of homocysteine by N5-methyltet- rahydrofolate (CH3FH4) (Fig. 1). This paper deals with studies on the regulation of the subpathway leading to homocysteine. Three enzymes catalyze the sequential reactions unique to this pathway: homoserine O-trans- succinylase, forming O-succinylhomoserine from succinyl coenzyme A (CoA) and homo- serine (25); cystathionine -y-synthase, forming cystathionine from O-succinylhomoserine and cysteine (4, 5); and f,-cystathionase, forming homocysteine from cystathionine (4). The un- linked structural genes for the enzymes are metA, metB, and metC, respectively. Previous studies, reviewed by Smith (32), have shown that there is feedback control of the first en- zyme (20) and noncoordinate repression by ' Presented in part at the 62nd Annual Meeting of the American Society of Biological Chemists, San Francisco, Calif., 13-18 June 1971 (Fed. Proc. 30:1344, 1971). methionine of synthesis of the second and third enzymes (18), the actual corepressor pos- sibly being S-adenosylmethionine (SAM) or something derived from it (8). Because of the lack of an assay suitable for use in crude ex- tracts of Salmonella, little is known about the repressibility of the first enzyme. Lawrence (17) recently reported experiments showing that the first enzyme is derepressed in methio- nine regulatory mutants known to have dere- pressed second and third enzymes (18). How- ever, he assayed the enzyme by measuring the appearance of free CoA in the reaction mix- ture, a procedure which is subject to interfer- ence in crude extracts (see below). The discovery that N5-methyltetrahydro- folate polyglutamates (CH.FH4Gn) are allosteric activators of cystathionine -y-syn- thase in Neurospora crassa (29) prompted us to investigate the possibility of a regulatory role for folate derivatives in Salmonella. No regula- tory function for folates was found. However, in the course of these studies we developed 547 on May 12, 2020 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACTERIOLOGY, Aug. 1972, p. 547-556Copyright 0 1972 American Society for Microbiology
Vol. 111, No. 2Printed in U.S.A.
Regulation of Homocysteine Biosynthesis inSalmonella typhimurium1
MICHAEL A. SAVIN, MARTIN FLAVIN, AND CLARENCE SLAUGHTER
Laboratory of Biochemistry, National Heart and Lung Institute, Bethesda, Maryland 20014
Received for publication 10 April 1972
The regulation of the homocysteine branch of the methionine biosyntheticpathway in Salmonella typhimurium has been reexamined with the aid of a
new assay for the first enzyme. The activity of this enzyme is subject to syner-
gistic feedback inhibition by methionine plus S-adenosylmethionine. The syn-
thesis of all three enzymes of the pathway is regulated by noncoordinate re-
pression. The enzymes are derepressed in metJ and metK regulatory mutants,suggesting the existence of regulatory elements common to all three. Experi-ments with a methionine/vitamin B,2 auxotroph (metE) grown in a chemostaton methionine or vitamin B12 suggested that the first enzyme is more sensitiveto repression by methionine derived from exogenous than from endogenoussources. metB and metC mutants grown on methionine in the chemostat didnot show hypersensitivity to repression by exogenous methionine. Therefore, itappears that the metE chemostat findings are peculiar to the phenotype of thismutant; such evidence suggests a possible role for a functional methyltetra-hydrofolate-homocysteine transmethylase in regulating the synthesis of thefirst enzyme. Thus there appear to be regulatory elements which are common
to the repression of all three enzymes, as well as some that are unique to thefirst enzyme. The nature of the corepressor is not known, but it may be a deriv-ative of S-adenosylmethionine. metJ and metK mutants of Salmonella have a
normal capacity for S-adenosylmethionine synthesis but may be blocked insynthesis or utilization of a corepressor derived from it.
Methionine is synthesized in Salmonellatyphimurium by a highly branched pathway,the two terminal branches converging in themethylation of homocysteine by N5-methyltet-rahydrofolate (CH3FH4) (Fig. 1). This paperdeals with studies on the regulation of thesubpathway leading to homocysteine. Threeenzymes catalyze the sequential reactionsunique to this pathway: homoserine O-trans-succinylase, forming O-succinylhomoserinefrom succinyl coenzyme A (CoA) and homo-serine (25); cystathionine -y-synthase, formingcystathionine from O-succinylhomoserine andcysteine (4, 5); and f,-cystathionase, forminghomocysteine from cystathionine (4). The un-linked structural genes for the enzymes aremetA, metB, and metC, respectively. Previousstudies, reviewed by Smith (32), have shownthat there is feedback control of the first en-zyme (20) and noncoordinate repression by
' Presented in part at the 62nd Annual Meeting of theAmerican Society of Biological Chemists, San Francisco,Calif., 13-18 June 1971 (Fed. Proc. 30:1344, 1971).
methionine of synthesis of the second andthird enzymes (18), the actual corepressor pos-sibly being S-adenosylmethionine (SAM) orsomething derived from it (8). Because of thelack of an assay suitable for use in crude ex-tracts of Salmonella, little is known about therepressibility of the first enzyme. Lawrence(17) recently reported experiments showingthat the first enzyme is derepressed in methio-nine regulatory mutants known to have dere-pressed second and third enzymes (18). How-ever, he assayed the enzyme by measuring theappearance of free CoA in the reaction mix-ture, a procedure which is subject to interfer-ence in crude extracts (see below).The discovery that N5-methyltetrahydro-
folate polyglutamates (CH.FH4Gn) areallosteric activators of cystathionine -y-syn-thase in Neurospora crassa (29) prompted us toinvestigate the possibility of a regulatory rolefor folate derivatives in Salmonella. No regula-tory function for folates was found. However,in the course of these studies we developed
assays for the transsuccinylase which, in con-junction with the use of a chemostat, made itpossible to show that the repression of thisenzyme is not only noncoordinate with that ofthe second and third enzymes, but probablymakes use of a different mechanism.
MATERIALS AND METHODSMaterials. The preparation has been described of
succinyl CoA (31) and O-succinylhomoserine (6). L-Cystathionine, L-homoserine, 3H-L-homoserine,SAM, L-cystine, L-homocystine, and lactate dehy-drogenase (L-lactate: nicotinamide adenine dinucleo-tide oxidoreductase, EC 1.1.1.27) were obtained fromCalbiochem. Vitamin B,2, pyrdidoxal phosphate,and reduced nicotinamide adenine dinucleotide(NADH) were from Sigma Chemical Co., and L-me-thionine was from Nutritional Biochemicals Corp.Materials for the SAM synthetase assay included re-duced glutathione from Nutritional BiochemicalsCorp., adenosine triphosphate (ATP) from P-L Labo-ratories, and "C-ATP from Calatomic. Methyltetra-hydrofolate monoglutamate (CH.FH4GJ), digluta-mate (CH3FH4G,), triglutamate (CH,FH4G.), andmethylcobalamin were the generous gifts of W.Sakami (folates) and T. C. Stadtman.
Bacterial strains. The following Salmonellastrains were obtained from K. E. Sanderson ofthe University of Calgary, Canada: Lt-2 (wild type),metA 15, metB16, metB36, metC50, metE47,metF96, and metG419. The phenotypes are shown inFig. 1; metE47 lacks the B,1-independent homocys-tine transmethylase (1), and metG419 has a defect(increased Km) in methionyl transfer ribonucleic acid(tRNA) synthetase (9). The following are either dele-tions or had zero reversion (33): metA15, metB36,metC50, and metE47. Other Salmonella strains werethe generous gift of D. Smith of the University ofBirmingham, England: metJ713 and metK721, bothmethionine analogue-resistant mutants which over-produce methionine and have nonrepressible levelsof cystathionine -y-synthase and 6-cystathionase (18);and the double mutant metE205, metH465, whichlacks both BW,-dependent and B11-independenthomocysteine transmethylases (2). R. C. Greene ofDuke University generously provided the followingE. coli strains: K-12, wild type; E31 (metJ), a regu-latory mutant with nonrepressible levels of SAMsynthetase, cystathionine 'y-synthase, and fB-cysta-thionase (12); and E40 (metK), a mutant deficient inSAM synthetase with nonrepressible levels of cysta-thionine -y-synthase and ,B-cystathionase (8).
Bacterial cultures. The minimal medium used inall experiments was Vogel and Bonner's medium E,which contains (in grams per liter): K,HPO4, 10;NaNH4HPO4, 3.5; citric acid-1H,O, 2; MgSO4.7H2O, 0.2; and glucose, 0.2. For cultivation of auxo-trophs and studies of repression, various aminoacids or vitamin B1, were added in the concentrationsindicated in the tables. For flask culture experiments,50 ml of medium in a Klett side arm flask was firstinoculated from a slant. After overnight growth on arotary shaker at 37 C, the contents were used to
inoculate 500 ml of the same medium in a Fernbachflask. The cells were incubated at 37 C on a rotaryshaker, harvested by centrifugation at mid-log phase(approximately 170 Klett units), and then washedand extracted as described below. When enzymelevels were measured in stationary-phase cells (seeTable 5), the cultures were incubated for 24 hr, i.e.,about 20 hr after cessation of exponential growth.The methods used for the chemostat cultures were
modeled after those of Novick and Szilard (24), withthe apparatus of Tabor and Tabor (35). Thegrowth vessel was a 1-liter bottle with a side arm atthe 700-ml level. The afternoon before beginning eachexperiment, a 700-ml Fembach flask culture wasgrown to late log phase and then stored overnight at2 C. The flask culture medium contained the limitingnutrient at the same concentration to be used in thechemostat. At the beginning of the experiment, theflask culture was used to inoculate the growth vessel.Fresh sterile medium was then pumped into thegrowth vessel at a constant flow rate from a 10-literreservoir. Flow rates were adjusted to give the de-sired generation time for each experiment. Genera-tion time, r, is a chemostat function defined byNovick and Szilard (24) as the reciprocal of growthrate, a, where a = (1/n)(dn/dt), and n = number ofbacterial cells per ml. T is also equal to viw, where vis the chemostat growth vessel volume and w is therate of infusion of medium into the growth vessel.Doubling time is equal to r x In2. The growth vesselwas kept at 37 C in a constant temperature waterbath while air was bubbled through the medium tomaintain aeration and mixing. Growth medium andcells flowed out of the chemostat through the sidearm into a cylinder immersed in an ice bath. Theeffluent was discarded until at least three chemostatvolumes (2 liters) had passed through the side arm;the effluent cells were then harvested periodically bycentrifugation and, at the end of the experiment,were combined with the cells remaining in the chemo-stat growth vessel. In a few experiments, the latterwere harvested and extracted separately.
Cell density was routinely measured by absorb-ance at 550 nm in a cuvette of 1-cm light path, usinga Zeiss spectrophotometer, after the absorbance hadbeen calibrated against both chamber counts andviable colony counts. Cell densities were controlledat about 108 cells per ml by limiting the concentra-tions of nutrients in the reservoir to 12 x 10-I to 14x 10-6 M in the case of L-methionine, or 3 x 10-11 to5 x 10- II M in the case of vitamin B2. The rate ofaeration was shown not to limit growth under theseconditions.
Limitation of vitamin B12 in flask cultures did notyield a sharp end point of growth, but a transitionpoint, beyond which progressively slower growthcontinued until the maximum vitamin concentationper cell reached between 10 and 100 molecules percell. However, in chemostat cultures it was possibleto maintain a constant cell density throughout eachexperiment by vitamin limitation.Enzyme preparations and assays. Cells were
harvested by centrifugation and then washed in sev-eral volumes of the following buffer: potassium phos-
phate, pH 7.3, 50 mM; ethylenediaminetetraaceticacid, 1 mM; and dithiothreitol, 1 mm. Washed cellswere suspended in two or more volumes of the samebuffer and extracted with a Branson LS75 sonifier infour 30-sec bursts. Small volumes (1 ml) were ex-tracted with a Branson W185 sonifier by using themicrotip and four 15-sec bursts. The crude extractswere centrifuged at 15,000 x g for 20 min, and thesupernatant fluid was applied to a column of Seph-adex G25 (coarse beads, bed volume 10 times thesupernatant volume), which had been equilibratedand was then eluted with the above buffer. The en-tire procedure was carried out at 2 C, and the gel fil-tered protein fractions were either assayed at once orstored at -20 C.
Assay procedures were as previously described forcystathionine -y-synthase (assay A, reference 13), ,-cystathionase (10), and SAM synthetase (8); theelimination rates observed in the cystathionine syn-thase assay were multiplied by five to give the cor-rect rates for cystathionine synthesis (13).Two assays devised for the homoserine transacet-
ylase of Neurospora (23) were adapted to the bac-terial homoserine O-transsuccinylase, with somewhatunsatisfactory results. In the first assay, the enzymewas incubated with 3H-L-homoserine and succinylCoA. The O-succinylhomoserine synthesized wasconverted to N-succinylhomoserine by alkali treat-ment and then applied to a Dowex-50 (H+) columnas previously described (23). In the second assay,which is based on the ability of the enzyme to cata-lyze the exchange between homoserine and O-succi-nylhomoserine, O-succinylhomoserine was substi-tuted for succinyl CoA. In both assays, the separa-tion of 3H-O-succinylhomoserine from 3H-homo-serine is achieved because N-succinylhomoserine isnot retained by Dowex-50 (H+), whereas the proton-ated amino acid is. The reaction mixtures for bothassays contained, in 0.5-ml volume: 50 Amoles ofpotassium phosphate, pH 7.5; 1.5 ,umoles of succinylCoA or 5 ,moles of O-succinyl-L-homoserine; 2,gmoles of 3H-L-homoserine; and sufficient extract toyield 0.1 to 0.3 Amole of labeled succinylhomoserinein 20 min at 37 C.Much better results were obtained by coupling the
transsuccinylase with a large excess of purified (11,14) cystathionine y-synthase, to give the overall re-action: L-homoserine + succinyl CoA - a-ketobu-tyrate + succinate + CoA + NH3. The reactionmixtures contained, in a final volume of 1 ml: 1.5,umoles of succinyl CoA; 2.0 nmoles of L-homoserine;0.25 to 1.0 mg of extract protein; and 1.2 units ofpurified cystathionine -y-synthase. One unit of thepurified cystathionine synthase is the amount cata-lyzing the formation of 1 lmole of a-ketobutyrate bygamma elimination from O-succinylhomoserine in 1min. After 5 min at 37 C, the reactions were stoppedby the addition of 0.1 ml of 1.5% trichloroacetic acid.After centrifugation, samples of the supernatantfluid were assayed for a-ketobutyrate with lactatedehydrogenase (14). Each reaction mixture wasmatched with a blank lacking succinyl CoA. Theassay gave rates which were linear over a wide rangeof reaction time and over a range of transsuccinylase
concentration catalyzing the synthesis of from 8 to240 nmoles of O-succinylhomoserine per min. Therates were about 10 times higher than those reportedby Schlesinger (28). The difference may be due toend-product inhibition of the enzyme by O-succi-nylhomoserine, since rates similar to Schlesinger'swere found when the purified cystathionine y-syn-thase was not added until after the completion of thereaction or when the radioactive synthesis assay wasused. Most extracts were assayed by both the ex-change assay and the coupled synthesis assay, andthe rate ratio was 1: 8.We found that the enzyme could not be reliably
assayed in crude extracts by measuring the libera-tion of free CoA as described by Lawrence (17). Themethod gave very high blanks due to enzymatic hy-drolysis of succinyl CoA and to spontaneous hydrol-ysis and succinylation of the amino group of homo-serine. The different extraction procedures used byLawrence may have altered the portion of the blankwhich is due to enzymatic hydrolysis of succinylCoA.
Proteins were determined by the method of Layne(19). All enzyme activities are expressed as nano-moles of product formed per minute per milligram ofprotein.
RESULTSStudies of a regulatory role for folate de-
rivatives in homocysteine biosynthesis. In N.crassa, the second enzyme in the homocysteinepathway, cystathionine y-synthase, is thetarget for allosteric regulation, being inhibitedby SAM and activated by 5N-methyltetra-hydrofolate polyglutamates (CH3FH4Gn)(29). In E. coli, it is the first enzyme, homo-serine O-transsuccinylase, which is regulated,being subject to synergistic inhibition by me-thionine plus SAM (20). Table 1 shows thatthe same is true for Salmonella although thesynergism appears much less marked. Law-rence has recently reported similar findings forthis enzyme (17). The synergistic inhibition ofthe Salmonella transsuccinylase was observedonly with the direct synthesis assays and notwith the assay based on the exchange of homo-serine into O-succinylhomoserine. This raisesthe question whether the exchange reaction iscatalyzed by transsuccinylase or by another en-zyme. We believe both reactions are catalyzedby the same enzyme since the rates changed inparallel in all of the flask culture experimentsreported here.We next tested the effect on transsucci-
nylase activity of adding CH3FH4G3 to a gel-filtered ammonium sulfate fraction of thisenzyme from the wild type; there was no acti-vation (Table 1). The small inhibition observedis encountered with many enzymes whentreated with polyglutamate derivatives of
aEnzyme preparation used was slightly purified(approximately threefold) by ammonium sulfate pre-cipitation. Homoserine O-transsuccinylase activitywas measured by coupling it to cystathionine y-syn-thase as described.
b SAM, S-Adenosylmethionine; CH,FH4Gs, meth-yltetrahydrofolate triglutamate.
c Expressed as nanomoles of product formed perminute per milligram of protein.
levels of cystathionine y-synthase and (3-cys-tathionase, an unexplained phenomenon thatwe have made use of for many years in puri-fying these enzymes (10, 14).metE mutants will respond alternately to
methionine or vitamin B 2. Extracts of cellsgrown on non-growth-limiting levels (7.4 x 10- 9
M) of the vitamin had high transsuccinylaseactivity (Table 2). This observation, somewhatreminiscent of an earlier one in the argininepathway (30), suggested that exogenous andendogenous methionine might affect the re-pression of the transsuccinylase differently.The conclusion was not supported by thefinding that transsuccinylase was still absentfrom extracts of metB and metC grown onhomocystine instead of methionine (Table 2).Since the intracellular concentration of nu-trients cannot be controlled by limiting themin flask cultures, we examined this questionfurther with the use of a chemostat.
folate (B. T. Kaufman, unpublished data).Since gel filtration does not always separatepolyglutamates from protein (29), we alsoexamined a metF mutant, blocked in the syn-thesis of CHSFH, (Fig. 1). We were encouragedby finding no transsuccinylase activity (Table2) in extracts from flask cultures grown on lim-iting methionine (0.1 mM limits growth; 0.5mM does not). However, activity was not re-stored by any of the following: CH,FH,G1,CHSFH4G2, CH3FH4G,, N'-formyltetrahydro-folate monoglutamate, methylcobalamin (10-'M), or boiled extract of wild type. Moreover,none of these additions affected the activitiesof cystathionine 'y-synthase or #cystathionasein the metF extract, the levels of the latter twoenzymes being similar to wild type (Table 2).Thinking that the activation of transsucci-
nylase by folates might be able to be mani-fested only in vivo, we next examined a metEmutant. This mutant should accumulate largeamounts of methylfolates since in the absenceof B12 it is blocked in the methylation of homo-cysteine (Fig. 1). However, extracts of thisstrain grown on limiting methionine were alsofound to have no transsuccinylase, and levelsof the other two enzymes were comparable towild type (Table 2). In fact, transsuccinylasecould not be detected in any methionine auxo-troph (Table 2) grown on low levels of methio-nine which only partially reduced the enzymelevel in wild type. The levels of the other twoenzymes were little affected by methionine.Table 2 also shows that extracts of metA15mutants grown on low methionine have high
Homoserine
Met-A Succinyl CoA
O-Succinyl
Met-B
Cystat
Met-C
iomoserine+
teinehionine
Homocysteine
Methionine
At
S-Adenosylmethi
FH4GI
Glutamate
FH4 Gn
Serine
CH2 = FH4 Gn
Met-F
CH3 - FH4 GnMet-E
B12 Met-H
CH3 - FH4 GIrpilonine
FIG. 1. Pathway of methionine biosynthesis inSalmonella. The structural gene designations andtheir respective enzymes are as follows: metA, ho-moserine 0-transsuccinylase; metB, cystathionine-y-synthase- metC, ,5-cystathionase; metE, non-B12methyltetrahydrofolate-homocysteine transmethyl-ase; metF, methylenetetrahydrofolate reductase;and metH, B1,-dependent methyltetrahydrofo-late-homocysteine transmethylase. Not shownin the figure are metG, methionyl-tRNA synthetase;and metJ and metK, methionine regulatory genes.The following abbreviations are used in the figure:FH4G1, tetrahydrofolate monoglutamate; FH4Gn,tetrahydrofolate polyglutamate; CH2=FH4Gn,methylenetetrahydrofolate polyglutamate: CH3-FH4G1, methyltetrahydrofolate monoglutamate; andCHsFH4G,, methyltetrahydrofolate polyglutamate.
metE 47 L-Methionine 0.05 0 75 18Vitamin B,2 7.4 x 10' 21 25 16
metF 96 L-Methionine 0.05 0 50 24
metE, 205 L-Methionine 0.05 0 165 34metH 465
a Expressed as nanomoles of product formed per minute per milligram of protein.
Chemostat experiments. Figure 2 shows thelevels of the three enzymes of homocysteinebiosynthesis in metE47 grown in a methioninechemostat, plotted as a function of generationtime (defined above). The longer the genera-
tion time, the lower the intracellular concen-
tration of methionine (24). The shortest gener-
ation time used, 75 min, corresponds to a dou-bling time of 52 min (doubling time = r x
1n2) which exceeds the maximum doublingtimes of this strain in flask culture of 35 minon methionine and 40 min on vitamin B12. Asexpected, the synthesis of the second and thirdenzymes was progressively repressed inparallel as the generation time was increasedup to 4 hr. However, it was virtually impossibleto elicit any synthesis of the transsuccinylaseby lowering the intracellular concentration ofmethionine, a very low level of this enzymefinally appearing at a generation time greaterthan 5 hr. Figure 3 shows the results obtainedwith the same mutant in a vitamin B12 chemo-stat. The pattern seems to recapitulate that ofFig. 2 with everything displaced towardsshorter generation times. But the most strikingresults were that all three enzymes were fullyderepressed at generation times between 2 and
3 hr, and that there was no measurable trans-succinylase at short generation times, althoughthe mutant could not grow on vitamin B12 with-out the functioning of this enzyme. The resultsso far supported the conclusion that exogenousmethionine was more effective than endog-enous in repression of the transsuccinylase.At very long generation times the levels of
all three enzymes declined in both the methio-nine and vitamin B12 chemostats. Possibleexplanations for this decline are: (i) harvestingof cells before they reached a steady state; (ii)decreased protein content in general; (iii) lossof viability; and (iv) genetic change to counter-vene the possibly noxious accumulation of highconcentrations of methionine precursors. Noneof these possibilities has been specifically ruledout. In several experiments, the proportion ofviable cells was checked by comparing plateand chamber counts; the proportion was thesame in these experiments. Reversion to proto-trophy was never observed except at thelongest generation time (5.8 hr) in the methio-nine chemostat when 0.25% prototrophs werefound.
Similar experiments were next undertakenwith nonreverting (33) metB and metC mu-
FIG. 2. Enzyme levels in metE47 grown in themethionine chemostat. Methods used for growth ofchemostat cultures, enzyme preparations, and assaysare described in the text. Derepression, plotted as
relative activity, is shown as a function of generationtime, a chemostat function inversely proportional togrowth rate. A = homoserine O-transsuccinylase(first enzyme); B = cystathionine y-synthase(second enzyme); and C = ,8-cystathionase (thirdenzyme).
3 4GENERATION TIME, T (hours)
FIG. 3. Enzyme levels in metE47 grown in thevitamin B12 chemostat. Procedures are as describedin the legend to Fig. 2 and the text. A = homoserineO-transsuccinylase (first enzyme); B = cystathioniney-synthase (second enzyme); and C = ,-cysta-thionase (third enzyme).
tants (Table 3); metC mutants are leaky (7),but the doubling time of metC50 in minimalmedium is 5 hr. It was not possible to compareendogenous and exogenous methionine withthese mutants because the methionine precur-sors which they can utilize would not sustaingrowth in the chemostat at reasonably shortgeneration times. In the case of homocystine,this was apparently due to low affinity forsome rate-limiting step and, in the case of cys-
tathionine, to low velocity of growth. The re-sults with methionine (Table 3) show that inboth mutants transsuccinylase was fully dere-pressed at a generation time of 2.5 hr, at whichthe enzyme was fully repressed in metE47.This result suggested that the apparent ex-treme repressibility of transsuccinylase byexogenous methionine might be peculiar to, orin some way related to, the specific phenotypeof the metE mutant.Corepressor for the enzymes of homocys-
teine biosynthesis. The chemostat experi-ments with metE47 suggested that the core-pressor for transsuccinylase might be differentfrom the corepressor for the other two enzymesand more immediately derived from exogenousmethionine. metG419, a Km mutant for meth-ionyl-transfer ribonucleic acid (tRNA) synthe-tase, has been reported to have normally re-pressible cystathionine y-synthase and ,-cys-tathionase (9). The results in Table 4 indicatethat methionyl-tRNA is also not part of therepressor for transsuccinylase, which was nor-mally repressed by methionine in this mutant.metJ713 and metK721 were isolated as
methionine analogue-resistant mutants andwere found to have nonrepressible levels ofcystathionine 'y-synthease and ,-cystathionase(18), as well as of the methionine-specific ,B-aspartokinase (L-aspartate 4-phosphotrans-ferase, EC 1.2.1.11) and homoserine dehydro-genase (L-homoserine: NAD oxidoreductase,EC 1.1.1.3) (27). The results of Table 4 showthat transsuccinylase levels in these mutantsparallel those of the other enzymes of homo-cysteine biosynthesis. Lawrence has also re-cently reported derepression of transsucci-nylase in these mutants (17).
Greene et al. (8) have isolated E. coli mu-tants with nonrepressible levels of cystathio-nine y-synthase and ,-cystathionase. One ofthem, E40, mapped in the region of the metKgene of Salmonella although its exact geneticidentity has not been established (34; R. C.Green, personal communication). Transsucci-nylase was also derepressed in this mutant(Table 4). Greene et al. (8) found that themutant had an extremely low level of SAMsynthetase, suggesting that SAM, or somethingderived from it, might be a corepressor. Table5 shows that, in contrast to this E. coli mu-tant, Salmonella metK721 had wild-type levelsof SAM synthetase. Salmonella metJ713 hadderepressed levels of SAM synthetase (Table5) like E. coli E31 (metJ) (12).
Salmonella transsuccinylase was found todiverge from the other enzymes of homocys-teine biosynthesis in one other respect, in that
a Conditions for growth of organisms in the chemostat, extraction of cells, and enzyme assays are describedin the experimental procedures.
° Expressed as nanomoles of product formed per minute per milligram of protein.c Generation time is a chemostat function which is the reciprocal of growth rate (see text).
TABLE 4. Enzyme levels in methionine regulatory mutants of Salmonella and Escherichia coli grown in flaskculture
E. coli Wild type (K-12) 0 25 10 55metKc E40 0.05 320 300 210
a Expressed as nanomoles of product formed per minute per milligram of protein.b metG419 is a mutant with a defective methionyl-tRNA synthetase (Km for methionine 100 times higher
than wild type). The remaining Salmonella mutants have methionine regulatory defects.c Although E. coli strain E40 maps in the same region as the metK gene of Salmonella, the exact genetic
identity of this mutant is uncertain (26).
it fell to very low levels after the wild type hadentered resting phase (Table 6). However, inresting phase cultures of both metJ713 andmetK721 transsuccinylase was derepressed inparallel with the other enzymes (Table 6). Ourobservation that the first enzyme is repressedin resting phase cells of the wild type differsfrom the results reported by Lawrence (17).However, his results are based on an assaysubject to high nonenzymatic blanks (seeabove) and may not have revealed the low ac-tivities.
DISCUSSIONMany amino acids have additional functions
besides serving as building blocks for protein,but methionine is conspicuous in this respect.It contributes to membrane phospholipid (the
methyl groups of choline), is a source of poly-amines, a general methylating agent (viaSAM), and the initiator of protein synthesis(via a particular species of methionyl-tRNA).It would, then, not be surprising if the regula-tion of its synthesis were to reveal some un-usual features. Methionine synthesis is also ofinterest as an example of the general problemof regulation of the synthesis of a group of se-quen%jal enzymes coded by unlinked genes.Feedback control and the possible role of
folate derivatives in regulating homocys-teine biosynthesis. In N. crassa the con-verging pathways leading to the synthesis ofhomocysteine and of the methyl group are co-ordinated by the balance between two allo-steric controls: one is activation of cystathio-nine 'y-synthase, the second enzyme of the
Salmonella Wild type (Lt-2) 0 1.82metJ 713 0.10 3.67
0.50 6.87metK 721 0 1.20
0.50 1.43
a Cells were grown in flask culture and harvestedduring mid-log-phase growth as described in theexperimental procedures. S-adenosylmethionine syn-thetase was assayed as described by Greene et al.(15), except that desalted extracts rather than tolu-enized cells were used.
"Concentration of L-methionine in the growthmedium.
cExpressed as nanomoles of product formed perminute per milligram of protein.
TABLE 6. Enzyme levels in Salmonella methionineregulatory mutants after cessation of growth in flask
a Cultures were incubated at 37 C for 24 hr (ap-proximately 16 hr after cessation of exponentialgrowth). Harvesting and extraction of cells and as-says used are described in the text.
Expressed as nanomoles of product formed perminute per milligram of protein.
homocysteine pathway, by CH,FH4Gn, theend product of the methyl pathway; and theother is the feedback inhibition of cystathio-nine y-synthase (as well as methylenetet-rahydrofolate reductase in the methyl path-way) by SAM (29). In E. coli it is the first en-zyme of the homocysteine pathway, homo-serine O-transsuccinylase, whose activity isregulated through synergistic feedback inhibi-tion by SAM plus methionine (20). The sameis true in Salmonella although the synergismseems to be weaker (Table 1). There is also
marked inhibition of the Salmonella transsuc-cinylase by its product, O-succinylhomoserine.We have found no evidence that the activity ofthis enzyme, or of the other two, is affected byfolate derivatives (Table 1). It also appearsthat CH3FH, plays no role in regulating thesynthesis of the three enzymes since repressionby methionine was the same in both metEand metF mutants (Table 2). Thus, the coor-dination of the converging pathways must beaccomplished in some other way.
Repression of enzyme synthesis. Repres-sion of the synthesis of the three enzymes isnoncoordinate, with much more facile repres-sion of transsuccinylase than of cystathioniney-synthase and somewhat less marked repres-sibility of ,B-cystathionase than of the latter.Stringent repression of the transsuccinylase isillustrated by the fact that no activity of thisenzyme could be shown in extracts of anymethionine auxotroph grown in flask cultureon methionine, even at growth-limiting con-centrations (Table 2). The mere absence of thisenzyme from metA extracts is accordingly noproof that this gene codes its structure. Thereseems little doubt from other types of evidence(25, 26) that it does.Evidence for common elements in repres-
sion of all three enzymes. The strongest indi-cation of elements common to the repressionmechanism of all three enzymes is that all theenzymes are fully derepressed in the analogue-resistant mutants, metJ713 and metK721(Table 4), and none is derepressed in themethionyl-tRNA synthetase mutant, metG419.An E. coli mutant tentatively designatedmetK has been shown to have a defectiveSAM synthetase, suggesting that SAM orsomething derived from it is a corepressor forall three enzymes. There is some uncertaintyabout this conclusion because the SalmonellametK mutant that we studied appears to havea normal SAM synthetase (Table 5). Thisproblem may be clarified if it can be deter-mined whether the latter mutant is blocked inthe conversion of SAM to another productwhich is the actual corepressor.The selective disappearance of transsucci-
nylase activity from wild-type Salmonella inresting phase may also reflect hypersensitivityto a common mechanism, particularly since itdoes not occur in either metJ or metK mu-tants (Table 6). Differing sensitivities to acommon repression mechanism could also ex-plain the noncoordinate pattern of repressionof the three enzymes. The high levels of cysta-thionine y-synthase and ,8-cystathionase inmetA mutants (Table 2) may be another mani-
festation of a common control mechanism.This finding is reminiscent of proposals madefor the histidine (15, 16) and arginine (21)pathways, that the first enzyme of the pathwaymight participate directly in the repressorcomplex.Evidence for elements in repression not
shared by all three enzymes. The possibilitythat regulation of transsuccinylase synthesismight involve elements not shared with theother two enzymes was raised by chemostatexperiments with metE47 (Fig. 2 and 3). Theseexperiments at first suggested that exogenousmethionine might selectively promote the re-pression of the transsuccinylase since it wasvirtually impossible to derepress this enzymeby limiting intracellular methionine derivedfrom the medium (Fig. 2), whereas it could bederepressed by limiting vitamin B,2 (Fig. 3).However, when metB and metC mutants weregrown in a methionine chemostat, transsucci-nylase was easily derepressed (Table 3). Theseresults are limited since it was not possible tocultivate metB and metC mutants in a chemo-stat limiting endogenous methionine synthesiswith methionine precursors as nutrients. Butthey do suggest that the superrepression oftranssuccinylase in the experiments of Fig. 2 isa function of the metE phenotype rather thanof a difference in effectiveness of methioninederived from exogenous and endogenoussources.At the two shortest generation times used in
the vitamin B12 chemostat (Fig. 3), no trans-succinylase activity could be detected at all,which seems curious since all of the enzymesof methionine biosynthesis are required forgrowth to occur under these conditions. Inaddition, methylenetetrahydrofolate reductaseis almost completely repressed by vitamin B2(22); some investigators believe this finding isnot unreasonable (3).
In the vitamin Bl2 chemostat (Fig. 3), thedirect effect of shortening the generation timeis to reduce the intracellular concentration of aholoenzyme (the B 2-transmethylase) ratherthan of methionine or a methionine precursor.We assumed that any effects on the synthesisof the homocysteine biosynthetic enzymeswould result from the consequent decrease inthe intracellular concentration of "endoge-nously formed" methionine, but this may notbe so. It has recently been proposed that thefunctional holoenzyme of the B12-transmethyl-ase is itself a component of the repressionapparatus for methylenetetrahydrofolate re-ductase and the non-B l2-transmethylase (seeFig. 1; reference 3, 22; H. Kung, C. Spears, R.
C. Greene, and H. Weissbach, personal com-munication). One unique characteristic ofmetE mutants grown on methionine is thatthey contain no functional transmethylase atall. The Bl2-transmethylase is presumablypresent (22; Kung et al., personal communica-tion), but only in apoenzyme form.One could speculate that the presence of a
functional transmethylase is somehow neces-sary for induction of the synthesis of transsuc-cinylase, thereby preventing the overproduc-tion of homocysteine when it cannot be meth-ylated. Transmethylase itself might be a struc-tural component of an inductive regulatorycomplex (3, 22; Kung et al., personal commu-nication). A more effective mechanism for co-ordinating the synthesis of homocysteine withthat of the methyl groups would be afforded ifthe transmethylases could methylate a com-pound other than homocysteine, producing aproduct necessary for the induction of trans-succinylase synthesis.
ACKNOWLEDGMENTSWe would like to thank Herbert Tabor of the National
Institute of Arthritis and Metabolic Diseases for his helpfuldiscussions of the chemostat data.
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