regulation glycolysis phosphotransferase activities ... · regulation ofglycolysis and...

Vol. 154, No. 2JOURNAL OF BACTERIOLOGY, May 1983, p. 819-8300021-9193/83/050819-12$02.00/0Copyright © 1983, American Society for Microbiology

Regulation of Glycolysis and Sugar PhosphotransferaseActivities in Streptococcus lactis: Growth in the Presence of

2-Deoxy-D-GlucoseJOHN THOMPSON* AND BRUCE M. CHASSY

Microbiology Section, Laboratory of Microbiology and Immunology, National Institute of Dental Research,Bethesda, Maryland, 20205

Received 10 December 1982/Accepted 8 February 1983

Streptococcus lactis Kl has the capacity to grow on many sugars, includingsucrose and lactose, in the presence of high levels (>500 mM) of 2-deoxy-D-glucose. Initially, growth of the organism was transiently halted by the addition ofcomparatively low concentrations (<0.5 mM) of the glucose analog to the culture.Inhibition was coincident with (i) rapid accumulation of 2-deoxy-D-glucose 6-phosphate (ca. 120 mM) and preferential utilization of phosphoenolpyruvate viathe mannose:phosphotransferase system, (ii) depletion of phosphorylated glyco-lytic intermediates, and (iii) a 60% reduction in intracellular ATP concentration.During the 5- to 10-min period of bacteriostasis the intracellular concentration of2-deoxy-D-glucose 6-phosphate rapidly declined, and the concentrations of glyco-lytic intermediates were restored to near-normal levels. When growth resumed,the cell doubling time (Td) and the steady-state levels of 2-deoxy-D-glucose 6-phosphate maintained by the cells were dependent upon the medium concentra-tion of 2-deoxy-D-glucose. Resistance of S. lactis Kl to the potentially toxic analogwas a consequence of negative regulation of the mannose:phosphotransferasesystem by two independent mechanisms. The first, short-term response occurredimmediately after the initial "overshoot" accumulation of 2-deoxy-D-glucose 6-phosphate, and this mechanism reduced the activity (fine control) of the mannose:phosphotransferase system. The second, long-term mechanism resulted in repres-sion of synthesis (coarse control) of enzyme 11mannose. The two regulatorymechanisms reduced the rate of 2-deoxy-D-glucose translocation via the mannose:phosphotransferase system and minimized the activity of the phosphoenolpyr-uvate-dependent futile cycle of the glucose analog (J. Thompson and B. M.Chassy, J. Bacteriol. 151:1454-1465, 1982). Phosphoenolpyruvate was thusconserved for transport of the growth sugar and for generation of ATP requiredfor biosynthetic and work functions of the growing cell.

In a recent study (23), we showed that growthof Streptococcus lactis 133 on sucrose or lactosewas inhibited by relatively low concentrationsof 2-deoxy-D-glucose (2DG). In S. lactis allthree sugars are translocated simultaneouslywith phosphorylation by the multi-component,phosphoenolpyruvate (PEP)-dependent phos-photransferase system (PTS) (for reviews, seereferences 2, 4, and 11). We suggested thatbacteriostasis was a consequence of futile recy-cling of the glucose analog by the followingsequence of vectorial reactions:

2DGoUt + PEPi mannosePTS

2DG-6Pin + Pyruvatein (1)

2DG-6P1n + H20 hexose 6P:phosphatase

2DGin + Pii. (2)

Ellman or permease2DGin +

Net: PEPin + H20-2DGout (3)

Pyruvatein + Pii. (4)

where 2DG-6P is 2-deoxy-D-glucose 6 phosphateand EIIman is the enzyme II component of themannose:PTS.The futile cycle caused the dissipation of PEP

and the generation of ATP via pyruvate kinase(ATP:pyruvate 2-0-phosphotransferase, EC2.7.1.40) was reduced to a rate incompatiblewith the energy requirements for normal growth.When a genetic lesion was introduced into theenzyme II component of the mannose:PTS,stage 1 of the cycle was eliminated, and themutant strain (S. lactis 133 man A) becameresistant to the glucose analog.

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820 THOMPSON AND CHASSY

In a continuation of this work, we found thatsome wild-type strains were resistant to highlevels of2DG even though such cells contained afunctional mannose:PTS. For example, growthof S. lactis Kl in medium containing 10 mMsucrose was not significantly inhibited by >500mM 2DG. In this communication we describethe resistance phenomenon and suggest mechan-ism(s) for bacterial immunity to the potentiallytoxic analog. Our data provide additional insightinto in vivo regulation of glycolysis and su-gar:PTS activities in growing cells of S. lactis.

MATERIALS AND METHODSOrganism. S. lactis Kl was obtained from the cul-

ture collection of the New Zealand Dairy ResearchInstitute, Palmerston North, New Zealand.

Culture nnanve and growth studies. Cultureswere maintained, and the organism was grown incomplex medium containing 28 mM hexose or 14 mMdisaccharide as described previously (24). Growthexperiments were performed with 3-mi volumes ofculture in sterile, screw-top cuvettes, and growth ofcells (absorbance at 600 nm) was followed by using aGilford 300N microspectrophotometer adapted to ac-cept the growth cuvettes (23). Accumulation of[14C]2DG by growing cells was monitored as describedby Thompson and Chassy (23).

Determination of mannose:PTS activity in vivo. Cellsof S. lactis Ki were grown, harvested, and washed aspreviously described (21). The starved cells, whichcontain an endogenous reserve of PEP (10, 25), weresuspended at a concentration of 200 ,ug (dry weight) ofcells per ml in 50 mM potassium phosphate buffer (pH7) containing 1 mM MgCI2 (KPM buffer) and 5 mMsucrose. After 10 min of incubation at 30°C, 0.2 mM[14C]2DG (specific activity, 0.2 pCi/p.mol) was addedto the cell suspension, 1.0-ml samples were withdrawnat intervals of 5, 10, 15, 20, and 30 s, and cells werecollected by membrane filtration. The initial rates of2DG-6P accumulation were derived from tangentsdrawn to each progress plot. The concentrations ofintracellular 2DG-6P were calculated on the basis that1 g (dry weight) of S. lactis Kl cells was equivalent to1.67 ml of intracellular (protoplast) fluid (25).

Identifcation of 14C-labeled glycolytic intermediates.The effects of 2DG accumulation upon the concentra-tions of intracellular glycolytic intermediates weredetermined by modification of a previously describedprocedure (23). Cells of S. lactis Kl were grown incomplex medium containing 10 mM [U-14C]sucrose(specific activity, 0.2 p.Ci/Ipmol) and the appropriateconcentrations of 2DG. In the exponential phase ofgrowth (absorbance at 600 nm, 0.5 or 0.75; Gilford 300N spectrophotometer), 30 ml of culture containing 4.7or 7 mg total dry weight of cells was rapidly filteredonto a 47-mm-diameter membrane filter (MilliporeCorp.; type HA, 0.22-p.m pore size). The filter with thecells was immediately transferred to 5 ml of boilingwater for 5 min, and intrac0llular metabolites wereextracted. The cell extract was clarified by centrifuga-tion at 13,000 x g for 30 min at 4°C. The supernatantfluids were removed, lyophilized, and reconstitutedwith 50 p,l of distilled water. Radiolabeled glycolyticintermediates present in 5-,ul volumes of the cell

J. BACTERIOL.

extracts (ca. 80,000 to 100,000 cpm) were separatedand identified by polyethyleneimine-cellulose thin-lay-er chromatography (21, 23). Intracellular concentra-tions of glycolytic intermediates were calculated fromthe radioactivity of each metabolite as described previ-ously (23).Enzyme assays. Cells of S. lactis Kl were permeabi-

lized with a mixture of toluene-acetone, and PTSactivities were determined by the in vitro spectropho-tometric method of St. Martin and Wittenberger (19).ATP was determined by using firefly luciferase-lucifer-in according to the procedure described in the lumines-cence reagent kit obtained from Packard InstrumentCo. Determinations were made with an Aminco Chem-Glow (model J4-7441) photometer and an integratortimer (model J4-7462-A). Samples of reconstituted cellextracts were diluted 100-fold, and 10 ,ul of dilutedsolution was used for ATP analysis. The final reactionvolume was 400 ,ul, and calibration curves of 100 to800 pmol of ATP were prepared from triplicate stan-dards. Protein was determined by the Coomassiebrilliant blue dye-binding assay of Bradford (1) withequine gamma globulin as the protein standard.

In vitro PTS complementation assay. Cells of S.lactis Kl were grown in 3.2 liters of complex mediumcontaining 14 mM sucrose, or 14 mM sucrose and 10mM 2DG (22). The cells were harvested, washed, anddisrupted in a Bead-Beater (Biospec Products, Bar-tlesville, Okla.) as described previously (22). Celldebris and residual whole cells were removed bycentrifugation at 42,000 x g for 30 min, and thesupernatant fluid was further centrifuged for 120 minat 220,000 x g. The high-speed supernatant fluid wasconcentrated to 20 ml in an Amicon pressure ultrafil-tration cell (UM-05 membrane). The pellet obtained byhigh-speed centrifugation was suspended in 10 ml 0.1M HEPES (N-2-hydroxyethylpiperazine-N-2'-ethane-sulfonic acid) buffer (pH 7.5) containing 1 mM dith-iothreitol. The formation of ["4C]2DG-6P was followedby its binding to DEAE-cellulose (DE-81) filter circlesby the method of Sherman (18). The PTS assaycontained 100 mM HEPES buffer (pH 7.5), 1 mMdithiothreitol, 5 mM PEP, 10 mM MgCl2, 10 mM NaF,5 mM [14C]2DG (specific activity, 0.2 AlCi/4umol), 0 to25 1l of high-speed centrifugation supernatant, and 0to 50 ,ul of high-speed pellet membranes in a finalreaction volume of 100 ,ul. Incubation was at 37C for20 min. Samples (30 to 50 ,ul) of the reaction mixturewere transferred to DE-81 filter circles, washed free ofunreacted [14C]2DG, and air dried. [14C]2DG-6P re-tained on the filter was determined by liquid scintilla-tion counting.

Reagents. Chemicals and reagents were purchasedfrom the Sigma Chemical Co., St. Louis, Mo. Radiola-beled compounds, [1-14C]2DG and [1-14C]2DG-6P (di-sodium salt), were obtained from New England Nucle-ar Corp., Boston, Mass. Luminescence reagents(Picozyme Kits, I, II, III, IV) were purchased fromPackard Instrument Co., Inc., Downers Grove, Ill.

RESULTSEffect of 2DG on growth of S. lactis Kl. The

doubling time (Td) of S. lactis Kl in complexmedium containing 14 mM sucrose was approxi-mately 44 min (Fig. 1.). When 2DG was added toa series of actively growing cultures at concen-


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SUGAR TRANSPORT AND METABOLISM IN S. LACTIS

0.8-C ~~~~~~~~~2

0.6 -

0.C nm h) II

.~0.4

0.2-

012 3 4 50.1 lifme (hr) I

0 1 2 3 4 5

Time (hr)FIG. 1. Effect of increasing concentrations of 2DG upon growth of S. lactis Kl in complex medium

containing 14 mM sucrose as the energy source. At the midlog phase of growth (absorbance at 600 nm, 0.5), 2DGwas added to the cultures to obtain the following (millimolar) concentrations: (0) control (no 2DG): (0) 0.05; (U)0.5; (A) 1; (O) 10. Inset, Accumulation and regulation of intracellular [14C]2DG-6P by cells of S. lactis Kl.[14C12DG (0.5 mM; specific activity, 0.2 ,uCi/,Lmol) was added to the culture, and uptake of [14C]2DG-6P duringthe initial overshot, transient inhibition, and resumption ofgrowth was followed as described in the text. Samplesfrom a duplicate system containing ["4C]sucrose were removed at times indicated by arrows 1, 2, and 3, andextracts were analyzed for "C-labeled glycolytic intermediates as described in the legend to Fig. 2.

trations of >0.05 mM, an immediate, but tran-sient, inhibition of growth occurred. After ashort period of bacteriostasis (ca. 5 to 10 min),growth was resumed at a rate dependent uponthe concentration of the analog in the medium(e.g., Td at 10 mM 2DG was approximately 65min).Accumulation of 2DG and identification of in-

tracellular products. Previously we showed thatinhibition of growth of S. lactis 133 occurredsimultaneously with the accumulation and main-tenance of 2DG-6P to an intracellular concentra-tion of >120 mM (23). It was important, there-fore, to determine whether the analog was alsoaccumulated by the 2DG-insensitive strain. Theaddition of 0.5 mM [14C]2DG to a culture of S.lactis Kl caused an immediate inhibition ofgrowth, coincident with accumulation of[14C]2DG-6P to ca. 130 mM (Fig. 1, inset).Approximately 95% of the radioactive materialextracted from the cells cochromatographedwith authentic 2DG-6P, and the remainder was[14C]2DG. Within 2 to 3 min of attainment ofmaximum 2DG-6P uptake, the intracellular con-centration of the derivative rapidly declined, andgrowth was resumed. After 25 min the 2DG-6P

concentration had decreased by approximately70% and throughout subsequent growth wasmaintained at ca. 25 mM. The major intracellularderivative was [14C]2DG-6P, but additional prod-ucts (-5% of the total radioactive material) weredetected by paper chromatography. One deriva-tive was tentatively identified as 6-phospho-2-deoxygluconate, and a second compound re-mained at the point of origin, but was notidentified. The steady-state concentration of2DG-6P (ca. 25 mM) maintained by S. lactis Klduring growth (Fig. 1, inset) was four- to fivefoldlower than that maintained by S. lactis 133during bacteriostasis (23).2DG-6P accumulation and depletion of glyco-

lytic intermediates. The experiment shown inFig. 2 was conducted to study the effect of 2DGuptake upon the concentrations of intracellularglycolytic intermediates. Cells of S. lactis Klwere grown (Fig. 1.) in medium containing[U-14C]sucrose, and extracts were preparedfrom cells sampled before (Fig. 1, arrow 1) andat intervals of 3 and 30 min after the addition of2DG to the culture (Fig. 1, arrows 2 and 3,respectively). Radiolabeled glycolytic interme-diates in the three extracts were quantitatively

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S.

lactate

G6Pj

F6P

DHAP

2-PG

3 i-PI

PEP

A-.-

Qriqin

FIG. 2. Intracellula14C-labeled glycolytictis Kl before (laneA1)(lane 3) after the ad(culture. The cells wer[U-14C]sucrose, andindicated by arrows 1,Atpi-thtriA uwfzrp enerrn

crose, and after 10 min, when steady-state gly-colysis had been attained, [14C]2DG was addedto the suspension. Within 15 min the resting cellsaccumulated [14C]2DG-6P to ca. 100 mM (Fig.3), but in contrast to growing organisms this high

5.1 0.5 525 steady-state level of sugar phosphate was main-51 ' tained for the duration of the experiment. When

this experiment was conducted with resting cells5 1 0.0 3A grown previously in the presence of 2DG, the

concentration of 2DG-6P maintained at thesteady state was only 30% of that maintained bycells grown in the absence of the analog (Fig. 3).

1.6 0 4 1 X In neither case did the addition of the complexmedium components elicit a reduction in intra-cellular 2DG-6P concentration (data not shown).However, accumulation and rapid expulsion of2DG(-6P) was found in cells whose growth hadbeen terminated by chloramphenicol (Fig. 4).This result showed that the short-term capacity

20.0 0.9 of S. lactis Kl to regulate the intracellularconcentration of the glucose analog was inde-pendent of de novo protein synthesis.

Short-term regulation of intracellular 2DG-6Pby growing cells. The resumption of growth afterexposure of the cells to 2DG (Fig. 1) resulted

r (millimolar) concentrations of from a regulatory mechanism which reduced theintermediates present in S. lac- steady-state concentration of 2DG-6P. Thisand 3 min (lane 2) and 30 min short-term response could have been achieveddition of 0.5 mM 2DG to the by either (A) an increased rate of dephosphory-re grown in medium containing lation and expulsion of 2DG or (B) a reduction incells were collected at times the rate of 2DG-6P accumulation by the man-2, and 3 in Fig. 1. Intermediates nose:PTS. Possibility (A) seemed unlikely fromeP fnazht (QAM;P eohirntwu;Wsv;Wsu we;rc;. SUV;rose o-pnuspnate koor), glucose

6-phosphate (G6P), fructose 6-phosphate (F6P), dihy-droxyacetone phosphate (DHAP), 2- and 3-phospho-glyceric acids (2-PG and 3-PG), PEP, and fructose 1,6-diphosphate (FDP).

identified by thin-layer fluorography (Fig. 2).The control cells contained ca. 20 mM fructose1,6-diphosphate, 10mM of a mixture ofPEP and2- and 3-phosphoglyceric acids, and lower con-centrations of hexose 6-phosphate and sucrose6-phosphate (Fig. 2, lane 1). Within 3 min after2DG addition (i.e., at the point of growth inhibi-tion and maximum uptake of 2DG-6P) the cellswere essentially depleted of glycolytic interme-diates (Fig. 2, lane 2). When growth resumed(Fig. 1, arrow 3) the concentrations of themetabolites were restored and began to ap-proach those present in control cells (Fig. 2, lane3). At 3 min after the addition of 2DG to theculture, the intracellular ATP concentration hadfallen from 4.4 to 1.8 mM (a decrease of approxi-mately 60%), and when growth resumed theATP concentration (1.4 mM) was still considera-bly lower than the concentration in control cells.

Accumulation of 2DG by resting and chloram-phenicol-treated cells. Cells of S. lactis K) weresuspended in buffered medium containing su-

zc-

100

80 -

60 -

40

20

OfI0- -1 1

0 5 10 15 20 25 30TIME (minutes)

FIG. 3. Accumulation of intracellular [4C]2DG-6Pby resting cells of S. lactis Kl grown previously in thepresence (0) or absence (-) of2DG. Cells were grownin complex medium containing 14 mM sucrose and,when necessary, 10 mM 2DG. The cells were collectedand suspended at a concentration of 200 ,uig (dryweight) per ml in 0.1 M KPM buffer (pH 7.0) contain-ing 5 mM sucrose. After 10 min of incubation, 0.2 mM[14C]2DG (specific activity, 0.2 ,uCiVI.mol) was addedto each suspension, and accumulation of ['4C]2DG-6Pby the cells was followed as described in the text.

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SUGAR TRANSPORT AND METABOLISM IN S. LACTIS

EO0.8 -

c

~0.6-

0~~~~~~~~~~~~~~~8z

04 0.4-7

0E

FIG. 4. Regulation of 2DG-6P accumulation by chloramphenicol-treated cells of S. lactis Ki. Growth

conditions were as described in Fig. 1. Chloramphenicol (0) was added to the culture (arrow 1) to a final

concentration of 25 ,ug ml-l. When growth had ceased (absorbance at 600 nm, 0.7), 10 mM sucrose was added

(arrow 2) to ensure continued glycolysis, and min later 0.5 mM (14C]2DG (specific activity, 0.2 p.Ci4Lumol) wasadded to the cuvette (arrow 3). Intracellular concentrations of [14C]2DG-6P (inset) were determined by standard

procedures.

an energetic stand point, because this would notreduce the rate of PEP dissipation by the futilecycle. Possible mechanisms for (B) could in-clude: (i) inactivation or reduced synthesis of themannose:PTS, (ii) changes in kinetic parametersof this PTS toward 2DG, (iii) reduced levels ofintracellular PEP or the phosphorylated histi-dine-containing phosphocarrier protein of thePTS (HPr - P), or (iv) product inhibition ofmannose:PTS activity by intracellular 2DG-6P.The four possibilities were considered in thefollowing experiments.

(i) Activity of the mannose:PTS during growthin the presence of 2DG. The data presented inTable 1 show that mannose:PTS activity re-mained constant (throughout at least four dou-blings of cell mass) after addition of 2DG to themedium. These results discounted the first pos-sibility.

(ii) Kinetic parameters of the mannose:PTS.The effect of 2DG upon the kinetic characteris-tics of the mannose:PTS in vivo was studiedduring growth of the organism in medium con-taining increasing concentrations of [14C]2DG.The data (Fig. SA) showed that after transient

growth inhibition, the intracellular [14C]2DG-6Pconcentrations and the cell doubling times weredependent upon the concentration of the analogin the medium and, therefore, upon the activity

TABLE 1. Mannose:PTS activity' of cells of S.lactis Kl during exponential growth' in the presence

(or absence) of 2DG

Absorbance (600 nm) Mannose:PTS activityof growing cultures Control (no 2DG) +2DG

0.1 45.3 36.8c0.2 42.2 44.20.4 50.7 43.50.8 52.2 43.51.6 54.8 38.0

aExpressed as micromoles of 2DG-6P accumulatedper gram (dry weight) of cells per minute. Cells wereremoved from the culture at the designated absor-bance, harvested, and washed, and the initial rate of[14C]2DG uptake was determined.

I Cells were grown in complex medium containing14 mM sucrose.

c 2DG (1 mM) was added to the culture immediatelyafter removal of this first sample (at an absorbance at600 nm of 0.1).

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J. BACTERIOL.824 THOMPSON AND CHASSY

A 1

40

E

-J

w _

- 10 -

zr

56 Ew

F

52z

00ax

EXTRACELLULAR [2DG1 mM

-101 mm-,

[2DGJexFIG. 5. (A) Effect of extracellular (medium) concentration of 2DG upon cell doubling time (0) and

intracellular steady-state concentration of 2DG-6P (0) maintained by cells of S. lactis Kl. (B) Double-reciprocalplot of intracellular steady-state 2DG-6P concentration maintained by growing cells versus concentration of2DGin the medium.

of the mannose:PTS and rate of PEP utilization.In the present experiments the half-time (T112) of2DG efflux from growing cells was ca. 5 s, andthe rate constant k for efflux was 8.3 min-(Thompson and Chassy, unpublished data).

The intracellular 2DG-6P concentrations main-tained by S. lactis Kl during exponential growthrepresented a steady-state condition (Fig. 5A) inwhich the rate of accumulation of 2DG-6P viathe mannose:PTS was equal to the rate of de-


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SUGAR TRANSPORT AND METABOLISM IN S. LACTIS 825

phosphorylation and efflux of 2DG. Therefored[2DG-6P],n/dt = 0, and in the steady state (5):

Vrm,c [2DGIex = k [2DG-6P]in (5)[2DGIex + Km'

entry = effluxKXyn is the apparent "affinity" constant of themannose:PTS for 2DG, Vemnx is the maximumrate of uptake of the analog, and k is the first-order rate constant for dephosphorylation and2DG exit. [2DG]ex and [2DG-6P]Jn represent theextracellular and intracellular concentrations ofthe free and phosphorylated analog, respective-ly. Rearrangement and transposition of equation(5) yields the linear equation

1 kKm 1 k= x + (6

[2DG-6P]in Vzax [2DG]ex Vmax

The reciprocal plot of the data from Fig. 5A,according to this equation, and the slope

S.F.

Lactate

S6P

G6P JF6P

2-PG3-PGPEP

FP

5.4

*: :::.:: : : .....

5 O .....: :. ......... .... : .*: .:

..

.:

:...

:.

............

.::.::.:. . . ....... i j .

... i.

: j>_,..-i-11.::4^ ^ ^-| | | | wIU.O sW,,.. Xi ,.. r...

i...

..Origin

2

(kK ,/V,',,) and intercept (kIV,nna,) values areshown in Fig. SB. V,,,n (600 ,umol of 2DG-6Paccumulated per g [dry weight] of cells per min)was determined by substitution for k = 8.3min1 in the intercept formula. A second substi-tution for VenaX into the slope formula yields avalue for Kn of 0.13 mM. These kinetic parame-ters determined from steady-state data in grow-ing cells are in reasonable agreement with previ-ous data (Ken, 0.08 mM; and VenaX, 360 pmol of2DG-6P accumulated per g [dry weight] of cellsper min) determined from initial rate studies of2DG uptake by PEP-preloaded, starved cells(21). The kinetic parameters of the mannose:PTS toward 2DG were not changed significantlyduring growth of S. lactis Kl in the presence of2DG.

(iii) Possibility of regulation of 2DG-6P uptakeby PEP limitation. The possibility of regulationof 2DG-6P uptake by PEP limitation was investi-gated in the experiment described in Fig. 6. Cells

4.8

... ...... .....

4.8 *: . .... : : :: : .:::: ::

*: :... : !.: '::: :: .:.. . :.: . . : . ...

:: ..:....j... ..: ... :: ::.:

.. ::*:: . ' j .::

.:: ..... .:. :,.,.'..

9.3 _

:....

183 _ |,, ,!.

*:, .X ..:'@! .:. .: :.:8 ,, .:.: :'.:.

::: : ... :* ' i, '.'#2oi.E *oGb s

3

6.1* .. O j ........... ..... ............... .

;. .....

4.8 o 4.8. , j . .* .. . . ., . ... ; : ....... .. ........

... ..

w t 0...........: .< ^^ ;?:: ::. g :

* iv s<i iotvf'.:^h::,.: .. .... g.'wy_ ..e:: ::!v.:.

.:.jR §

*C.,. ::..... _ M...:: _s_ Fg_s Z _ _

fiS N -Q:: .. ....::

_̂.':12_ _. j

17.0 |-1 11;.3.F ,*.?.*i.. i... ^*.

.i.i R'__L-_i_

4

6.8

12.8

5

FIG. 6. Identification and intracellular (millimolar) concentrations of "C-labeled glycolytic intermediatespresent in growing cells of S. lactis Kl. Cells were grown in complex medium containing ["4C]sucrose andincreasing (micromolar) concentrations of 2DG: 0, control lane 1; 25, lane 2; 75, lane 3; 150, lane 4; and 500, lane5.

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10F

j8

4

0

.

6

5

4 f

3I

0 0.1 0.2 0.3 0A 0.5

Ex~aceIu [2DGI mMFIG. 7. Relationship between the medium concentration of 2DG, and the intracellular concentrations of (0)

FDP and (0) PEP plus 2- and 3-phosphoglyceric acids maintained by growing cells of S. lactis Kl. The data forthis figure and the ratios of the concentrations (0) were obtained from Fig. 6.

of S. lactis Kl were grown (Fig. 5A) in mediacontaining [14C]sucrose and increasing concen-trations of 2DG. Glycolytic intermediates pre-sent in the growing cells were determined afterextraction and thin-layer fluorography (Fig. 6).The intracellular sucrose 6-phosphate andhexose 6-phosphate levels remained generallyconstant, but the concentrations of fructose 1,6-diphosphate, 3-phosphoglycerate, 2-phospho-glycerate, and PEP decreased with increasing2DG level in the medium. Since the glycolyticreactions from fructose 1,6-diphosphate to PEPare reversible and close to equilibrium, a reduc-tion in PEP concentration would be readilytransnmitted to the first metabolite in the se-quence (fructose 1,6-diphosphate) so that: (i)intracellular fructose 1,6-diphosphate, 3-phos-phoglycerate, 2-phosphoglycerate, and PEPconcentrations would decrease simultaneously,and (ii) the ratio of the concentrations of thesemetabolites would remain constant (Fig. 7). Thedata in Fig. 6 and 7 support both predictions.The intracellular ATP levels in control cells andin cells growing in the presence of 25, 75, 150,and 500 ,uM 2DG were 4.7, 3.3, 4.4, 4.6, and 4.8mM, respectively. The cells metabolized su-crose at near-normal rates and maintained anintracellular PEP level 60 to 65% of that found incontrol cells. It is unlikely that decreased uptakeof 2DG-6P (after the initial accumulation; Fig. 1)can be attributed to limitation of intracellularPEP (Fig. 2, lane 3). When cells were grown in

the presence of 2DG, fermentation of sucrosebecame increasingly heterolactic and, besideslactic acid, ethanol and acetate were detected inthe medium (Thompson and Chassy, unpub-lished data).

(iv) Effect of 2DG-6P on mannose:PIS activity.In vitro complementation studies with cell ex-tracts (Table 2) showed that 25 mM 2DG-6P hadlittle inhibitory effect upon mannose:PTS activi-ty. Furthermore, extracts prepared from cellsgrown in the presence of 2DG did not containany factor which alone or in combination with2DG-6P was able to inhibit the mannose:PTS invitro.Long-term repression of mannose:PTS synthe-

sis. In the short term, the addition of 2DG to agrowing culture of S. lactis Ki produced nosignificant repression of the mannose:PTS (Ta-ble 1). However, growth for several generationsin medium containing 10 mM 2DG did result inrepression of mannose:PTS synthesis, and boththe initial rate and the maximum level of 2DG-6Paccumulated by the cells were approximately70%6 lower than those observed in control cells.(Fig. 3). This result was confirmed by in vitroassay of PTS activities in permeabilized cells(Table 3), where it was found that PEP-depen-dent phosphorylation of mannose:PTS sub-strates (glucose, mannose, 2DG, glucosamine,and N-acetylglucosamine) was reduced by 80%o.In vitro complementation studies showed thatthe reduced mannose:PTS activity was due to

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SUGAR TRANSPORT AND METABOLISM IN S. LACTIS 827

TABLE 2. Effect of 2DG-6Pz upon the activity of the mannose:PTS in vitro

HSS HSP Sp actcHSSb sample Source of sample

vol (1J) HSPd vol (,u) No 2DG-6P +2DG-6P

S. lactis Kl 40 S. lactis Kl 10 1.43 1.49S. lactis Kl 40 S. lactis Kl 10 0.75 0.60(2DG)e (2DG)e

S. lactis Kl 20S. lactis Kl 10 1.60 1.28S. lactis Ki 20

(2DG)ea 2DG-6P was present in the assay at a final concentration of 25 mM.b HSS, High-speed supernatant fluid.c Expressed as nanomoles of [14C]2DG-6P formed per milligram of high-speed supernatant fluid protein per

minute. The assays contained ca. 28 ,ug of high-speed pellet protein, and other conditions were as described inthe text.

d HSP, High-speed (membrane) pellet.e Designates preparations derived from S. lactis Kl grown in the presence of 2DG.

decreased levels of the membrane-associatedEIIman (data not shown).

DISCUSSIONWhen sucrose is the sole PTS sugar pres-

ent during growth of S. lactis Kl, intracellularPEP is consumed by the sucrose:PTS and viaATP:pyruvate 2-O-phosphotransferase (pyru-vate kinase) for ATP generation. Throughoutexponential growth the rate of sucrose transportmust be closely linked to and controlled byglycolytic activity (7, 8, 13). In addition, thismetabolic cycle (Fig. 8, route 1) must be regulat-ed so that sucrose is translocated only as fast asit can be metabolized by the growing cell. Onemight expect that the addition to the medium ofa second sugar (2DG}-which is both non-meta-bolizable and a substrate of a high-affinity PTS-would disturb the energetic balance of the grow-ing cell. We attribute the initial inhibitory effectof the glucose analog to competition betweenthe sucrose-EIIsuc and 2DG-EIIman complexesfor the common high-energy phosphoryl donor(HPr-P) (2, 4, 17). When 2DG was added to theculture, the analog was rapidly accumulated (as2DG-6P) via the mannose:PTS with concomitantdepletion of PEP and other glycolytic intermedi-ates. At the same time growth was transientlyhalted. Previously we suggested that growthstasis in S. lactis 133 resulted from the dissipa-tion of intracellular PEP after the establishmentof an energetically wasteful 2DG futile cycle (23)(Fig. 8). It is clear from the data in Fig. 1 andFig. 2 (lane 2) that, upon first exposure of S.lactis Kl to 2DG, intracellular PEP and HPr-Pwere utilized preferentially for translocation of2DG via the mannose:PTS (Fig. 8, route 2). S.lactis Kl had the potential to establish, but didnot sustain, a high level of futile cycle activity.Within minutes of attainment of maximum intra-cellular 2DG-6P (ca. 120 mM), a regulatoryresponse occurred which reduced the activity of

the futile cycle. This response resulted in (i) arapid decrease in intracellular 2DG-6P, (ii) rees-tablishment of normal concentrations of glyco-lytic intermediates, and (iii) resumption ofgrowth at a rate dependent upon the concentra-tion of 2DG in the growth medium (Fig. 5A) andupon the degree of saturation of the man-nose:PTS. The most significant feature of thisshort-term response was that PEP and HPr-Pwere no longer consumed preferentially by themannose:PTS (Fig. 8, route 2), but were insteadutilized primarily for translocation of the growthsugar (Fig. 8, route 1).We have examined several possible mecha-

nisms for this short-term regulatory response,

TABLE 3. PTS activitiesa in permeabilized cells inS. lactis Kl after prolonged growth' in the presence

(or absence) of 2DG

PTS activitycSugar tested Control Cells grown

cells with 2DG

Glucose 161.7 <11.5Mannose 160.6 13.72DG 98.7 22.3Glucosamine 61.9 <11.5N-Acetylglucosamine 52.8 <11.5Sucrose 94.0 86.8Gulosed <11.5 <11.5

a Determined by in vitro spectrophotometric assay(19) with permeabilized cells.

b Cells were serially transferred and finally grownovernight in complex medium containing 14 mM su-crose and, when required, 10 mM 2DG.

c Expressed as micromoles ofNADH oxidized (i.e.,sugar phosphate formed) per gram (dry weight) of cellsper minute. Glucose, mannose, 2DG, glucosamine,and N-acetylglucosamine are transported by the man-nose:PTS in S. lactis (24).

d Gulose (C-3, C-4 epimer of glucose) is not trans-ported by the mannose:PTS, and this assay provided ameasure of the endogenous rate of NADH oxidation.

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FIG. 8. Translocation and accumulation of sucrose and 2DG by S. lactis Kl. Abbreviations: X, unidentified2DG exit system; PK, pyruvate kinase; LDH, lactate dehydrogenase. ElISUc and EII"af are enzyme IIcomponents of the sucrose:PTS, and mannose:PITS, respectively. EL-P and HPr-P are soluble, phosphorylatedderivatives of enzyme I and the histidine-containing phosphocarrier protein of the PTS, respectively. Routes 1and 2 indicate phosphoryl transfer from HPr-P into the sucrose metabolic cycle and 2DG futile cyclerespectively, and + refers to enzyme activation by fructose 1,6-diphosphate.

but a satisfactory explanation is not yet at hand.Product (2DG-6P) inhibition of the man-nose:PTS at the inner surface of the cytoplasmicmembrane was a likely possibility, because inhi-bition of PTS activities by sugar phosphates hasbeen reported in intact cells (4, 11, 14, 15) and inmembrane vesicles (6). However, three observa-tions militate against this attractive proposal.First, 2DG-6P at a concentration of 25 mM didnot inhibit the mannose:PTS in vitro (Table 2).Second, from this mode of inhibition one wouldexpect the same pattern of rapid accumulationand efflux of2DG from resting cells, but this wasnot observed (Fig 3). Finally, 2DG-6P inhibitionof mannose:PTS activity in vivo would precludethe establishment of the PEP-dependent futilecycle in sensitive strains, e.g., S. lactis 133 (23).In the short term neither the synthesis nor thekinetic parameters of the mannose:PTS weresignificantly reduced by addition of 2DG to themedium. Furthermore, intracellular PEP does

not appear to be rate limiting for 2DG transloca-tion, because after a period of transient inhibi-tion, cells growing in the presence of 2DGcontained at least 60 to 65% of the PEP levelspresent in control cells (Fig. 6) and readilytransported the growth sugar via the su-crose:PTS.

It should be noted that the level of 2DG-6Paccumulated by S. lactis Kl is governed by theavailability of HPr-P, and if the short-termregulatory mechanism limits the supply of thishigh-energy phosphoryl donor to the man-nose:PTS, then 2DG-6P accumulation will bereduced accordingly. Evidence for such a regu-latory mechanism has recently been obtained byScholte and Postma (17) in Salmonella typhi-murium. These workers studied competition be-tween the two PNS systems (ElI-A/ElI-B andEllg/0III1c) which mediate glucose transloca-tion in S. typhimurium. They concluded that theflow of phosphoryl groups via EI and HPr,

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SUGAR TRANSPORT A?'iD METABOLISM IN S. LACTIS 829

(rather than PEP concentration per se) imposedthe rate limiting step for in vivo transport ofglucose via the preferred EIlglc/ElIlglc system.We believe that the regulatory response ob-served after the initial "overshoot" of 2DG-6Paccumulation by S. lactis Kl may result fromeither (i) limitation of the availability of HPr-Pfor the mannose:PTS or (ii) reduction of theaffinity of the mannose:PTS for HPr-P. Suchlimitations would reduce the accumulation of2DG-6P, and this could occur without significantchange in the kinetic parameters (Vmax, Kin) ofthe 2DG transport system.

Short-term resistance to 2DG is a conse-quence of fine control of activity of the man-nose:PTS. However, growth of S. lactis Kl forseveral generations in medium containing 2DGresulted in repression of synthesis (coarse con-trol) of EIIma, (Table 3); this also reduced thelevel of 2DG-6P uptake by the cell. The molecu-lar basis for inhibition of EIIm,n synthesis is notknown, but intracellular 2DG-6P may serve asan inhibitor of gene expression. London andHausman (9) have described a similar repressionof ElIxylitol synthesis by intracellular (and non-metabolizable) xylitol 5-phosphate in Lactoba-cillus casei. The net effect of short- and long-term regulation of the mannose:PTS is that bothmechanisms prevent excessive dissipation ofPEP by the 2DG futile cycle (Fig. 8). Energy istherefore conserved for biosynthetic and workfunctions of the growing cell.

In 2DG-sensitive strains, e.g., S. lactis 133,the short-term regulatory response does notoccur (23). However, 2DG-resistant mutants ofthese strains arise with high frequency and aredefective in the ElIman. By contrast, resistancein S. lactis Kl is achieved by modulation ofextant regulatory mechanisms rather than bymutation. Our studies with S. lactis, and previ-ous investigations with oral streptococci (e.g.,Streptococcus salivarius and S. mutans [3, 12,16, 20, 26]) have demonstrated that geneticallysimilar organisms may exhibit wide variation insensitivity to 2DG. The recent discovery of thePEP-dependent futile cycle in S. lactis (23) andthe results of the present study may provide abiochemical basis for 2DG sensitivity and resist-ance in the streptococci.

ADDENDUM IN PROOFAfter submission of this manuscript, Stock et al. (J.

Biol. Chem. 257:14543-14552, 1982) reported repres-sion of EIImn synthesis by 2DG in S. typhimurium.

ACKNOWLEDGMENTS

We thank Charles Wittenberger and Jack London for theirconstructive criticisms of the manuscript. We also thankEdward St. Martin for many helpful suggestions and StanleyRobrish for ATP analyses.

LITERATURE CITED1. Bradford, M. M. 1976. A rapid and sensitive method for

the quantitation of microgram quantities of protein utiliz-ing the principle of protein-dye binding. Anal. Biochem.72:248-254.

2. Dills, S. S., A. Apperson, M. R. Schmidt, and M. H. Saier,Jr. 1980. Carbohydrate transport in bacteria. Microbiol.Rev. 44:385-418.

3. Fitzgerald, R. J., and D. B. Fitzgerald. 1977. Inhibition ofcaries in hamsters by 2-deoxy-D-glucose J. Dent. Res.56:1431.

4. Hays, J. B. 1978. Group translocation transport systems,p. 43-102. In B. P. Rosen (ed.), Bacterial transport.Marcel Dekker, Inc., New York.

5. Hoffee, P., E. Englesberg, and F. Lamy. 1964. The glucosepermease system in bacteria. Biochim. Biophys. Acta79:337-350.

6. Kaback, H. R. 1969. Regulation of sugar transport inisolated bacterial membrane preparations from Escherich-ia coli. Proc. Natl. Acad. Sci. U.S.A. 63:724-731.

7. Kornberg, H. L. 1981. Formation and utilization of PEP inmicrobial carbohydrate transport. Curr. Top. Cell. Regul.18:313-327.

8. Kornberg, H. L., and M. C. Jones-Mortimer. 1977. Thephosphotransferase system as a site of cellular control, p.217-240. In B. A. Haddock and W. A. Hamilton (ed.),Microbial energetics. Symposium of the Society for Gen-eral Microbiology. Cambridge University Press, Cam-bridge.

9. London, J., and S. Hausman. 1982. A study of the xylitol-mediated transient inhibition of ribitol utilization by Lac-tobacillus casei. J. Bacteriol. 150:657-661.

10. Mason, P. W., D. P. Carbone, R. A. Cushman, and A. S.Waggoner. 1981. The importance of inorganic phosphatein regulation of energy metabolism of Streptococcus lac-tis. J. Biol. Chem. 256:1861-1866.

11. Postma, P. W., and S. Roseman. 1976. The bacterialphosphoenolpyruvate:sugar phosphotransferase system.Biochim. Biophys. Acta 457:213-257.

12. Roberts, K. R., and M. L. Hayes. 1980. Effects of 2-deoxy-D-glucose and other sugar analogues on acid productionfrom sugars by human dental plaque bacteria. Scand. J.Dent. Res. 88:201-209.

13. Roseman, S. 1969. The transport of carbohydrates by abacterial phosphotransferase system. J. Gen. Physiol.54:138-184.

14. Safer, M. H., Jr. 1980. Catalytic activities associated withthe enzymes II of the bacterial phosphotransferase sys-tem. J. Supramol. Struct. 14:281-294.

15. Safer, M. H., Jr., and E. G. Moczydlowski. 1978. Theregulation of carbohydrate transport in Escherichia coliand Salmonella typhimurium, p. 103-125. In B. P. Rosen(ed.), Bacterial transport. Marcel Dekker, Inc., NewYork.

16. Schachtele, C. F., and W. S. Leung. 1975. Effect of sugaranalogues on growth, sugar utilization, and acid produc-tion by Streptococcus mutans. J. Dent. Res. 54:433-440.

17. Scholte, B. J., and P. W. Postma. 1981. Competitionbetween two pathways for sugar uptake by the phospho-enolpyruvate-dependent sugar phosphotransferase sys-tem in Salmonella typhimurium. Eur. J. Biochem. 114:51-58.

18. Sherman, J. R. 1963. Rapid enzyme assay techniqueutilizing radioactive substrate, ion-exchange paper, andliquid scintillation counting. Anal. Biochem. 5:548-554.-

19. St. Martin, E. J., and C. L. Wittenberger. 1979. Charac-terization of a phosphoenolpyruvate-dependent sucrosephosphotransferase system in Streptococcus mutans. In-fect. Immun. 24:865-868.

20. Tanzer, J. M., and R. J. Fitzgerald. 1978. Lack of cariesinhibitory property of 2-deoxy-D-glucose in rats. J. Dent.Res. 57:440.

21. Thompson, J. 1978. In vivo regulation of glycolysis andcharacterization of sugar:phosphotransferase systems inStreptococcus lactis. J. Bacteriol. 136:465-476.

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22. Thompson, J., and B. M. Chassy. 1981. Uptake andmetabolism of sucrose by Streptococcus lactis. J. Bacte-riol. 147:543-551.

23. Thompson, J., and B. M. Chasy. 1982. Novel phospho-enolpyruvate-dependent futile cycle in Streptococcus lac-tis: 2-deoxy-i>glucose uncouples energy production fromgrowth. J. Bacteriol. 151:1454-1465.

24. Thompson, J., and M. H. Saler, Jr. 1981. Regulation ofmethyl-.-D-thiogalactopyranoside-phosphate accumu-

lation in Streptococcus lactis by exclusion and expulsionmechanisms. J. Bacteriol. 146:885-894.

25. Thompson, J., and T. D. Thomas. 1977. Phosphoenolpyruvate and 2-phosphoglycerate: endogenous energysource(s) for sugar accumulation by starved cells ofStreptococcus lactis. J. Bacteriol. 130:583-595.

26. Vadeboncoeur, C., D. Mayrand, and L. Trahan. 1982. Acomparative study of enzymes involved in glucose phos-phorylation in oral streptococci. J. Dent. Res. 61:60-65.

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