JOURNAI. OF BACTERIOLOGY, Apr. 1974, p. 250-258 Vol. 118, No. 1Copyright © 1974 American Society for Microbiology Printed in U.S.A.

Glucose Transport in Brucella abortusRICHARD F. REST AND DONALD C. ROBERTSON

Department of Microbiology, The University of Kansas. Lawrence, Kansas 66045

Received for publication 14 January 1974

Brucella abortus British strain 19 transported glucose with an apparent Km of0.16 mM and an apparent Vmax of 250 nmol per min per mg of N. The onlycommon glucose analogue transported was 2-deoxyglucose (2-DOG), with anapparent Ki of 0.73 mM. Alpha- or beta-methyl glucosides and 3-0-methyl-glucose were not transported. Transport was linear for 70 to 90 s, depending onthe concentration of substrate used. 2-Deoxyglucose was transported as the freesugar and was not further metabolized once inside the cell. There was no glucosephosphoenolpyruvate phosphotransferase system (PEP-PTS) present, and therewere no inhibitors present in Brucella cell-free extract that inhibited theEscherichia coli glucose PEP-PTS. N-Ethylmaleimide (NEM) and p-chloromer-curibenzoate (pCMB) completely inhibited transport of glucose and2-DOG. Glutathione, dithiothreitol, and f3-mercaptoethanol reversed the effectsof pCMB but not of NEM. A pH optimum of 7.2 and a temperature optimum of37 to 45 C were observed for both Km and Vmax. The glucose transport systemappeared to be constitutive for glucose transport in cells grown on fructose,galactose, erythritol, or glucose. The electron transfer inhibitors carbonylcyanide, m-chlorophenylhydrazone, NaN3, 2,4-dinitrophenol, and KCN inhib-ited 2-DOG transport to a greater extent than did the metabolic energy inhibitorsNaAsO4, iodoacetate, KF, and 2-heptyl-4-hydroxyquinoline-N-oxide. Dicy-clohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphospha-tases, inhibited transport by 100%.

Intracellular parasites, such as Brucella andMycobacterium tuberculosis, cause chroniclong-term debilitating diseases due to parasiticsurvival and growth within mononuclear leuko-cytes. Most pathogenic microorganisms arekilled after phagocytic ingestion; however,Brucella and M. tuberculosis become depend-ent upon the leukocyte for nutrients and protec-tion. The mechanisms by which parasites adaptto intracellular growth are unknown, but it isobvious that they must be able to utilize themetabolites normally present within the leuko-cyte. Unique transport mechanisms may haveevolved in intracellular parasites that favortheir survival.Most of the work on transport mechanisms

described in recent literature (3, 12, 20) hasdealt with facultative anaerobes. Transport inthese bacteria occurs via the phosphoenolpyru-vate phosphotransferase system (PEP-PTS),referred to as group translocation, and by activetransport systems energized by metabolic (18),oxidative (20), or chemiosmotic (3) energy.Work that has been done with the obligateaerobes Mycobacteria (2, 36) and Pseudomonas

(11, 26) has shown that active transport isinvolved in a number of metabolite transportsystems and that the PEP-PTS does not appearto function in sugar transport.

Brucella strains, too, are obligate aerobes, butthey do not possess a classical glycolytic path-way. Glucose catabolism occurs via the hexosemonophosphate pathway operating in conjunc-tion with the tricarboxylic acid cycle (28). Thus,B. abortus may be a unique model useful instudying the coupling of active transport tometabolism. In this paper, we present datashowing that glucose uptake in B. abortus is anactive process, with no PEP-PTS involved. Thedata suggest that this active transport process iscoupled to more than one form of energy, basedon the use of metabolic, electron transfer, andionophore-type inhibitors.

MATERIALS AND METHODSMaterials. Tryptose, yeast extract, and potato

infusion agar were obtained from Difco Laboratories.Trypticase soy agar was purchased from BBL. Nico-tinic acid, niacinamide, biotin, thiamine, 2, 5-diphenyl-oxazole, N-2-hydroxyethyl-piperazine-N'-2'-ethane-.

250

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

GLUCOSE TRANSPORT IN B. ABORTUS

sulfonic acid (HEPES), triethanolamine hydrochloride(TEA), p-chloromercuribenzoic acid (pCMB), dithio-threitol (DTT), N-ethylmaleimide (NEM), carbonylcyanide, m-chlorophenylhydrazone (CCCP), 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), glutathione,,8-mercaptoethanol (,-ME), glucose-6-phosphate de-hydrogenase (G-6-PDH), adenosine 5'-triphosphate(ATP), and nicotinamide adenine dinucleotide phos-phate (NADP) were obtained from Sigma ChemicalCo. Dimethyl 1-4-bis-(5-phenyloxazolyl) benzene wasfrom Packard Instrument Co. Naphthalene (purified)was obtained from Mallinckrodt Chemical Works.Filters (HAWP 02500) were purchased from the Mil-lipore Corp. Dicyclohexylcarbodiimide (DCCD) wasobtained from Eastman Kodak Co. 2-Deoxyglucose(2-DOG) was obtained from Pfantsiehl. ['H ]Inulin(300 uCi//mol) and D- [U- "C ]glucose (311 uCi/Mmol)were purchased from Amersham/Searle. [1- 4C ]2-deoxyglucose (54.6 ,Ci/hmol) was obtained from NewEngland Nuclear Corp. All other chemicals were ofreagent grade and were purchased from commercialsources.

Bacterial strains and growth conditions. Bru-celia abortus British strain-19 was obtained from B.L. Deyoe, National Animal Research Laboratories,Ames, Iowa.The medium used contained tryptose, 20 g; yeast

extract, 5 g; NaCl, 5 g; K,HPO4, 100 mg;MgSO4 7H,O, 100 mg; FeSO4, 400 mg; MnSO4, 100jg; sodium citrate, 10 Hg; thiamine, 20 ,g; nicotinicacid, 40 gg; pantothenic acid, 20 Mg; and biotin, 0.1/lg, per liter of distilled deionized water. The mediumwas prepared as described by Robertson and McCul-lough (28). All cells were grown on 1% glucose, unlessotherwise indicated, at 37 C on a New Brunswickrotary shaker (300 rpm). Under these conditions, adoubling time of 150 min was obtained.

Cultures were transferred monthly by plating onTrypticase soy agar. After 5 days of incubation at37 C, colonies typical of the smooth phase (34) weretransferred to potato infusion agar slants. The slantswere incubated for 24 h at 37 C and then stored at 4 to6 C.

Cells were harvested in mid-log phase, correspond-ing to an optical density at 620 nm (OD,,20) of 3 to 4 ona Spectronic 20 spectrometer (Bausch and Lomb).Cells were centrifuged at 4,000 x g for 20 min in a Sor-vall RC-2B centrifuge (3 to 5 C), washed once in 0.1 MTEA-NH40H buffer, pH 7.2, and resuspended in 0.1M HEPES-NH40H buffer, pH 7.2, by swirling withglass beads. Bacterial suspensions were kept at 3 to5 C and used within 5 days. The transport capacity(measured as the concentrative ability) of cells heldfor up to 5 days at 3 to 5 C was essentially the same asthat of freshly harvested cells.

Cell water and quantitative determinations. Cellwater, as determined by the procedure of Winkler andWilson (35), was 3 Mlliters of intracellular water permg (dry weight) of cells, the dry weight of the cellsbeing 18 to 19% of the wet weight. Nitrogen contentwas determined by the micro-Kjeldahl method (15).Protein was determined by a modified microbiuretprocedure (4), using a standard curve prepared with

bovine serum albumin.A B. abortus suspension (1 ml), with an OD,,,0 of 20,

contained the equivalent of 7 mg of dry weight, 1 mgof nitrogen, and 21 Mliters of intracellular water.Measurement of substrate transport. Two

methods were used to measure substrate transport:the aliquot method and the batch method. Bothmethods give similar results and were used inter-changeably, depending on the nature of the experi-ment.The aliquot method used was that of Kaback (17),

with the following modifications. The reaction mix-ture, in a 13- by 100-mm tube, contained 25 ,uliters ofB. abortus (OD6,0 of 10, equivalent to 0.25 Mliters ofintracellular water), 10 Mliters of ["C ]2-DOG (1MCi/Mmol), 10 uliters each of any added components,and 0.1 M HEPES-NH4OH buffer, pH 7.2, to 100Aliters. HOQNO, DCCD or CCCP was dissolved indimethyl sulfoxide (DMSO) and added as 100-foldconcentrated solutions. Cells were equilibrated for 10min with stirring at 37 C before the addition oflabeled substrate, except where indicated.

Reactions were started by the addition of labeledsubstrate. Reactions were stopped by diluting 50-foldwith 0.1 M TEA-NH4OH buffer, pH 7.2, at roomtemperature, and were immediately filtered. Thefilter was then rinsed once with 5 ml of the samebuffer. Varying the temperature of the wash buffer (0to 37 C) made no difference in the data obtained.

Immediately after each aliquot was filtered, thefilter membrane was dissolved in 10 ml of scintillationfluid (8). Samples were counted on a Nuclear ChicagoIsocap 300 or on a Tri-Carb liquid scintillation spec-trometer (model 3375; Packard Instrument Co., Inc.)with efficiencies of 85 and 75%, respectively.The batch method consisted of removing 0.1-ml

aliquots from a reaction mixture with a 1-ml Vari-pet,automatic pipetter (Manostat Corp.), dispensing thealiquots onto the center of 0.45-Am filters, and wash-ing the filters twice with a 5-ml amount of 0.1 MTEA-NH40H, pH 7.2, at room temperature. Thevacuum remained on throughout the experiment. Thereaction mixture consisted of the same concentrationsand components as did the reaction mixtures of thealiquot method, except 6 to 10 times the volume wasused.Measurement of substrate efflux. Efflux experi-

ments were performed in 25-ml Erlenmeyer flasks at37 C in a gyratory water bath shaker (New BrunswickScientific Co., model G76). A suspension of B. abortus(0.25 ml; 0D,,0 of 10) was incubated with 1 mM["4C]2-DOG (1 MCi/Mmol) and at 10 min was dilutedwith 25 ml of 0.1 M HEPES-NH4OH (pH 7.2, 37 C)plus any additions. Samples of 2.5 ml were removed attimed intervals by using a Cornwall automatic pipet-ter (Becton, Dickinson and Co.) and were filtered asdescribed in the transport experiments above.Measurement of phosphorylation of glucose or

2-deoxyglucose. To identify transported and accu-mulated materials, a 1-ml reaction containing B.abortus (equivalent to 21 uliters of intracellularwater) in 0.1 M HEPES-NH4OH, pH 7.2, was incu-bated with 1 mM [14C 12-DOG (1 MCi/Amol) for 3 or 15

251VOL. 118, 1974

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

REST AND ROBERTSON

min and then centrifuged at 4,000 x g for 20 min. Thesupernatant was removed and the tube walls werewiped dry. The pellet was resuspended in 5 ml of 50%ethanol, boiled for 5 min, and centrifuged again. Thisprocedure extracted 98% of the total radioactivityfrom the cell pellet. The ethanol extract was concen-trated under vacuum and spotted on Whatman no. 1paper for descending chromatography. The originalsupernatant was also spotted. The solvent systemcontained 1-propanol-ethylacetate-water-25% am-monia (6:1:3: 1, vol/vol/vol/vol). The chromatographwas developed for 14 h and dried, and a vertical stripcontaining the ethanol extract was cut out. The stripwas scanned on a Packard Radiochromatogram scan-ner, model 7200. The remainder of the sheet wassprayed for reducing sugars (7) or for sugar phos-phates (5). Standards of 2-deoxyglucose-6-phosphate(2-DOG-P) and 2-deoxygluconic acid (2-DOG-COOH) were prepared by the methods of Eagon (11)and Moore (22), respectively.

Cell-free extracts of B. abortus were used to checkfor the presence of a PEP-PTS. Cells were broken in aBronwill MSK cell homogenizer by the method ofRobertson and McCullough (25), with the followingmodifications. B. abortus (18.5 ml; OD,20 of 40), 0.5ml of niacinamide (2 mg/ml), and 1 ml of DTT (2 x10' M) were added to a precooled (-5 C) 40-mlglass-stoppered bottle containing 6 g of 0.17- to0.18-mm glass beads (B. Braun Melsungen Ap-paratebau). Cells were homogenized for 4 min, thebeads and debris were allowed to settle, and thesupernatant was drawn off and centrifuged at 4,000 xg for 20 min. This removed whole cells and largeaggregated debris. The cloudy, straw-colored super-natant, hereafter referred to as cell-free extract(CFE), was dialyzed against 50 volumes of 0.05 MHEPES-NH4OH, pH 7.2, at 4 C for 6 h.Two methods were used to characterize the phos-

phorylation of glucose and 2-DOG by CFE of B.abortus. Method one was the radiochemical kinaseassay of Newsholme (24), with the following modifica-tions. The reaction contained 50 Aliters of CFE, 5uliters of MgCl2 (0.1 M), 25 gliters of either PEP (0.02M) or ATP (0.02 M), and 20 uliters of either["C ]glucose (0.01 M) or ["C 12-DOG (0.01 M) at 37 C.At timed intervals between 0 and 2.5 min, 25-glitersamples were removed, diluted 1:1 with boiling 95%ethanol, and centrifuged. The supernatant (40 ,1iters)was spotted on the center of Whatman diethylamino-ethyl (DEAE) filters (DE 21, 2.4 cm, Reeve Angel),air-dried for 0.5 h, and washed with 100 ml ofdistilled, deionized water.Method two used a coupled enzymatic assay,

measuring the formation of glucose-6-phosphate(G-6-P) and reduced NADP with G-6-PDH on aGilford 240 recording spectrophotometer. The reac-tion mixture contained 50 ,uliters of B. abortus CFE,2.5 pliters of G-6-PDH (7 units), 100 Isliters ofHEPES-NH4OH (0.1 M, pH 7.2), 20 Mliters of MgCl,(0.1 M), 20 Mliters of NADP (0.02 M), 20 Mliters ofglucose (0.01 M), 25 gliters of either PEP (0.02 M) orATP (0.02 M), and water to 300 gliters. Reactionswere started by the addition of substrate and were run

at room temperature. When Escherichia coli B and B.abortus CFE were combined, 50 gliters of each wasused.

RESULTSIdentif'ication of non-metabolizable glucose

analogues for transport. A number of glucoseanalogues were tested to find a substrate thatcould be transported but not metabolized by B.abortus. A substrate with such properties wasneeded to obtain accurate kinetic data on thetransport process. 2-Deoxyglucose was the onlycommon glucose analogue transported by the B.abortus transport system. B. abortus did nottransport alpha- or beta-methyl glucosides or3-O-methylglucose. Glucose was transportedwith an apparent Km of 0.16 mM and anapparent Vmax of 250 nmol per min per mg of N(Fig. 1). 2-Deoxyglucose was a direct competi-tive inhibitor of the glucose transport systemand was shown, through the use of a Lineweav-er-Burk plot (9), to have an apparent Ki for2-DOG of 0.73 mM (Fig. 2). 2-Deoxyglucosetransport was linear for 70 to 90 s and was fullyconcentrated between 90 and 120 s. B. abortusconcentrated 0.1 mM external 2-DOG, 25- to30-fold, to an internal concentration of 2.5 to 3.0mM. At 0.01 mM external 2-DOG, an internaiconcentration of 1 mM was realized, equal to a100-fold concentration of substrate.To determine the fate of transported 2-DOG,

descending paper chromatography was per-formed on extracts of whole cells that had beenpreincubated with ["IC ]2-DOG. 2-Deoxyglucosewas transported as the free sugar and wasresistant to further metabolism. When incuba-tion of cells with 2-DOG was terminated at 2.5min, only one peak was observed on the radio-chromatographic scans (Fig. 3). This peak (R,

3. 0

2.5

2.0028000

_60

E Y ~~~ ~~~~~~~~~~~~40t,1.0 -/\

20-\

0. 5 - \/ ° ~~~~~0200 400 600 800

00 2 34M

FIG. 1. Uptake of glucose over the concentrationrange of 0.1 mM to 5.0 mM in B. abortus (0). Insert,Hofstee plot used to determine Km and Vmax (A).Points represent samples taken at 30 s.

252 J. BACTERIOL.

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

GLUCOSE TRANSPORT IN B. ABORTUS

1 -1

FIG. 2. Lineweaver-Burk plot of glucose transport(-) and glucose transport inhibited by 1 mM 2-DOG(A) in B. abortus. Points represent samples taken at30 s. The Km for glucose is shown. The Kg for 2-DOGwas calculated from the formula: K, = [iI/I(Kp/K.)-11.

0.68) corresponded to the standard peak of2-DOG (R, = 0.69). No peaks corresponding to2-DOG-P (R, = 0.22) or 2-DOG-COOH (R, =0.54) were observed. When the supematant ofthe original incubation mixture was spotted,again only one spot was observed, which corre-sponded to 2-DOG (data not shown). In experi-ments terminated at 15 min, a major and aminor peak were observed upon scanning thechromatographs of the concentrated ethanolextract. The major peak corresponded to 2-DOG, and the minor peak corresponded to2-DOG-COOH. However, the 2-DOG-COOHpeak was only 10% the area of the 2-DOG peakas measured with a Hruden planimeter (Plan-chets Lab Products). Since the time for maxi-mal substrate accumulation to occur is between90 and 120 s, we believe this oxidation productof 2-DOG found at 15 min to be insignificant.

Experiments employing CFE were used toverify that 2-DOG was not phosphorylated oroxidized. Short-term (0 to 2.5 min) experimentswere performed by using a radiochemical kinaseassay to determine whether any ionic productsof 2-DOG were formed upon the addition ofPEP or ATP to CFE in the presence of [14C ]2-DOG. The ionic forms of 2-DOG, including2-DOG-P and 2-DOG-COOH, adhere to theDEAE pads, whereas the free sugar, 2-DOG,does not. No counts above background werefound on the filters after washing with distilledwater, showing the lack of any ionic products.Lack of a phosphoenolpyruvate phospho-

transferase system. In the presence of PEP, no

ionic products of glucose were found upon theaddition of [14C]glucose to B. abortus CFE, aswas the case with [14C ]2-DOG. The results (Fig.4) obtained by using DEAE filter disks stronglyindicate the absence of a PEP-PTS specific forglucose in Brucella. The presence of a solublehexokinase was observed upon incubation of B.abortus CFE with [14CJglucose and ATP. Alinear assay was observed with a rate of 11,200dpm per mg of protein, corresponding to 4.48nmol per min per mg of protein.

Coupled spectral assays were used to gainadditional evidence concerning the presence orabsence of a PEP-PTS for glucose in B. abortus(Table 1). With glucose as substrate, the forma-tion of G-6-P was observed only upon theaddition ofATP to the system, and not upon theaddition of PEP. In the presence of ATP, G-6-P

B

C

FIG. 3. Chromatographic scans of ethanolic ex-tracts of B. abortus incubated with [14C]2-DOG for2.5 min (B) and 15 min (C). Standards are shown inscan A. Arrow indicates origin.

15 K

5

00 1 2 3 4 5

TIME, (minutes)

FIG. 4. Kinase activity present in CFE of B. abor-tus, as measured by the radiochemical kinase assayusing ATP plus [14CJglucose (), ATP plus [14C12-DOG (0), PEP plus [14C glucose (A), and PEP plus[14C12-DOG (A). Each assay contained 0.24 mg ofCFE protein.

253VOL. 118, 1974

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

REST AND ROBERTSON

TABLE 1. Phosphorylation of glucose by A TP orPEPin the presence of B. abortus or E. coli CFE via

spectral methods

Extract Phosphate nmol per minExtract ~ donor per mg ofprotein

B. abortusa ATP 15.65PEP 0.27

E. coli B ATP 41.30PEP 3.30

B. abortus + E. coli B ATP ND"PEP 3.30

a Each assay contained 0.27 mg of B. abortus CFEprotein or 0.185 mg of E. coli B CFE protein, or both.

'Not determined.

was formed at the rate of 15.65 nmol per min permg of protein. These spectral assay data sup-port those of the CFE experiments using theradiochemical kinase assay and the whole cellexperiments using chromatographic techniques,which indicated the absence of a glucose PEP-PTS.

Extracts of E. coli B were prepared (exactlyas those of B. abortus) as a positive control forthe presence of a PEP-PTS. They were used toshow that no inhibitors were present in B.abortus CFE that would mask the presence ofthe PEP-PTS. As expected, the E. coli CFEphosphorylated glucose in the presence of PEP,at the rate of 3.30 nmol per min per mg ofprotein. When Brucella CFE was added to theE. coli extract, the phosphorylation of glucoseby PEP continued at the original rate. There-fore, there were no inhibitors present in the B.abortus CFE that inhibited the glucose PEP-PTS in extracts of E. coli.

Effects of various substrates on efflux of2-DOG. To more clearly define the specificity ofthe glucose transport system, we studied theeffects of various "cold" carbohydrates on theefflux of ["C ]2-DOG from preloaded cells of B.abortus. Dilution of preloaded cells with 10 mMglucose or 10 mM galactose caused a more rapidand more complete efflux of ["1C]2-DOG thandid dilution with HEPES-NH4OH or with 10mM fructose (Fig. 5). Galactose (1 mM) or 1mM 2-DOG caused a lesser degree of efflux thandid the 1 mM glucose or the 10 mM galactose,and 10 mM erythritol did not stimulate effluxabove the HEPES-NH4OH control (data notshown). When 10 mM 2,4-dinitrophenyl (DNP)or 25 mM KCN were used in conjunction with 1mM glucose to dilute preloaded cells, the effluxobserved was identical to that observed whenglucose alone was used as diluent. That is, DNPand KCN had no effect on efflux.

Effects of sulfhydryl inhibitors. Preincuba-tion of cells with 0.1 mM NEM orpCMB totallyinhibited the subsequent transport of glucose or2-DOG (Fig. 6). Background counts in theseexperiments (50 dpm) were lower than thosecounts observed when pCMB or NEM were

12

9

a-6

3

0

As

90 120

TIME, (seconds)

FIG. 5. Efflux of ["C ]2-DOG from preloaded B.abortus into 100 mM HEPES-NH4OH buffer, pH 7.2(A), or 10 mM fructose (A), 10 mMglucose (M), or 10mM galactose (0), all contained in 100 mM HEPES-NH4OH buffer.

3

2

0

-~ X

0l' ' ^=

TIME, (seconds)

FIG. 6. Reversal of 1 mM pCMB inhibition of 0.1mM 2-DOG transport by 10 mM f-ME (-). Similarcurves were obtained for 10 mM DTT and 10 mMglutathione. Irreversible inhibition of 1 mM NEM of2-DOG transport (A). Arrow indicates addition of,8-ME.

254 J . BACTrERIOL.

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

GLUCOSE TRANSPORT IN B. ABORTUS

omitted (300 dpm), suggesting that pCMB andNEM affect the active site of a binding proteinand thus affect binding of substrate to the cellsurface.The effect ofNEM on transport was irreversi-

ble upon the addition of 10 mM DTT or -ME.On the other hand, the effect of pCMB wasimmediately and almost completely reversedupon the addition of 10 mM DTT or ,-ME tocells incubated with ["C ]2-DOG. The shorttime required for reversal of the pCMB effectsuggests that an easily accessible sulfhydrylgroup, located near the cell membrane, is beingchemically modified.

Effect of pH, temperature, and ions. Theeffects of temperature are shown in Fig. 7. Theoptimum temperature of glucose and 2-DOGtransport, both for Km and Vmax, is 37 C. Nouptake occurred at 0 C. As the temperatureincreased from 0 to 37 C, both maximal concen-trative ability and maximal rate of entry in-creased. A wide plateau was reached at 37 to45 C, where the kinetics of uptake (both Km andVmax) remained constant. At 55 C, the ability ofB. abortus to concentrate 0.1 mM 2-DOGdropped to 20% of that at 37 C.When transport of 0.1 mM 2-DOG was mea-

sured at varying pH values, a sharp Km andVmax optimum was observed at 7.2. The concen-trative ability of the cells dropped 60% at onepH unit to either side of pH 7.2 (data notshown).

In the presence of 0.1 mM NaCl, KCl, orMgCl2, maximal uptake of 0.1 mM 2-DOG wasinhibited by 20%, and the rate of uptake was

decreased by 30%. The reason for this inhibitionis unknown.Constitutivity of transport. B. abortus cells

grown on glucose, fructose, galactose, or erythri-tol were used in standard uptake experiments,except the cells were preincubated for 20 minwith 100 ,ug of chloramphenicol (CAM) per ml.A control using glucose-grown cells with no

CAM was also performed. Cells grown on anyone of the carbohydrates were capable of trans-porting 2-DOG in the presence ofCAM with thesame kinetics as the glucose control cells.

Effect of energy and electron transportinhibitors. The effects of inhibitors on trans-port can be seen in Table 2. Two general groupsof inhibitors were used to characterize theenergy requirements of the Brucella glucosetransport system: electron transfer inhibitorsand metabolic energy inhibitors. The first groupcontained NaN,, KCN, CCCP, and DNP.These are known to interrupt electron transferthrough the cytochrome system or to uncoupleoxidative phosphorylation from electron trans-

port. The second group of inhibitors containedHOQNO, NaF, iodoacetic acid (IAA), arse-

nate, and arsenite, which inhibit succinic dehy-drogenase, enolase, glyceraldehyde-3-PO dehy-drogenase (G-3-PDH), energy production of theG-3-PDH step, and pyruvate dehydrogenase,respectively.

All of the above electron transfer inhibitorsblocked 2-DOG transport by 100%, at the con-

centrations used. DCCD, a potent inhibitor ofthe membrane-bound adenosine triphosphatase(ATPase), also inhibited transport by 100%.Conversely, the metabolic inhibitors seemed tohave little effect on 2-DOG transport.

DISCUSSIONThe glucose transport system of B. abortus

showed a uniquely high degree of specificity for

3

3~~~~~~~~~~~~~~~~~1z

0 9 7

0 30 60 90 120 150 180 210

TI ME, (seconds)

FIG. 7. Effect of temperature on 2-DOG transportin B. abortus. Transport at 0 C (a), 15 C (a), 25 C(0), and 37 C (A).

TABLE 2. Effect of metabolic and electron transferinhibitors on 2-DOG transport in B. abortus

Inhibitor Conc (mM) Inhibition (%) ofuptake at 60 s

CCCP 0.1 100DNP 10 100DCCD 0.1 80DCCD 0.5 100NaN, 10 75NaN3 25 100KCN 10 100HOQNO 0.1 30HOQNO 0.5 50NaHAs2O4 10 20IAA 10 25NaF 10 0DMSO (1%) 0

255VOL. 118, 1974

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

J. BACTERIOL.REST AND ROBERTSON

its substrate in that, of all analogues tested,only 2-DOG was transported. Most glucosetransport systems studied in bacteria and yeast(E. coli, Salmonella typhimurium, Bacillussubtilis, Saccharomyces cerevisiae, etc.) havebeen relatively nonspecific as to glucose ana-

logues accepted for uptake. The only otherbacterium to have such a high degree of speci-ficity was Pseudomonas aeruginosa (11).

In the Hofstee plot (10), it can be seen thattwo transport systems might exist for glucose(Fig. 1): an active system and a passive or

facilitated system. The secondary kinetics seemto be nonsaturable and nonactive. When energy

or electron transfer inhibitors were used thatblock active transport by 100%, the passivecomponent always remained. An explanation ofthe "passive," nonsaturable kinetics is not pos-

sible at this time. The most simplistic explana-tion would be the presence of a high Km and a

low Km system for glucose uptake. This idea isnot tenable with the kinetic data though, for a

second (high) Km system has not been observed.The concentrations used to obtain points near

the ordinate of the Hofstee plot (Fig. 2) were

greater than 100 times the Km for glucose. Datathus obtained might have been artifactual dueto (i) significant changes in osmotic strength,which changed conditions within the experi-mental system, (ii) trapping of substrate withinthe pericytoplasmic space that could not beremoved by washing, and (iii) the presence of a

nonsaturable system.We observed that galactose acted as a com-

petitive inhibitor for the "glucose" uptake sys-

tem. It is possible, then, that galactose sharessome part of the glucose transport system (orvice versa) and that the nonsaturable kineticsobserved for glucose are due to glucose beingtransported by the "galactose" part of thetransport system, which we have shown to benonconcentrative (data not shown). These ob-servations have been seen more clearly in effluxstudies. The results seen in Fig. 4 show thatgalactose caused the same extent of 2-DOGefflux as did glucose. Other carbohydratestested, erythritol and fructose, had no effect on

2-DOG efflux from preloaded cells.It is important to determine the fate of

transported substrate analogues once inside thebacterial cell (2, 11, 23, 30). We have shownconclusively that 2-DOG is not further metabo-lized once inside B. abortus. In short-termexperiments of 0 to 2.5 min, no metabolism of2-DOG was observed in B. abortus whole cells or

CFE, using three different analytical methods.We have shown an extremely active galactose

oxidase in B. abortus CFE (unpublished data).This might be the enzyme responsible for theoxidation of 2-DOG to 2-DOG-COOH observedin the experiments terminated at 15 min, sincewe have detected no membrane-bound glucosedehydrogenase.As proposed by Romano et al. (30), most obli-

gate aerobes do not possess a PEP-PTS. It istherefore not surprising that the PEP-PTS is

absent in B. abortus. Energy for transport can

be obtained through oxidative aerobic metabo-lism. Robertson and McCullough (29) haveshown that glucose catabolism in B. abortusproceeds via the hexose monophosphate path-way, operating in conjunction with the tricarbox-ylic acid cycle. A functional Embden-Meyerhof-Parnas pathway is not present; therefore, thereis no need to conserve phosphate bond energy

with the presence of a PEP-PTS.The acute sensitivity of transport to the

sulfhydryl inhibitors pCMB and NEM that we

have shown in Brucella has been shown in otherbacteria and yeast. The rapid reversibility ofthe pCMB effect by the sulfhydryl reagentsglutathione and d-ME indicates an easily acces-

sible sulfhydryl group. This sulfhydryl groupmight be located either within a "binding"protein of some kind, which is located on or near

the surface of the B. abortus cytoplasmic mem-brane, or within some membrane protein in-volved in energizing the transport process. At-tempts to isolate B. abortus "binding" proteinsin our laboratory have to date been unsuccess-

ful. It seems that the binding protein(s) of B.abortus is more tightly bound to the cell thanthose that have been reported for other bacteria(1, 25, 27, 33).Preliminary evidence has suggested that glu-

cose transport is constitutive in B. abortus. Thisis the case in most bacteria (32), with theexception of P. aeruginosa (15). The kinetics of2-DOG transport were identical for cells grown

on fructose, galactose, erythritol, or glucose. Forabsolute proof of the constitutivity, we realizethat mutant analysis must be performed, andwork is being done in our laboratory at thepresent time to identify transport and meta-bolic mutants of B. abortus.The electron transfer inhibitors NaN,, CCCP,

KCN, and DNP all effectively blocked activetransport of 2-DOG by 100%. DCCD, a mem-

brane-bound ATPase inhibitor, also inhibitedtransport by 100%. The metabolic energy inhib-itors NaAsO,, IAA, KF, and HOQNO did notaffect transport to any great extent. The effectsof azide and cyanide are well documented intheir ability to prevent reoxidation of the cyto-

256

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

GLUCOSE TRANSPORT IN B. ABORTUS

chrome chain. CCCP has been shown to inter-fere both with electron transfer and protonconductance. An explanation of all these inhibi-Lors can be found in Harold's review on energyand bacterial membranes (12).Because the effects of DNP on bacterial

energy metabolism are unclear, the strong inhi-bition by DNP on the B. abortus system isdifficult to interpret. DNP uncouples oxidativephosphorylation and inhibits substrate levelphosphorylation in Mycobacterium phlei (34).Since B. abortus might be similar to M. phlei inthat it is an aerobe and has evolved to the stateof facultative parasitism, one might then as-sume that the cytochrome systems of these twoorganisms might be similarly inhibited byDNP.In most bacteria, the cytochrome chain is a

"tight" system, so that a block anywhere in thechain will cause a cessation of electron flowthroughout the chain. Hence, with whole cellsthe exact site of energy coupling to activetransport by use of electron transfer inhibitors isimpossible to determine because of complica-tions involving cellular metabolism.

It is clear from the accumulated inhibitordata that B. abortus needs a functional, unin-terrupted cytochrome chain for active transportof 2-DOG. Any potent inhibitor of electrontransfer inhibited active transport of 2-DOG.(Work is now being done in our laboratoryconcerning the cytochrome system of B.abortus.) It is also clear that normal metabolicenergy inhibitors did not immediately affectactive transport. This might be explained bythe metabolic pattern of B. abortus. A numberof energy-yielding steps are accomplished be-fore glucose is catabolized to the 3-carbonintermediates of the lower glycolytic pathway.The effect of DCCD on transport in whole

cells of B. abortus is extremely complex. DCCDhas been shown: (i) to inhibit membrane-boundATPase in Streptococcus faecalis, and thustransport (3), (ii) to inhibit transport at highconcentrations (above 30 MM) in E. coli, and(iii) to stimulate transport in ATPase-deficientvesicles of E. coli at low concentrations (31).DCCD does not inhibit transport in E. coliwhole cells until the cell wall is first softened;however, transport is inhibited in S. faecalisand B. subtilis, without first softening the cellwall. It is surprising, then, that B. abortus, agram-negative bacterium, is so drastically af-fected by DCCD, without prior cell wall soften-ing. Mechanisms of energization of active trans-port and the interesting effects of DCCD ontransport are now being investigated in our

laboratory with the aid of membrane vesicles ofB. abortus.

ACKNOWLEDGMENTS

This investigation was supported by grants from theResearch Corporation (Brown-Hazen Fund), the Universityof Kansas General Research Fund, and by Public Healthtraining grant GM703 from the National Institute of GeneralMedical Sciences.

LITERATURE CITED1. Anraku, Y. 1968. Transport of sugars and amino acids in

bacteria. I. Purification and specificity of the galactoseand leucine binding proteins. J. Biol. Chem.11:3116-3122.

2. Asana, A., S. N. Cohen, R. F. Baker, and A. F. Brodie.1973. Orientation of the cell membrane in ghosts andelectron transport particles of Mycobacterium phlei. J.Biol. Chem. 248:3386-3397.

3. Asghar, S. S., E. Levin, and F. M. Harold. 1973.Accumulation of neutral amino acids by Streptococcusfaecalis. Energy coupling by a proton-motive force. J.Biol. Chem. 248:5225-5233.

4. Bailey, J. L. 1967. Techniques in protein chemistry, p.341. Elsevier Publishing Co., New York.

5. Bandurski, A., and B. Axelrod. 1951. The chromatographicidentification of some biologically important phos-phate esters. J. Biol. Chem. 193:405-410.

6. Barnes, E. M., and H. R. Kaback. 1971. Mechanisms ofactive transport in isolated membrane vesicles. I. Thesite of energy coupling between D-lactic dehydrogenaseand ,6-galactoside transport in Esherichia coli mem-brane vesicles. J. Biol. Chem. 246:5518-5522.

7. Bragg, P. D., and L. Hough. 1958. The oxidation ofproline, hydroxyproline, and N-methyl-glycine with-periodate. J. Chem. Soc. Part IV, p. 4050-4053.

8. Bruno, G. A., and J. E. Christian. 1961. Determination ofcarbon-14 in aqueous bicarbonate solutions by liquidscintillation counting techniques: application to bio-logical fluids. Anal. Chem. 33:1216-1218.

9. Dixon, M., and E. C. Webb. 1964. Enzymes, p. 54-166.Academic Press Inc., New York.

10. Dixon, M., and E. C. Webb. 1964. Enzymes, p. 315-359.Academic Press Inc., New York.

11. Eagon, R. G. 1971. 2-Deoxyglucose transportation viapassive diffusion and its oxidation, not phosphoryla-tion, to 2-deoxy-gluconic acid by Pseudomonasaeruginosa. Can. J. Biochem. 49:606-613.

12. Harold, F. M. 1972. Conservation and transformation ofenergy by bacterial membranes. Bacteriol. Rev.36:172-230.

13. Harold, F. M., and D. Papineau. 1972. Cation transportand electrogensis by Streptococcus faecalis. I. Themembrane potential. J. Membrane Biol. 8:27-44.

14. Hirata, H., K. Alterdorf, and F. M. Harold. 1973. Role ofan electrical potential in the coupling of metabolicenergy to active transport by membrane vesicles ofEscherichia coli. Proc. Nat. Acad. Sci. U.S.A.108:1804-1808.

15. Hylemon, P. B., and P. V. Phibbs. 1972. Independentregulation of hexose catabolizing enzymes and glucosetransport activity in Pseudomonas aeruginosa. Bio-chem. Biophys. Res. Commun. 48:1041-1048.

16. Kaback, H. R. 1968. The role of the phosphoenolpyru-vate-phosphotransferase system in the transport ofsugars by isolated membrane preparations of Esche-richia coli. J. Biol. Chem. 243:3711-3724.

17. Kaback, H. R., and E. M. Barnes, Jr. 1971. Mechanismsof active transport in isolated membrane vesicles. II.

257VOL. 118, 1974

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

REST AND ROBERTSON

The mechanism of energy coupling between D-lacticdehydrogenase and 3-galactoside transport in mem-

brane prepatations of Escherichia coli. J. Biol. Chem.246:5523-5537.

18. Klein, W. L., and P. D. Boyer. 1972. Energization ofactive transport by Escherichia coli. J. Biol. Chem.247:7257-7265.

19. Koch, F. C., and T. L. McMeekin. 1924. A new directNesslerization micro-Kjeldahl method and a modifica-tion of the Nessler-Folin reagent for ammonia. J. Amer.Chem. Soc. 46:2066-2069.

20. Lombardi, F. J., J. P. Reeves, and J. R. Kaback. 1973.Mechanisms of active transport isolated bacterialmembrane vesicles. XIII. Valinomycin induced rubi-dium transport. J. Biol. Chem. 248:3551-3565.

21. Midgley, M., and E. A. Dawes. 1973. The regulation oftransport of glucose and methyl a-glucoside inPseudomonas aeruginosa. Biochem. J. 132:141-154.

22. Moore, S., and K. P. Link. 1940. Carbohydrate characteri-zation. I. The oxidation of aldoses by hypoiodate in

methanol. J. Biol. Chem. 133:293-299.23. Mukkada, A. J., G. L. Long, and A. H. Romano. 1973.

The uptake of 2-deoxy-D-glucose by Pseudomonasaeruginosa and its regulation. Biochem. J. 132:155-162.

24. Newsholme, E. A., J. Robinson, and K. Taylor. 1967. Aradiochemical enzymatic activity assay for glycerolkinase and hexokinase. Biochim. Biophys. Acta132:338-346.

25. Pardee, A. B. 1966. Purification and properties of a

sulfate binding protein from Salmonella typhimurium.J. Biol. Chem. 241:5886-5892.

26. Phibbs, R. V., and R. G. Eagon. 1970. Transport andphosphorylation of glucose, fructose, and mannitol by

Pseudomonas aeruginosa. Arch. Biochem. Biophys.138:470-482.

27. Piperno, J. R., and D. L. Oxender. 1966. Amino acidbinding protein released from Escherichia coli byosmotic shock. J. Biol. Chem. 241:5732-5734.

28. Robertson, D. C., and W. G. McCullough. 1968. Theglucose catabolism of the genus Brucella. I. Evaluationof pathways. Arch. Biochem. Biophys. 127:263-273.

29. Robertson, D. C., and W. G. McCullough. 1968. Theglucose catabolism of the genus Brucella. II. Cell-freestudies with B. abortus (S-19). Arch. Biochem. Bio-phys. 127:445-456.

30. Romano, A. H., S. J. Eberhard, S. L. Dingle, and T. D.McDowell. 1970. Distribution of the phosphoenolpyru-vate: glucose phosphotransferase system in bacteria. J.Bacteriol. 104:808-813.

31. Rosen, B. P. 1973. Restoration of active transport in an

Mg2+-adenosine triphosphate-deficient mutant ofEscherichia coli. J. Bacteriol. 116:1124-1129.

32. Schneider, R. P., and W. R. Wiley. 1971. Regulation ofsugar transport in Neurospora crassa. J. Bacteriol.106:487-492.

33. Tsay, S., K. Brown, and E. Goudy. 1971. Transport ofglycerol by Pseudomonas aeruginosa. J. Bacteriol.108:82-88.

34. White, A., P. Handler, and E. L. Smith. 1968. Principalsof biochemistry, p. 375-376, 4th ed. McGraw-Hill BookCo., New York.

35. Winkler, H. H., and T. H. Wilson. 1966. The role ofenergy coupling in the transport of ,B-galactosides byEscherichia coli. J. Biol. Chem. 241:220-221.

36. Yabu, K. 1970. Amino acid transport in Mycobacteriumsmegmatis. J. Bacteriol. 102:6-13.

258 J. BACTERIOL.

on March 1, 2019 by guest

http://jb.asm.org/

Dow

nloaded from