Vol. 175, No. 6JOURNAL OF BACrERIOLOGY, Mar. 1993, p. 1682-16860021-9193/93/061682-05$02.00/0Copyright © 1993, American Society for Microbiology

Induction of the X Receptor Is Essential for Effective Uptakeof Trehalose in Escherichia coli

WOLFGANG KLEIN AND WINFRIED BOOS*Department of Biology, University of Konstanz, D-7750 Konstanz, Germany

Received 13 October 1992/Accepted 1 January 1993

Trehalose transport in Escherichia coli after growth at low osmolarity is mediated by enzyme IITI of thephosphotransferase system (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J.Bacteriol. 172:3450-3461, 1990). The apparent Km (16 IM) of trehalose uptake is low. Since trehalose is a goodsource of carbon and the apparent affinity of the uptake system is high, it was surprising that the disaccharidetrehalose [O-a-D-glucosyl(1-1)-a-D-glucosideI has no problems diffusing through the outer membrane at highenough rates to allow full growth, particularly at low substrate concentrations. Here we show that inductionof the maltose regulon is required for efficient utilization of trehalose. malT mutants that lack expression of allmaltose genes, as well as lamB mutants that lack only the A receptor (maltoporin), still grow on trehalose at theusual high (10 mM) trehalose concentrations in agar plates, but they exhibit the half-maximal rate of trehaloseuptake at concentrations that are 50-fold higher than in the wild-type (malT+) strain. The maltose system isinduced by trehalose to about 301% of the fully induced level reached when grown in the presence of maltosein a malT+ strain or when grown on glycerol in a maltose-constitutive strain [malT(Con)]. The 30%o level ofmaximal expression is sufficient for maximal trehalose utilization, since there is no difference in theconcentration of trehalose required for the half-maximal rate of uptake in trehalose-grown strains with thewild-type gene (malT+) or with strains constitutive for the maltose system [malT(Con)J. In contrast, when theexpression of the A receptor is reduced to less than 20%o of the maximal level, trehalose uptake becomes lessefficient. Induction of the maltose system by trehalose requires metabolism of trehalose. Mutants lackingamylotrehalase, the key enzyme in trehalose utilization, accumulate trehalose but do not induce the maltosesystem.

Metabolism of trehalose in Escherichia coli is complicatedby the fact that depending on the osmolarity of the growthmedium, different pathways are active. At high osmolarity,cells synthesize large amounts of internal trehalose as anosmoprotectant, independent of the carbon source. Thissynthesis requires the transfer of glucose from UDP-glucoseto glucose-6-phosphate to form trehalose-6-phosphate (Tre-6-P) and is mediated by Tre-6-P synthase, the product of theotsA gene product (16). Hydrolysis of Tre-6-P is by Tre-6-Pphosphatase, the product of the otsB gene. otsB and otsAform an operon (21) whose expression is osmoregulated andunder the control of RNA polymerase programmed with thestarvation-phase sigma factor e (20). Despite the fact thattrehalose is synthesized internally at high osmolarity, thecells can still use external trehalose effectively as a carbonsource. The use of external trehalose is accomplished by aperiplasmic trehalase which is induced under conditions ofhigh osmolarity (4, 18) and whose regulation is also at leastpartly under the control ofM (20). Thus, periplasmic treha-lose is hydrolyzed to glucose and taken up by glucose-specific systems.At low osmolarity, the presence of trehalose induces a set

of genes whose products mediate the uptake and metabolismof trehalose (5). The transport system consists of enzyme IIof the phosphotransferase system, specific for trehalose,which in combination with enzyme IIGlc, mediates uptakeunder simultaneous phosphorylation to Tre-6-P. Uptake oftrehalose by enzyme II e-mediated transmembrane phos-phorylation exhibits high affinity, with 16 RM as the apparentKm of uptake. The breakdown of internal Tre-6-P is medi-

* Corresponding author.

ated by a catabolic Tre-6-P phosphatase to free trehalose,which is subsequently hydrolyzed by amylotrehalase. Thisenzyme releases from trehalose one molecule of glucose,while the other is transferred to an as yet unidentifiedpolysaccharide, which in turn is most likely degraded by aphosphorylase, yielding glucose-i-phosphate. The inducerof these genes, located at 96.5 min on the E. coli chromo-some, is Tre-6-P (23).The rate of growth with trehalose as the carbon source is

only slightly less than that with maltose, a disaccharide thatis isomeric with trehalose. The utilization of maltose andmaltodextrins in E. coli is mediated by a set of four enzymesand a high-affinity binding-protein-dependent transport sys-tem (9, 19). The degradation of maltose and maltodextrins bythe key enzymes amylomaltase (26) and maltodextrin phos-phorylase (34) yields glucose and glucose-i-phosphate,which after transformation into glucose-6-phosphate by thehousekeeping enzymes glucokinase and phosphoglucomu-tase, enter glycolysis. The regulation of the maltose systemis mediated by the mal gene activator MalT, which incombination with the inducer maltotriose (27) and the cyclicAMP-catabolite gene activator protein complex is requiredfor the expression of all mal genes (30). In addition, theregulation of mal gene expression is also influenced by thelevel of MalK (24, 29), a subunit of the transport system, aswell as by MalY (28), with the presence of both resulting inthe repression of the maltose system. Maltotriose, whichinduces the maltose system (27) is produced endogenouslyeither by gluconeogenesis or by degradation of glycogen (10,11).There are some connections between the maltose and

trehalose systems. It has been known for some time thattrehalose can induce the maltose system (33, 35), even

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FUNCTION OF E. COLI X RECEPTOR 1683

TABLE 1. Bacterial strainsa

Strain Known genotype source

UE14 Same as MC4100 but with treA::TnlO 5UE15 Same as UE14 but with treA Tets 5JB3018-2 Same as MC4100 but with malT(Con)l 7ME810 F' (lacIq A(lacZ)M15 proAB traD36) endA glpR A(lac-pro) lamB::TnlO sbcB strA supE thiA M. EhrmannMM165 F- ara-14 his-4 lacYleuB6 malT(Con)l mtd-I rbsL136 thi-1 thr' tonA31 tsr-i tsx-78 xyl zjb-729::TnlO 7Rim9O Same as UE15 but with malT::TnlO This studyUE48 Same as MC4100 but with treC::lacZ 5WK129 Same as JB3018-2 but with treA::TnlO This studyWK145 Same as Rim9O but with malT Tet' This studyWK146 Same as WK145 but with zjb-729::TnlO This studyWK147 Same as UE15 but with lamB::TnlO This study

a All strains are derivatives of E. coli K-12.

though malT mutants can still grow on trehalose. There is nocross specificity by the two transport systems (5, 22). Yet,trehalose chemotaxis is defective in malT mutants (4), andthe end products of maltose and trehalose metabolism are

the same, glucose and glucose-i-phosphate.The apparent Km of maltose uptake by the maltose trans-

port system is 1 to 2 ,uM (37), about 10-fold lower than thatfor trehalose by the trehalose transport system. The rate ofdiffusion through the outer membrane of each disaccharideat a concentration near the Km of their respective transportsystem will be approximately the same. To satisfy thedemand for substrate in the periplasm to attain the half-maximal rate of maltose transport at maltose concentrationsin the medium around the Km of uptake, the cells need thepresence of the X receptor in the outer membrane (36). Lackof the X receptor requires a 100- to 500-fold increase in themaltose concentration to reach the half-maximal rate ofuptake (37). In this case, maltose enters the periplasm by thegeneral diffusion porins, OmpF and OmpC, for which therate of diffusion is strictly proportional to concentration.By reducing the amount of X receptor in the outer mem-

brane without reducing the level of the binding-protein-dependent transport machinery, one can observe that a

reduction to 50% of the wild-type level is without effect,while a reduction to 20% manifests itself in the reduction oftransport at micromolar maltose concentrations (6). Onewould thus conclude that at a maltose concentration aroundthe Km of the uptake system proper, the amount of X

receptor slightly exceeds the requirements of maltose diffu-sion through the outer membrane.No specific outer membrane diffusion pore for trehalose

has been found so far. Yet, from the high rate of transport(4.5 nmol per min per 109 cells) at its apparent Km of uptake,one would expect that a specific porin exists, since thediffusion properties of the disaccharide trehalose through thegeneral porins OmpC and OmpF should be similarly unfa-vorable as for maltose. Since the overall Vm, of trehalosetransport is of the same order as that of maltose (9 versus 20nmol per min per 109 cells [5, 15]), it follows that thehalf-maximal rate of uptake in the absence of any specificporin could be achieved only with a trehalose concentrationfrom 0.1 to 0.5 mM.The in vitro analysis of the diffusion properties of the X

receptor in the black lipid assay (2) had shown that itssubstrate specificity was not restricted to maltose and mal-todextrins. Other mono- and disaccharides inhibited malto-porin-dependent salt conductivity with a K1 that is of thesame order as that for maltose. In particular, trehalose

appeared to be a substrate of the X receptor. This finding wasconsistent with our earlier conclusion that trehalose chemo-taxis is needed for the presence of the X receptor (4).

In this report, we present evidence that the X receptor isindeed needed for the efficient uptake of trehalose at lowsubstrate concentrations and that trehalose metabolism in-duces the maltose system, including the X receptor.

MATERIALS AND METHODS

Bacterial strains are described in Table 1. They weregrown under aeration in Luria broth (LB) or in minimalmedium A (MMA) with 0.2% carbon source (25). Twocarbon sources were used at concentrations of 0.2% each.Strain constructions were done by P1 vir-mediated transduc-tion by the method of Miller (25). Selection for Tetr was donewithout phenotypic expression plating on DYT (25) contain-ing 5 ,ug of tetracycline per ml. Selection for loss of Tetr wasdone by the method of Bochner et al. (3). Uptake of[14C]trehalose and [14C]maltose was measured after growingthe cells overnight in MMA containing either glycerol,maltose, or trehalose (or combinations) at a concentration of0.2%. To measure maltose transport, the cultures werewashed three times in MMA and resuspended in the samebuffer without a carbon source to an optical density at 578nm (OD578) of 0.2. To measure uptake of trehalose, lamB+strains were used at an OD478 of 1.0 and lamB strains wereat an OD578 of 2.0. [14C]trehalose and ['4C]maltose wereadded at a final concentration (each) of 23 nM, and 0.5-mlsamples were filtered at different time intervals (0.45-,umpore size; Schleicher & Schuell ME25 filters). After thefilters were washed with 10-ml portions of MMA, the radio-activity of the filters was determined in a scintillationcounter. For measuring the half-maximal rates of uptake, thesame procedure was repeated in the presence of increasingconcentrations of unlabeled substrate. Care was taken tomix labeled and unlabeled substrates prior to their additionto the cells. Similarly, when the inhibition of trehalosetransport by maltodextrins was measured, 23 nM [14C]trehalose (final concentration) was mixed with differentconcentrations of unlabeled maltodextrins prior to theiraddition to the cells.

RESULTS

malT mutants exhibit strongly defective kinetics of trehaloseuptake. In order to test for the presence of the trehalosetransport system, we routinely assay the abilities of treA

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FIG. 1. Inhibition of uptake of 23 nM ['4C]trehalose by increas-ing concentrations of unlabeled trehalose. The initial rate of uptakeof 23 nM ['4C]trehalose (equal to 100% in the figure) was measuredin the presence of increasing concentrations of unlabeled trehalosegiven in moles per liter. From the Michaelis-Menten equation, itfollows that the concentration of unlabeled trehalose that yields a50% reduction in the rate of ['4C]trehalose uptake is equal to the Kmof the uptake system. The following strains were used after growthon MMA with 0.2% trehalose as the carbon source: UE14 (0);WK129 (A); WK146 (l); Rim9O (O); WK147 (x).

mutants lacking the periplasmic trehalase (4) to take up[14C]trehalose at submicromolar concentrations after thecells were grown either on trehalose or on glycerol in thepresence of trehalose. We noticed that mutants lacking malTand therefore all proteins of the maltose system behavedsimilarly to trehalose transport mutants (treB). Yet, the tregenes were fully induced, as measured by the activity ofamylotrehalase, the gene product of treC (data not shown).The explanation for this phenomenon was that the kinetics oftrehalose transport in malT mutants were entirely differentthan in a malT+ strain. The half-maximal rate of transport inthe malT+ strain occurred at 16 ,uM (5), while in the malTmutant it was estimated to be 0.55 mM, whereas the extrap-olated Vm. in both cases was the same. These findings areshown in Fig. 1. The initial rate of uptake of 23 nM['4C]trehalose (equal to 100%) was measured in the presenceof increasing concentrations of unlabeled trehalose. Underthe prerequisite of transport being mediated by a singlesaturable transport system and substrate concentrations of[14C]trehalose far below the Km of the system, it followsfrom the Michaelis-Menten equation that the concentrationof unlabeled trehalose that yields a 50% reduction in the rateof [14C]trehalose uptake is equal to the Km of the uptakesystem. This finding also allows us to calculate (simply bymultiplying the amount of [14C]trehalose taken up with thedilution factor of the added unlabeled trehalose) the half-maximal rate of trehalose uptake. The calculated values aregiven in Table 2. Whereas the mal+ strain shows half-maximal saturation at 11 ,uM, consistent with the previouslydetermined apparent Km of uptake of 16 ,uM (5), the malTstrain shows half-maximal saturation only at 0.55 mM. Incontrast, the rate at half-maximal saturation of uptake is ofthe same order of magnitude in both strains.The lack of X receptor (maltoporin) is responsible for the

alteration in the uptake kinetics of trehalose. Using a lamBmutant that lacks the receptor in the above analysis, weobtained the same results as with the malT mutant. There-fore, it is clear that K-receptor-mediated diffusion of treha-

TABLE 2. Trehalose concentrations and half-maximal rates ofuptake in strains with various amounts of X receptor'

Strain (relevant genotype or Trehalose Uptake ratecharacteristic) concn (,uM) (nmol/min/109 cells)

UE14 (malTf) 11 1.8WK129 [malT(Con)] 11 2.0WK146 (reduced LamB) 55 4.2WK147 (lamB::TnlO) 550 5.3Rim9O (malT: :TnJO) 550 3.1

a The values were calculated from the data given in Fig. 1. Cells were grownon trehalose.

lose through the outer membrane is required for the efficientuptake of trehalose in the micromolar substrate range. Asseen in Fig. 1, a malT(Con) strain did not exhibit a higheraffinity for trehalose uptake than a malT' strain, demon-strating that the amount of X receptor present in trehalose-grown cells is sufficient for free access of trehalose to theuptake system located at the inner membrane.We would like to emphasize that the substrate concentra-

tion for the half-maximal rate of uptake is not identical withthe Km value for the transport system. These two valueswould be identical only if the outer membrane does not actas a diffusion barrier for the substrate. We believe this to bethe case in trehalose-grown malT' cells, but clearly not instrains lacking the A receptor. In this case, the overall rate oftransport at low substrate concentrations is determined bythe linear dependency of diffusion through OmpC andOmpF, while at higher concentrations uptake will be limitedby the saturation of the transport system proper, located inthe inner membrane. This phenomenon would result in anonlinear Lineweaver-Burk plot, preventing the extrapola-tion of a meaningful Km (6). Therefore, we use the concen-tration of substrate at the half-maximal rate of uptake as ameasure for the affinity of trehalose uptake.The uninduced level of the A receptor is insufficient for

maximal transport of trehalose. The observation that aftergrowth on trehalose, malT' and malT(Con) strains exhibitthe same high apparent affinity of trehalose transport indi-cated that either the uninduced level of X receptor is suffi-cient for effective trehalose uptake or that trehalose inducesthe A receptor to high enough levels for effective trehaloseuptake. Brass et al. (7) have isolated a TnlO insertionpositioned between malK and lamB so that the expression oflamB is under the control of the pout promoter of TnlO. Theamount of A receptor synthesized in this mutant is only about20% of fully induced wild-type levels but is clearly more thanthe level found in wild-type cells grown on glycerol. Usingthis TnlO insertion for expression of A receptor in trehalose-grown cells, we found that uptake of trehalose exhibited alower affinity than when the K receptor is expressed from itsnatural promoter (Fig. 1).

Trehalose metabolism induces the maltose system. We usedthe assay of maltose transport at low substrate concentra-tions as a measure for the level of induction of the maltosesystem. Table 3 shows that growth in the presence oftrehalose induces the maltose system to about 25 to 30% ofthe level obtained with maltose as the sole carbon source. Incontrast, with a treC-lacZ mutant lacking amylotrehalase,the key enzyme of trehalose metabolism, no induction of themaltose system was observed, even though trehalose stillaccumulated in this mutant (5). Thus, trehalose does causeinduction of the maltose system but metabolism of trehaloseis required.

percent of remaining[14C]_trdGjcse uptake

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TABLE 3. Induction of the maltose transportsystem by trehalose

Rate of uptake with the following carbon source(s) (0.2%)a:Strain Glycerol Trehalose Glycerol plus Glycerol

trehalose plus maltose

MC4100 0.65 2.45 10.5UE14 0.50 1.80 1.50 9.5UE15 0.60 1.75 8.5UE48 0.65 0.65 12.4

a The rates of [14Cjmaltose uptake were measured at 23 nM substrateconcentration and are given in picomoles per minute per 108 cells. The cellshad previously been tested for their ability to transport trehalose.

Transport of trehalose is inhibited by maltodextrins. Theaffinity of X receptor for maltose and maltodextrins is low butbecomes higher as the length of the maltodextrin chainincreases. By testing the different maltodextrins as inhibitorsfor trehalose uptake at different concentrations (0.1, 1, and10 mM), we observed an inhibition of trehalose transport atsubmicromolar concentrations (Table 4) that was compatiblewith the dissociation constant of X receptor for these malto-dextrins (1, 2, 12, 13).

DISCUSSION

We have demonstrated in this report that the effectiveuptake of the disaccharide trehalose depends on the partialinduction of the maltose system and that it is the X receptorwhich is responsible for this maltose system dependency.From the mutant analysis, it could not be excluded thatmalM, the gene distal to lamB (coding for the X receptor) isresponsible for the diffusion of trehalose. We feel thatpossibility is unlikely for the following two reasons. (i) ThemalM gene product, even though its function remains un-known, has been characterized as a periplasmic component(17). Therefore, the malMgene product should not be able toovercome the diffusion barrier of the outer membrane forany substrate. (ii) The ability of maltodextrins to inhibittrehalose transport reflects the affinity of the X receptortoward these maltotextrins.

It is expected that an inner membrane transport systemwith an affinity in the micromolar range that exhibits a Vm.sufficient to maintain growth should depend on a specificouter membrane porin. This expectation is particularly truefor carbon sources of disaccharide size and larger. Besidethe well-known case of maltose and maltodextrin diffusionby maltoporin through the outer membranes of severalgram-negative enteric bacteria (14), there is the sucrosesystem that depends on a specific outer membrane porin for

TABLE 4. Remaining uptake of trehalose at 23 nM substrateconcentration in the presence of increasing

concentrations of maltodextrinsa

% Uptakea of ['4C]trehalose with theMaltodextrin following concn (mM) of maltodextrin:

0.1 1.0 10.0

Maltose 95 95 70Maltotriose 95 50 15Maltopentaose 60 25 10Maltoheptaose 50 20 10

a Relative to the percent uptake in the absence of maltodextrins.

effective diffusion (31, 32). On the other hand, the E. colilactose system, which also can sustain sufficiently high flowrates of carbon, apparently does not depend on a specificouter membrane porin. Obviously, the drawback for notspending energy on making a specific porin is low affinity ofthe transport system. Thus, for the low-affinity lactosesystem, the diffusion rate of lactose through the nonspecificporins OmpC and OmpF is high enough to satisfy thedemand of the inner membrane system, even at concentra-tions below the Km of the transport system.The case of monosaccharides is interesting. There are

several transport systems for monosaccharides such asgalactose, ribose, or arabinose that exhibit an apparent Kmof uptake in the micromolar range. Most of these systemscan sustain growth at an reasonable rate. Yet, the functionsof specific porins in these systems have not been demon-strated. Either these monosaccharides are small enough toexhibit, even at micromolar concentrations, a high enoughdiffusion rate through the unspecific porins OmpC andOmpF or they also rely on the presence of the X receptor, asis the case for the trehalose system. In this case, it is likelythat even the uninduced level of the maltose system wouldbe sufficient to allow the flow rates at low substrate concen-trations.As we have shown in this report, for maximal efficiency of

trehalose utilization, the maltose system is induced bytrehalose metabolism to about 30% of the levels obtainedwith maltose as an inducer. This level is necessary andsufficient for maximal trehalose uptake. We have recentlyshown that glucose in combination with glucose-i-phos-phate, when produced internally, will induce the maltosesystem and will form internal maltose and maltotriose, whichinduces the maltose system (10). Internal glucose and mostlikely glucose-1-phosphate are the primary degradationproducts of trehalose metabolism. Thus, induction by treha-lose metabolism is not surprising.There is another feature of the maltose system that is

relevant in this context. The maltose system exhibits arelatively high uninduced level of expression that is due toendogenous induction by the formation of glucose andglucose-i-phosphate via gluconeogenesis and thus maltoseand maltotriose (10). Obviously, this level of induction willbe influenced by carbon sources other than trehalose, whichin turn regulate the expression of X receptor.The uninduced and thus endogenously induced level of

mal gene expression is subject to osmoregulation (8). Ex-pression is high only when the medium osmolarity is low andturned down upon the addition of 300 mM NaCl. Thus, ifsystems other than that for trehalose depend for theiroptimal efficiency on the uninduced level of X-receptorexpression, they will be affected at a high osmolarity of themedium. It is interesting to note that the trehalose-utilizingsystem also can no longer be induced by trehalose at highosmolarity (5, 23). Thus, not only is the trehalose uptakesystem repressed by high osmolarity but also the flow oftrehalose through the outer membrane will be curtailed bythe limitation of X receptor.

ACKNOWLEDGMENTS

We gratefully acknowledge the stimulating discussions with T.Ferenci on the diffusion properties of the X receptor.We received financial support from the Deuschte Forschungsge-

meinschaft (Sonderforschungsbereich 156) and the Fond der Deut-schen Chemischen Industrie. W.K. is the recipient of a fellowshipfrom the Cusanuswerk.

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