JOURNAL OF BACTERIOLOGY, Sept. 1993, p. 5655-56650021-9193/93/175655-11$02.00/0Copyright © 1993, American Society for Microbiology

Maltose and Maltotriose Can Be Formed Endogenously inEscherichia coli from Glucose and Glucose-i-Phosphate

Independently of Enzymes of the Maltose SystemKATJA DECKER, RALF PEIST, JOACHIM REIDL, MARINA KOSSMANN, BETTINA BRAND,

AND WINFRIED BOOS*

Department ofBiology, University ofKonstanz, 78434 Konstanz, Gennany

Received 8 March 1993/Accepted 6 July 1993

The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyzethe uptake and utilization of maltose and a,1-4-linked maltodextrins. The presence of these sugars in thegrowth medium induces the maltose system (exogenous induction), even though only maltotriose has beenidentified in vitro as an inducer (0. Raibaud and E. Richet, J. Bacteriol., 169:3059-3061, 1987). Induction isdependent on MalT, the positive regulator protein of the system. In the presence of exogenous glucose, themaltose system is normally repressed because of catabolite repression and inducer exclusion brought about bythe phosphotransferase-mediated vectorial phosphorylation of glucose. In contrast, the increase of free,unphosphorylated glucose in the cell induces the maltose system. A ptsG ptsM glk mutant which cannot growon glucose can accumulate ['4CJglucose via galactose permeases. In this strain, internal glucose is polymerizedto maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled. Thispolymerization does not require maltose enzymes, since it still occurs in malT mutants. Formation ofmaltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lackingphosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-l-phosphate entering the cells via a constitutive glucose phosphate transport system. malQ mutants, which lackamylomaltase, are constitutive for the expression of the maltose genes. This constitutive nature is due to theformation of maltose and maltodextrins from the degradation of glycogen.

The Escherichia coli maltose system consists of a malto-dextrin-specific pore (encoded by lamB) (17, 30) in the outermembrane and a binding-protein-dependent transport sys-tem in the cell envelope (encoded by malE malF malGmalK) (38), as well as one periplasmic enzyme (encoded bymalS) (35) and three cytoplasmic enzymes (encoded bymalQ, malP, and malZ) (27, 34, 41). Expression of all malgenes depends on the positive regulator MalT (33).The transport system (11, 12, 20, 24, 40) can recognize and

accumulate maltose and linear a,1-4-linked maltodextrins upto a chain length of seven glucose units (15). The majorenzymes of the system (see Fig. 1) are the cytoplasmicamylomaltase (MalQ) (42) and maltodextrin phosphorylase(MalP) (34). Amylomaltase recognizes maltotriose and largermaltodextrins (donors), cleaving off the reducing glucoseresidue and transferring the remaining dextrinyl residue ontothe nonreducing end of maltodextrin (acceptors), includingmaltose and glucose. With maltotriose, the smallest donorsubstrate, as well as with longer linear maltodextrins, amy-lomaltase thus produces glucose and longer maltodextrins(26). Maltodextrin phosphorylase subsequently releases glu-cose-i-phosphate from the nonreducing end of maltodextrinswith a minimal chain length of five glucose residues (37). Theglucose and glucose-l-phosphate are both transformed intoglucose-6-phosphate by glucokinase (encoded byglk) (9) andphosphoglucomutase (encoded by pgm) (1), respectively.The third cytoplasmic enzyme of the maltose system, mal-todextrin glucosidase (MalZ) (41), recognizes maltotrioseand longer maltodextrins up to maltoheptaose. It releases

* Corresponding author.

glucose from the reducing end of the dextrin chain. The endproducts of the degradation by maltodextrin glucosidase are

always glucose and maltose (41). Surprisingly, malQ strains,which lack amylomaltase, are unable to grow on maltotriose,despite the presence of maltodextrin glucosidase, even whenit is overexpressed; malZ mutants, which lack maltodextringlucosidase, grow normally on maltose and maltodextrins(41).The last enzyme of the maltose system, the product of the

malS gene, is the periplasmic a-amylase. This enzymepreferentially cleaves maltohexaose from the nonreducingend of longer maltodextrins. It also recognizes with lessefficiency smaller maltodextrins down to maltotriose thatcleave off maltose, maltotriose, maltotetraose, and mal-topentaose. ot-Amylase is not essential for growth on mal-tose or small dextrins but allows slow growth on longdextrins (16, 17).

In two respects, Fig. 1 is metabolically incomplete. First,for growth on maltose itself, a primer donor for the amylo-maltase reaction (maltotriose or a longer maltodextrin) isrequired. Second, the only coactivator of MalT in vitro ismaltotriose (29). The endogenous source of maltotriose isunknown. One possible clue was that malK mutants expressother mal genes constitutively, as if MalK might have a rolein eliminating the endogenous inducer (7). Furthermore,mutants in malI that no longer expressed malK-lacZ consti-tutively were isolated (32). MalI proved to be a repressor foran adjacent operon containing the genes malX and malY.While it is clear that only the overexpression ofmaWY affectsmal gene expression, the mechanism of repression is unclear(31). The present paper explores the issue of the source of

5655

Vol. 175, No. 17

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

5656 DECKER ET AL.

Maltose

Glucose A7

Maltose Il-altotriosema/Uk lucose A

Glucose Maltotetroseo Maltose -oe s+ 6lucose-i-P

ma/U,)k 1lucose /. /2 ma/PL.

GlucoseM._ MaltopenteoseMaltose - + ]Glucose-I-P

ma/UY Glucose So1MJPGlucose Maltohexeose

Maltose L. 6-4Gucose-l-Plucose z-,

GT lucose 4Maltoheptaose

ADP 4.-fi Glucose-6-P

Glucose-6-P

Itg sis

Glgcolgsis

FIG. 1. Maltose degradation by the maltose enzymes. The en-zymes amylomaltase (malQ), maltodextrin phosphorylase (malP),and maltodextrin glucosidase (malZ) are indicated by designation oftheir genes. After uptake, a maltosyl, maltotriosyl, or maltotetraosyl(and so on) residue is transferred from maltotriose, maltotetraose, or

maltopentaose (and so on) onto the incoming maltose, releasingglucose in the process. Maltopentaose and longer maltodextrins arerecognized by maltodextrin phosphorylase, forming glucose-i-phos-phate (P) and a maltodextrin that is smaller by one glucosyl residue.Maltodextrin glucosidase recognizes maltotriose and longer malto-dextrins (up to maltoheptaose), releasing glucose consecutivelyfrom the reducing end of the maltodextrins. This scheme demon-strates that maltose degradation by the maltose enzymes requiresthe endogenous presence of a maltodextrin primer with the mini-mum size of maltotriose. The activity of the last maltose enzyme,the periplasmic ot-amylase, is not considered in this scheme.

maltotriose and points neither to MalK nor to MalY as beinginvolved in inducer degradation.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterialstrains and plasmids used are described in Table 1. Precul-tures were grown in Luria broth, and then the main culturewas grown in minimal medium A (MMA) (25) with 0.4%carbon source. When the activity of the malK-lacZ fusionwas assayed (see Tables 6 and 8), the cultures were grown insixfold-diluted MMA. Indicator media were used as de-scribed by Miller (25). 5-Bromo-4-chloro-3-indolyl-3-D-ga-lactopyranoside (X-Gal) was used at 50 ,uM (final concentra-tion). Chloramphenicol, kanamycin, and tetracycline wereused at 25, 50, and 5 ,ug/ml, respectively. To induce themaltose system with glucose in strain UE26 and its deriva-tives, strains were grown in MMA containing 0.4% glycerolas the carbon source in the presence of 1 mM D-glucose and1 mM D-fucose. The latter was necessary for the induction ofthe galactose transport system needed for the uptake ofglucose.

Genetic techniques. Plvir transductions were done asdescribed by Miller (25). To construct strains with particulargenotypes, TnlO or TnS insertions within genes (malT,malQ, malP, gigA, glgC, and pgi) or next to desired muta-tions (malZ, pgm, glk, and uhpT) were used in Plvir-mediated transductions. For the removal of TnlO insertions,the technique described by Bochner et al. (3) was used.

Plasmid preparations and transformation were done accord-ing to the method described by Maniatis et al. (23).Enzymatic assays. ,-Galactosidase was assayed according

to the method described by Miller (25). Specific activity (inunits per milligram of protein) is given in micromoles ofortho-nitrophenyl-3-D-galactopyranoside (ONPGal) hydro-lyzed per minute per milligram of protein at room tempera-ture. Protein concentration was taken from the opticaldensity at 578 nm (OD578) of the bacterial culture, with thecorrelation that the OD578 of 1 is equivalent to 107 ,ug ofprotein per ml (25).The activity of oa-amylase (MalS enzyme) was measured

according to the procedure described by Freundlieb et al.(17) in intact cells (1 ml of bacterial suspension with anOD578 of 5.0) with 1 mM (final concentration) paranitrophe-nyl-a-maltohexaoside (PG6). Specific activity is given inpicomoles of paranitrophenol liberated per minute per 109cells at room temperature. The number of cells was calcu-lated from the OD578 of the culture as described above.Phosphoglucomutase activity was measured by the detec-

tion of glucose-6-phosphate formed from glucose-i-phos-phate with glucose-6-phosphate dehydrogenase. The assaymixture contained a 1-ml solution of 50 mM Tris-HCl (pH7.6), 10 mM MgSO4, 300 ,uM NADP, 0.2 mg of glucose-6-phosphate dehydrogenase (Boehringer GmbH, Mannheim,Germany), and 2mM glucose-i-phosphate. The reaction wasstarted by the addition of 10 to 50 ,ul of crude cellular extract(4 to 6 mg of protein per ml), and the increase in absorptionat 340 nm was recorded.Phosphoglucoisomerase was assayed in the same way as

that described for phosphoglucomutase activity, except thatglucose-i-phosphate was replaced by 1 mM fructose-6-phosphate. The specific activities (in units per milligram ofprotein) of both enzymes are given in micromoles of glucose-6-phosphate oxidized per minute per milligram of protein atroom temperature. Crude cellular extracts were obtained bythe use of a French pressure cell with an operating pressureof 16,000 lb/in2 and then by centrifugation at 30,000 x g for30 min. The supernatant was used without further treatment.

Radioactively labeled sugars. [14C]maltose was from Am-ersham. It was uniformly labeled and had a specific radio-activity of 589 mCi/mmol. [14C]glucose was also from Am-ersham. It was uniformly labeled and had a specificradioactivity of 270 mCi/mmol. [14C]maltotriose was labeledexclusively in the reducing glucose moiety. It was synthe-sized as described previously (41). Its specific activity wasnot determined.Transport of ['4Cjmaltose and [14CJglucose. Cultures were

grown overnight in MMA with 0.4% glycerol as the carbonsource. Optionally, 1 mM D-fucose, 1 mM D-glucose, or 1mM glucose-i-phosphate was added. The cultures werewashed three times with MMA and resuspended in MMA toan OD578 of 0.2 to 0.5. To 3 ml of the bacterial suspension,[14C]maltose or [14C]glucose was added, resulting in a finalconcentration of 60 nM (for maltose) or 120 nM (for glucose).Samples (0.5 ml) were withdrawn after different time inter-vals, filtered through Millipore filters with pore sizes of 0.45,um, and washed with 10 ml of MMA, and the radioactivitywas determined in a liquid scintillation counter. The rate ofuptake was determined from the linear increase in theaccumulated radioactivity and is given as picomoles ofsubstrate taken up per 10' cells.

Analysis of incorporated labeled sugars. Cultures preparedfor the transport assay as described above were resuspendedin MMA to an OD578 of 1. To 1 ml of the culture, [14C]glu-cose was added at a final concentration of 0.6 ,uM (in the

J. BACTERIOL.

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SYNTHESIS OF MALTOTRIOSE IN E. COLI 5657

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Known genotypea Origin orpasmi OW~~~~~~~~~~~~~~~~~~~~~~~~~~~~~referenceStrainsMC4100

BRE1162CB39CB17HS3084HS3166UE26 (ZCS112)ME250ME429REI7PGM1RP10RP11RP12RP13RP14RP15RP20RP40RP41KM500KM505KM506KM507KM510KM512KM513KM514KD27KD260KD261KD262KD263KD265KD266KD267KD268

F- araD139 A(araF-lac) U169 flbBS301 ptsF25 rbsR deoCI relAlrpsL150

MC4100; +(malK::lacZ) hyblll3MC4100; malQ::TnlOUE26; treA::TnlOMC4100; +(malK+-lamB-lacZ)42-1 (derived from strain SE1050)MC4100; malQ'F- glk-7pstG2 ptsMl rpsL150BRE1162; malP::TnlOBre1162; malI::TnlOME429; malI maLX malY TetsHfr3000; pgm-lRE17; pgm-1 zbf-3057::TnlORP10; TetsRP11; malP::TnlORP11; malT::TnlORP11; glgA::TnlORP11; glgC::TnlORE17; pgi::TnlOHS3084; pgm-1 zbf-3057::TnlOHS3084; pgi::TnlOHS3166; glgA::TnlOKD265;pgm-1 glk+ zfc-765::TnlOHS3166; malZ zaj::TnlOHS3166; malZ zaj::TnlO + (glgA-lacZ)RP11; malZ zaj::TnlORE17; glgA::TnlORP11; malQ::TnlOHS3166; amyA::KanUE26; recA srl::TnlOUE26; galU trpB::TnlOUE26; glgA::TnlOUE26; glgC::TnlOUE26; pgm-1 zbf-3057::TnlOKD263; pgin-1 Tet'KD265; glgA::TnlOKD265; uhpr KanrKD266; uhp7T Kanr

10

65This study14H. Shuman9732321This studyThis studyThis studyThis studyHengge-AronisHengge-AronisThis studyThis studyThis studyThis studyThis studyThis studyThis study41This studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis study

Cmr Tetr

pACYC184; malK+Ampr lacIqpTrc99; malYt

822231

a The uhpTh mutation was mobilized by Plvir transduction from a strain carrying a kanamycin resistance cassette in a deletion of the uhpB and uhpC genesand which had been selected for growth on fructose-6-phosphate. It probably carries a mutation in uhpA encoding the gene activator of the uhp operon resultingin the constitutive expression of the uhpT gene (18a).

case of maltotriose, the molarity could not be determined).After different time periods (1 to 60 min), the cells werecentrifuged in an Eppendorf centrifuge, and the supematantwas carefully removed. The pellet was resuspended in 15 plof 15% trichloroacetic acid (TCA) and kept on ice for 10 min.The suspension was centrifuged, and the supernatant wasapplied on thin-layer chromatography (TLC) plates (Kiesel-gel 60; Merck) and developed with butanol-ethanol-water(5:3:2 [vol/vol/vol]). After drying, the TLC plates wereautoradiographed, with times of exposure ranging from 3days to 2 weeks. When ['4C]maltose and [14C]maltotriosewere analyzed after accumulating in malQ strains (see Fig.9), 0.5-ml samples of cultures with an OD578 of 2.0 weretaken. The concentration of [(4C]maltose was 0.3 ,uM; thatof ['4C]maltotriose could not be determined. The incubationtimes were 5, 20, and 50 min.

In the experiments shown in Fig. 3 to 5, paper chroma-tography instead of TLC was used. For this purpose, theclarified TCA extract was diluted with water to 200 p,u1mixed-bed ion-exchange resin (about 100 to 150 ,ul) wasadded, and the supernatant was applied on Whatman 3MM.The solvent was also butanol-ethanol-water (5:3:2). Afterdrying, the paper was autoradiographed. For isolating themaltotriose-like compound, a long stripe containing thecompound was cut out and eluted with water by using thesame chromatography setup. The first 5 drops contained allof the radiography. The solution was rechromatographedonce to obtain the compound analyzed in Fig. 4. Thiscompound was analyzed by incubation with maltodextringlucosidase (MalZ) (see Fig. 4) in a manner analogous to thatused for the hydrolysis of maltotriose with this enzyme aspublished previously (41).

PlasmidspACYC184pMR11pTrc99pJR115

VOL. 175, 1993

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

5658 DECKER ET AL.

80 -

0 60 -

9, 40-

20-

0

D 60 120 180time lsect

0E

E°'8_xcs

0 60 120time lsecl

180

FIG. 2. Accumulation of ['4C]glucose and ["4C]maltose in aptsGptsM glk mutant. Strain CB17 (ptsG ptsM glk treA::TnlO) wasgrown with glycerol as a carbon source in the presence of 1 mMD-fucose (triangles), 1 mM D-fucose plus 1 mM D-glucose (squares),and 10 mM maltose (diamonds), and without additions icircles). (A)Uptake of 0.12 ,uM [ 4C]glucose; (B) uptake of 60 nM [ 4C]maltose.The results are given in picomoles of glucose or maltose taken up

per 109 cells. The correlation OD578 of 1.4 equal to 109 cells per ml(25) was used.

RESULTS

The induction of the maltose system by exogenous glucose.AptsGptsMglk mutant, lacking the glucose specific enzymeII's of the phosphotransferase system (PTS) as well asglucokinase (9), cannot grow on glucose but can accumulateglucose after induction of the galactose transport systemswith D-fucose (Fig. 2A). Under these conditions, with glyc-erol as a carbon source, the maltose system becomes in-duced (Fig. 2B; Table 2) by glucose. The induction variesfrom three- to fivefold over the uninduced (growth onglycerol) level, corresponding to about 10 to 20% of the fullinduction obtained with maltose (Fig. 2B). The minimalglucose concentration needed to induce the maltose systemin overnight cultures was 0.6 mM. Induction by glucoseindicated either that glucose activated MalT itself or that itwas transformed into the inducer, presumably maltotriose,even though it cannot be used as a carbon source under theseconditions.We tested whether internal free glucose could be the

substrate to synthesize maltotriose in this strain. UE26 (ptsGptsMglk) was grown on glycerol containing 1 mM D-fucose.

TABLE 2. Induction of the maltose transport system by glucoseinptsG ptsMglk mutantsa

Transport activity under the

Relevant

Stygenotypecerol Glycerol plus

glucose

UE26 3.0 12.7CB17 treA::TnlO 1.4 14.2KD260 galU 1.8 5.3KD262 glgC::TnlO 1.7 3.6KD261 glgA::TnlO 0.16 0.19KD27 pmnalK 0.26 0.33KD27 Vector 2.6 11.8UE26 pmalY NT 0.47UE26 Vector NT 9.0

a Transport activity is expressed in picomoles taken up per minute by 109cells at a maltose concentration of 60 nM at room temperature. All strainscarry the mutationsptsG, ptsM, and gik and were grown in the presence of 1mM D-fucose to induce the uptake of glucose. NT, not tested.

FIG. 3. Formation of [(4C]maltose and ["4C]maltotriose from[14C]glucose. Strain UE26 (ptsG ptsM giK) (A) was grown on

glycerol in the presence of 1 mM D-fucose. The cultures were notinduced with glucose. Isopropyl-o-D-thiogalactopyranoside (1 mM)was present in addition during growth of strain UE26 harboringplasmid pJR115 for induction of malY (B). The cultures were

incubated with 0.6 F.M [14C]glucose for the following lengths of time:lanes 1, 2 min; lanes 2, 5 min; lanes 3, 10 min; lanes 4, 20 min; lanes5, 30 min; lanes 6, 40 min; and lanes 7, 60 min. Cells were

centrifuged and extracted with TCA. The clarified and deionizedextracts were chromatographed on Whatman 3MM (solvent, buta-nol/ethanol/water [5/3/2]) and autoradiographed (incubation time, 3days). The controls were as follows: G, [1 C]glucose; M, [14C]mal-tose; T, ['4C]maltotriose.

The culture was given 0.6 PM [14C]glucose, and the accu-mulated radioactive products were separated by paper chro-matography. Figure 3 shows that label derived from glucosecould be incorporated into compounds that showed thechromatographic properties of maltose, maltotriose, andlarger maltodextrins. To identify the maltotriose-like com-pound, this spot was eluted from the chromatography paper.When treated with E. coli maltodextrin glucosidase (41), itwas hydrolyzed to [14C]glucose but not to [14C]maltose (Fig.

G M T 1 2

FIG. 4. Analysis of the ['4C]maltotriose formed by maltodextrin

glucosidase and paper chromatography. Lane 1, maltotriose-like

compound, eluted from the chromatogram shown in Fig. 3 and twice

rechromatographed; lane 2, the same compound incubated for 5 minwith maltodextrin~ lucosidase (MaIZ-enzyme). G, [14C]glucose; M,

I14C]maltose; T, [14C]maltotriose. The appearance of glucose in lane

2 demonstrates that lane 1 is indeed maltotriose and that only the

reducing glucose moiety of maltotriose is radiolabeled. The chro-

matography was as described in the legend to Fig. 3.

/ >r

J. BACTERIOL.

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SYNTHESIS OF MALTOTRIOSE IN E. COLI 5659

G M T 1 2 3 4 5 6 7 8 9 10FIG. 5. Effect of glgA::TnlO on the formation of ["4C]maltose

and ["4C]maltotriose from ["4C]glucose. Strain UE26 (ptsG ptsMglk) (lanes 1 to 5) and strain KD261 (ptsG ptsM glk glgA::TnlO)(lanes 6 to 10) were grown on glycerol in the presence of 1 mMD-fucose plus 1 mM D-glucose as an inducer. The washed cultureswere incubated with 0.6 pLM [14C]glucose for the following lengths oftime: lanes 1 and 6, 1 min; lanes 2 and 7, 4 min; lanes 3 and 8, 10 min;lanes 4 and 9, 30 min; lanes 5 and 10, 60 min. G, [14C]glucose; M,[14C]maltose; T, [(4C]maltotriose. The cells were treated as de-scribed in the legend to Fig. 3. The time of exposure of theautoradiogram was 10 days.

4). Since maltodextrin glucosidase (MalZ) cleaves glucoseresidues exclusively from the reducing end of maltodextrinsand since maltose is not cleaved by MalZ (41), it follows thatthe eluted compound is indeed maltotriose, and it is alsoclear that the formation in the cell of [14C]maltotriose from[14C]glucose took place by the addition of nonlabeled glu-cose moieties to the nonreducing end of [14CJglucose, form-ing first maltose and then maltotriose. At least the firstreaction could not be catalyzed by amylomaltase (MalQ).The presence or absence of glucose during growth affects

the efficiency with which glucose was used up (or maltotri-ose was formed) (compare Fig. 3A with Fig. 5, lanes 1 to 5).Glucose was used up faster and more maltotriose wasformed when the culture was preinduced with glucose.ADP-glucose or UDP-glucose is not involved in the forma-

tion of maltose and maltotriose from glucose. Using galU,glgC::TnlO, or malT derivatives of UE26 that lack UDP-

glucose pyrophosphorylase (galU), ADP-glucose pyrophos-phorylase (glgC), or all of the maltose enzymes (malT),respectively, we found that the same products were pro-duced from ["4C]glucose as those in UE26 (data not shown)and that the induction of the maltose transport system withglucose was comparable to that in UE26 (with the exceptionof the malT::TnlO derivative). Thus, the formation of mal-todextrins was not dependent on UDP-glucose, on ADP-glucose, or on enzymes whose expression is dependent on

MalT.The data obtained with the glgA::TnlO derivative of

UE26, resulting in the loss of glycogen synthase and (by thepolar effect of the TnlO insertion) probably also glycogenphosphorylase (encoded by glgP and distal to glgA [44]),were more difficult to understand. Induction by glucose no

longer occurred (Table 2), even though the incorporation of[I4C]glucose into maltose and maltotriose could still beobserved. The only difference from thegigA + strain was therate of incorporation of label into the maltodextrins.Whereas in the glgA+ strain substantial amounts of maltosewere already formed after 1 min, there was no maltose or

maltotriose visible in the glgA::TnlO derivative at that timepoint (Fig. 5). From this comparison alone, one wouldconclude that the synthesis of glycogen or at least thepresence of glycogen synthase was required for induction ofthe maltose system by glucose. This is clearly not the case,as will be analyzed below.A pgm mutation prevents the induction of the maltose

transport system by glucose in UE26. Since mutations pre-venting the formation of UDP-glucose and ADP-glucose hadno effect on the ability to form maltose and maltotriose fromglucose, it became unlikely that nucleotide-activated sugarswere involved as glucosyl donors in the formation of malto-triose. Therefore, we tested the effect of apgm mutation onthe ability of glucose to induce the maltose system in UE26.Phosphoglucomutase, the enzyme encoded by pgm, cata-lyzes the reversible transformation of glucose-i-phosphateinto glucose-6-phosphate. The introduction of apgm muta-tion (1) into UE26 resulted in the loss of induction of maltosetransport by glucose, and it also reduced the uninduced levelobserved after growth in the presence of glycerol (Table 3).When the pgm mutant was analyzed for the formation ofmaltotriose from [14C]glucose, no maltotriose could be found(Fig. 6A). After 10 min of incubation under conditions inwhich free internal glucose was nearly used up, the forma-tion of small amounts of maltose could be observed (Fig.6B). We found consistently that thepgm strain did not takeup glucose as efficiently as thepgm+ strain.

TABLE 3. Induction of the maltose transport system by glucose in ptsG ptsMglk mutants is dependent on glucose-l-phosphatea

Transport activity under the following growth conditions

Strain ~~~RelevantGlcr,Strain genotype Glycerol Glycerol and Glycerol and glucose, dglucose Glc-1-P Glucse-an

UE26 pgm+ 3.0 12.7 NT NTKD263 pgm 0.7 1.1 1.5 1.2KD265 pgm 1.1 0.45 1.3 0.7KD267 pgm uhpJ2 0.98 0.83 2.9 4.7KD268 glgA pgm 1.2 1.1 6.75 9.45

uhp7-a All strains are derivatives ofUE26 and carry the mutationsptsG,ptsM, and glk. They had been grown on glycerol in the presence of 1 mM D-fucose to induce

the uptake of glucose. Transport activity is expressed in picomoles taken up per minute by 109 cells at a maltose concentration of 60 nM at room temperature.NT, not tested; Glc-1-P, glucose-i-phosphate.

VOL. 175, 1993

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

5660 DECKER ET AL.

A _B~~~~~a_T1 2 3 4

G 1 2 3 4 5 6 7 8MT

FIG. 6. ['4C]maltotriose is not formed from [14C]glucose in apgm mutant. Strain UE26 (ptsGptsMglk) (A) was grown on glycerol in thepresence of 1 MM D-fucose plus 1 MM D-glucose. The cultures were incubated with 0.6 PM [14C]glucose for various lengths of time, and thecells were harvested, extracted with TCA, chromatographed on TLC, and autoradiographed. The following incubation times were used: lane1, 1 min; lane 2, 2.5 min; lane 3, 3.5; and lane 4, 7 min. Strain K.D268 (ptsGptsMglkpgm) was treated in the same way, and the followingincubation times were used: lane 5, 1 min; lane 6, 2.5 min; lane 7, 3.5 min; and lane 8, 7 min. (B) Strain K.D268, grown under the conditionsdescribed for panel A, was incubated with 0.6 p.M ['4Cqglucose for 10 min (lane 1) and 20 min (lane 2). The same culture was incubated with0.6 P.M ['4Cqglucose plus 1 mM glucose-i-phosphate for 10 min (lane 3) and 20 min (lane 4). The exposure time of the autoradiogram in panelA was 3 days; in panel B, it was 2 weeks. In addition to maltose and maltotriose, whose formation appears to be dependent on the presenceof endogenous glucose-i-phosphate, several other compounds (for instance, compounds a and b) in panel B are formed from [14C]glucoseindependently of thepgm mutation. Compound a is formed only from uniformly labeled glucose or from glucose labeled in the C-6 position,but not from glucose labeled in the C-i position. The origin or mode of synthesis of these compounds is unknown. 0, [14C]glucose; M,[14C]maltose; T, [14C]maltotriose.

The loss of inducibility by glucose in thepgm mutant wasclearly due to the lack of glucose-l-phosphate, since theaddition of this compound to the medium again allowedinduction by glucose. For the transport of glucose-i-phos-phate into the cell, a uhpl' (19) mutation had to be intro-duced. Glucose-i-phosphate alone also induced the maltosesystem in this strain, indicating the formation of glucosefrom glucose-i-phosphate. Possibly, glucose is formed fromthe partial hydrolysis of glucose-i-phosphate in theperiplasm (10), or it is formed inside the cell. Consistent withboth possibilities was the observation that the addition of 1mM unlabeled glucose-i-phosphate together with [14C]glu-cose strongly reduced the incorporation of label from[14C]glucose in any compounds (Fig. 6B), indicating that freeunlabeled and competing glucose was released from glucose-1-phosphate. Figure 7 depicts a scheme for the novel mal-tose-maltotriose phosphorylase in terms of its function ofproviding maltotriose as a donor substrate for the utilizationof maltose by amylomaltase. Figure 7 also explains both theinduction of the mal system by internal free glucose and theroles of glucose-l-phosphate and gluconeogenesis in basal-level expression of the mal system.As shown above, the absence of glycogen synthase

WlgA::TnIO) prevented the induction by glucose in apgm+strain. However, when the glgA::TnlO mutation was intro-duced into thepgm uhpW mutant, this strain could also beinduced for maltose transport by glucose and glucose-i-phosphate or by glucose-i-phosphate alone but not by glu-cose alone (Table 3). This demonstrated that glycogen orglycogen synthase was not required for induction. Theobservation that a glgA mutant prevents induction is most

likely due to the accumulation of ADP-glucose and itsfeedback inhibition on the synthesis of glucose-i-phosphate.The galactose blue phenotype inpgm mutants and the role of

the maltose system in the utilization of galactose. Mutants inpgm exhibit a blue phenotype (i.e., they stain blue in thepresence of iodine) when grown on galactose or on anothercarbon source in the presence of galactose. This was ex-plained by the accumulation of glucose-i-phosphate fromgalactose and the subsequent formation of maltodextrins bythe reversal of the normal reaction of maltodextrin phos-phorylase (1). The authors discussed the observation thatpgm mutants were still able to grow on galactose, eventhough the established catabolic pathway of galactose re-quires the transformation of glucose-i-phosphate to glucose-6-phosphate by phosphoglucomutase; the leakiness of thepgm mutation, the possibility of isoenzymes of phosphoglu-comutase, and the presence of phosphatases were men-tioned (1).We observed that growth on galactose in thepgm mutant

was dependent on the maltose system but that the galactoseblue phenotype was not. Table 4 lists the galactose growthphenotype ofpgm mutants carrying different mal mutations.Strains defective in the expression of all maltose genes(malT::TnlO) were unable to grow on galactose. Similarly, amutant defective in malP (malP::TnlO) or malQ (malQ::TnlO) was unable to grow on galactose. Also, when a malZmutation was introduced, growth on galactose was stronglyreduced, even though not as strongly as in the malP andmalQ or malT derivatives.To exclude the possibility that the synthesis of glycogen

was necessary for growth on galactose in the pgm mutant,

J. BA=rRIOL.

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SYNTHESIS OF MALTOTRIOSE IN E. COLI 5661

MlhltotetraosePi

fMaltotriose 6c-I-PPi

talmtotrios etialtose Sic-1-P

Pi

Glucose c4cC-l-P -+ ADP-61cc-A 6icogen/ t

PermeossATP PPI

/ 161c-6-P

Awl Glgcolgsis

Fru-6-P

6luconisogenesis I41~

tiPvruvote

FIG. 7. Endogenous formation of maltose and maltotriose andinduction of the maltose system by glucose. ADP-glucose pyrophos-phorylase (glgC), glycogen synthase (glgA), phosphoglucomutase(pgm), and phosphoglucoisomerase (pgi) are indicated by theirgenetic designations. MP, the novel maltose-maltotriose phosphor-ylase proposed in this paper. In contrast to maltodextrin phosphor-ylase and glycogen phosphorylase, this enzyme recognizes freeglucose and maltose and is responsible for the formation of shortmaltodextrins, including maltose and maltotriose. At present, it isunclear how free glucose is generated from glucose-l-phosphate.Endogenously formed maltotriose is used as a primer for maltosedegradation; it acts as an endogenous inducer of the maltose genesand is thus responsible for the relatively high uninduced level of themaltose system in a wild-type strain. An alternate way of formingmaltotriose is via the degradation of glycogen. This is most apparentin a malQ mutant. At present, it is unclear how glycogen is degradedto small maltodextrins. The function of the maltose enzymes seemsnot to be required.

we introduced a glgA::TnlO lacking glycogen synthase. Toour surprise, this also prevented growth of thepgm mutanton galactose (Table 4). However, it was clear that glycogensynthase is not required for growth; when glgC::TnlO wasintroduced into the pgm mutant, growth on galactose wasnormal. The glgC::TnlO mutation results in the lack ofADP-glucose pyrophosphorylase and, by polar effect on thedistal gigA gene, supposedly also glycogen synthase, thegene product ofglgA. We believe that it is the accumulationof ADP-glucose in the glgA mutant that becomes growthinhibiting when the mutant is exposed to galactose.As shown above, growth of thepgm mutant on galactose

required the presence of the cytoplasmic maltose enzymes.Aside from the formation of maltodextrins by amylomaltase(and recycling to glucose-i-phosphate by maltodextrin phos-phorylase), the only metabolically usable end product that isformed by these enzymes is glucose. Thus, subsequentphosphorylation of glucose to glucose-6-phosphate shouldbe necessary for growth on galactose in a pgm mutant.Indeed, apgm mutant defective inptsG,ptsM (encoding thetwo known enzyme II's of the PTS specific for glucose), andglk (encoding glucokinase) was unable to grow on galactose;however, the introduction of a wild-type glk+ gene restoredgrowth on galactose (Table 4). Thus, the utilization ofgalactose in a pgm mutant occurs via glucose-i-phosphate

TABLE 4. Growth of apgm mutant on galactose is dependent onthe maltose systema

Growth on Galactose blueStrain Relevant genotype gaaos phntegalactose phenotype

REI7 pgm+ +++RP10 pgm +++ ++RP11 pgm (Tets) +++ ++RP12 pgm malP - +KM513 pgm malQ - Mucoid growthRP13 pgm malT - ++KM510 pgm malZ + ++RP14 pgm glgA - ++RP15 pgm glgC +++ + +KD265 pgm glkptsGptsM - NTKM505 pgm glk+ ptsG ptsM + + NT

a All strains, with the exception of KD265 and KM505, carry the malK-lacZfusion of strain REI7 and are constitutive for the mal system. Growth ongalactose in these strains is scored after 2 days. The strains carrying the malP,malQ, malZ, and malT mutations develop outgrowing papillae on the galac-tose plates. The galactose blue phenotype is scored by the blue that developsafter exposure of colonies to iodine vapor. The strains were grown on minimalglucose plates in the presence of galactose. The use of glycerol as a carbonsource in the presence of more than 0.2mM galactose caused strong inhibitionof growth and the formation of outgrowing derivatives when thepgm mutationwas carried in strain REI7 (malK-lacZ, constitutive for the maltose system)and its derivatives. The nature of this growth inhibition is unclear, particularlysince some of the strains (those that are not defective in the mal enzymes)grow well on galactose alone. The growth inhibition by galactose was notobserved when the pgm mutation was carried in UE26 and its derivatives,KD265 and KM505 (see also Table 5). NT, not tested.

by the formation of maltodextrins and their degradation toglucose which can then be phosphorylated by glucokinase toglucose-6-phosphate, which then enters glycolysis.

Since growth on galactose of thepgm mutant was depen-dent on the maltose system, it was reasonable to assume thatthe galactose blue phenotype was also dependent on themaltose system. The best condition under which to observethe galactose blue phenotype ofpgm mutants was to growthe strains on MMA glucose plates in the presence ofgalactose. We found that the absence of maltose enzymesdid not prevent the galactose blue phenotype (Table 4). Thisindicated that maltodextrins, the cause for iodine blue stain-ing, were still formed from glucose-i-phosphate, presumablyby the same enzyme that forms maltose and small maltodex-trins from [14C]glucose and glucose-i-phosphate, as pro-posed in Fig. 7.

In the presence of the maltose enzymes, these maltodex-trins will then serve as primers for amylomaltase and malto-dextrin phosphorylase, leading to the formation of longermaltodextrins and glucose. The maltodextrins will be de-graded by amylomaltase and maltodextrin glucosidase toglucose, which will enter glycolysis after phosphorylation toglucose-6-phosphate.To demonstrate that galactose in apgm mutant can induce

the maltose system, we measured the specific activity of theperiplasmic a-amylase in aptsGptsMglkpgm mutant (Table5). Similarly, a malK+-lacZ fusion in which P-galactosidaseis fused via a short polypeptide of LamB to the last aminoacid of MalK (MalK-LamB-LacZ tribrid protein) and whichstill exhibits MalK activity (14) was measured for the induc-tion by galactose in apgm mutant (Table 6).

Clearly, galactose and, less effectively, lactose were ableto induce the maltose system in thispgm mutant. Inductionbecame less prominent in a strain (KM505) that did containa functional glucokinase, pointing to the role of free internalglucose for endogenous induction. Also, Table 5 shows that

VOL. 175, 1993

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

5662 DECKER ET AL.

TABLE 5. Induction of a-amylase by galactose and lactose in a pgm mutant"

Activity under the following growth conditionsStrain Relevant

genotype Glycerol Glycerol and Glycerol and Glycerol andgalactose lactose maltose

UE26 glkpgm' 4.5 2.8 7.9 58.0KD265 glkpgm 1.4 37.8 12.2 189KM505 glk+ pgm 1.1 5.4 8.0 50.0

a All strains are defective in PTS-mediated glucose uptake (ptsG ptsM). a-Amylase activity is given in picomoles of PG6 hydrolyzed per minute by 109 cells.

the presence of apgm mutation reduces the uninduced levelof mal gene expression that is seen with glycerol as a carbonsource, pointing to the role of endogenous induction for theuninduced state of the maltose system.

Glycogen is the source of endogenous inducer in malQmutants. When a malQ mutant was grown on glycerol, themaltose transport system was found to be expressed consti-tutively (Table 7). When a glgA::TnlO or a glgC::TnlOinsertion was introduced into the malQ mutant (Table 7),maltose transport was no longer constitutive but becameinducible by maltose. Thus, the formation of maltotriosefrom glycogen (13) must be responsible for the constitutivenature observed in malQ mutants. Furthermore, the actionof amylomaltase on maltotriose must be responsible for theloss. of endogenous induction. It is unclear how glycogen isdegraded to small inducing maltodextrins. Introduction of a

malZ or an amyA mutation, encoding maltodextrin glucosi-dase (41) and the newly discovered cytoplasmic amylase(28), respectively, did not lead to inducibility. Thus, theseenzymes are not involved in the production of maltotriosefrom glycogen.

Maltose and maltotriose are effective in vivo inducers of themaltose system. The dependence of induction in a malQgigAdouble mutant on different concentrations of maltose andmaltotriose is shown in Fig. 8. Surprisingly, maltose inducedeffectively at the micromolar level. Maltotriose resulted in a

somewhat higher level of induction, and its threshold levelwas somewhat lower than that for maltose. The fates of theaccumulated [14C]maltose and [14C]maltotriose in differentmalQ strains are indicated in Fig. 9. Accumulated [14C]mal-tose was not significantly transformed into maltotriose whenMalZ was present, irrespective of the presence of glycogensynthase. When MalZ was absent, the formation of malto-triose and maltotetraose from maltose could easily be ob-served. The formation of these sugars was independent ofglycogen synthase. We conclude that the formation of thesesugars from maltose is due to the enzyme (MP in Fig. 7) thatis also responsible for the formation of maltose and smallmaltodextrins from glucose and glucose-i-phosphate, as

TABLE 6. Induction of MalK+-LacZ by galactose in a

pgm mutant"

Activity under the following growth conditions

StrainGlycerol Glycerol and Glycerol and

galactose maltose

HS3084 (pgm+) 0.032 0.021 0.8RP40 (pgm) 0.027 0.13 0.41RP41 (pgi) 0.016 NT NT

I All strains harbor the malK+-lacZ fusion that exhibits MalK activity andallows growth on and transport of maltose. ,-Galactosidase activity is given inmicromoles of ONPGal hydrolyzed per minute per mllhigram of protein. NT,not tested.

demonstrated above. In the presence of MalZ, these sugarsare probably also formed but effectively degraded to maltoseby MalZ. It is unclear whether the small amounts of malto-triose remaining under these conditions are sufficient forinduction or whether maltose itself is responsible for induc-tion. Since in vitro assays showed that MalT-dependenttranscription was stimulated by less than 10 ,uM maltotriose,the small amounts of maltotriose, barely visible in Fig. 9A(blocks 1 and 3), that are formed from maltose, even in thepresence of MalZ, may be sufficient for induction.The fate of [14C]maltotriose accumulated in the malQam

mutant revealed a different picture. The [14C]maltotrioseused is labeled exclusively in the reducing glucose residue(41). In the presence of MalZ, significant degradation ofI14C]maltotriose was observed without significant formationof radioactively labeled maltose. This is due to the removalof [14C]glucose at the reducing end by the action of malto-dextrin glucosidase (MalZ) (41), which is followed by degra-dation of [14C]glucose to CO2. In contrast, in the absence ofMalZ the formation of [14Cjmaltose and [14C]maltotetraosecould be observed (Fig. 9B). This demonstrates that theremoval (as glucose-i-phosphate) and likely also the additionof (unlabeled) glucose moieties by glucose-i-phosphate oc-curred at the nonreducing end of maltotriose. As withmaltose, the presence or absence of glycogen synthase didnot alter the formation of the maltodextrin pattern. Again,we conclude that formation of shorter and longer dextrinsfrom maltotriose is due to the presence of the postulatedmaltose-maltotriose phosphorylase that would establish anequilibrium among glucose-i-phosphate, Pi, and small mal-todextrins.As can be seen in Fig. 9A, when maltose is accumulated to

high internal levels, the formation of another compound thatmigrates on TLC plates ahead of maltose can be observed.

TABLE 7. Expression of the maltose transport system indifferent malQ mutants"

Transport activity under thefollowing growth conditions

Strain Relevant genotypeGlycerol Glycerol and

maltose

HS3166 malQam 36.1 46.8KM500 malQam glgA 4.5 41.9KM506 malQ"" malZ 45.2 45.1KM514 malQ"amyamA 30.8 NTKM507 malQ"" malZ glgA 6.4 33.1

a Transport activity is expressed in picomoles taken up per minute by 109cells at a maltose concentration of 60 nM at room temperature. The presenceof maltose in the growth medium of the nmalQ strain frequently resulted in theloss of transport activity, most likely by the formation of malK or malTmutations (18). Short exposure to maltose during growth (<2 h) did notinterfere with the constitutive expression of maltose transport and was usedfor induction. NT, not tested.

J. BACTERIOL.

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SYNTHESIS OF MALTOTRIOSE IN E. COLI 5663

0.1 1.0 10 100 1000Inducer concentration [microM]

FIG. 8. The induction of maltose transport in a malQglgA strainby maltose and maltotriose. Strain KM500(malQ"mglgA::Tn1O) wasgrown in glycerol and for 2 additional h in the presence of theindicated concentrations of maltose (asterisks) or maltotriose (trian-gles). Transport of ["4C]maltose was measured at a concentration of60 nM and is given in picomoles of maltose taken up per minute by109 cells.

Similarly, the presence of maltotriose inside the cell yields acompound moving ahead of maltotriose. These compoundsare the acetylated derivatives of maltose and maltotriose.They are formed by the action of maltose transacetylase, anenzyme encoded by mac that is not part of the maltoseregulon (5). Acetyl maltose and acetyl maltotriose are irrel-evant for the induction phenomenon, since mutants lackingthese enzymes are still normally induced by maltose (5).The constitutive nature of a malK-lacZ fusion depends on the

presence of neither glucose-l-phosphate nor glycogen. As wehave demonstrated above, endogenous inducer can beformed from glucose-i-phosphate or can be derived fromglycogen. It was therefore an obvious conclusion that theconstitutive expression of a malK-lacZ fusion is due toendogenous induction. Therefore, a pgm glgA double mu-tant should no longer be constitutive. The introduction ofpgm alone into a strain carrying malK-lacZ did not preventconstitutive expression; similarly, aglgA::TnlO insertion didnot affect the constitutive expression of malK-lacZ. Also,the pgm glgA double mutant was still constitutive. In addi-tion, the introduction of a pgi mutation (encoding glucose-6-phosphate isomerase) into the malK-lacZ-carrying straindid not prevent the constitutive expression of the malK-lacZfusion when the strain was grown on glycerol (Table 8). Thisindicates that the high level of expression of malK-lacZ isnot due primarily to endogenous induction but representsgenuinely constitutive expression due to the function ofMalT without MalK, even in the absence of an inducer. Thestrength of this conclusion depends on the tightness of thepgm and pgi mutations. Assays for the two enzymes withcrude extracts gave values of 0.003 U/mg for the phospho-glucomutase in the mutant (RP40) versus 0.16 U/mg in thewild type (HS3084) and 0.003 U/mg for the glucose-6-phosphate isomerase in the mutant (RP41) versus 2.36 U/mgin the wild type (HS3084).MalK and MalY do not prevent the induction of the mal

genes by degrading maltose or maltotriose. The ability, de-scribed above, to label internal maltose or maltotriose withexternal [14CJglucose allowed us to test whether the overex-

T 2 13

FIG. 9. The fate of [14C]maltose and (14C]maltotriose after accu-

mulating in malQ strains. A 0.5-ml volume of washed cell suspen-

sions (0D578 of 2.0) was incubated with 0.3 p.M [14C]maltose (A) or

[14C]maltotriose (B). After 5, 20, and 50 min (three lanes per block),the cells were centrifuged and extracted with TCA, the clarified

extract was chromatographed on TLC, and the dried plate was

autoradiographed. All strains carry the malQ"" mutation. Block 1,HS3166 (glgA4' malZ'); block 2, KM506 (glgA' malZ); block 3,KM500 (g1gA malZ'); block 4, KM507 (gigA malZ). In the first lane,a mixture of authentic ['4C]maltose and [14C]maltotriose was ap-plied. AM, acetyl maltose.

pression of MalK or MalY would degrade or remove malto-

triose as predicted by our model. In Fig. 3B, the experimentwith overexpressed malY is shown. The same [14C]-labeledmaltodextrins were formed in the presence and absence of

MalY. If anything, the amount and rate of formation' of

maltose and maltotriose are higher in the strain overproduc-

TABLE 8. Mutations in pgm and pgi do not affect the

constitutive nature of a malK-lacZ fusione

Straina Relevant genotype a GaactIvitydase

REI7 pgm+ 1.15RP11 pgm 1.13KM512 glg 2.00RP14 pgm glgA4 0.92RP15 pgn glgC 1.05RP20 pgm pgi 0.85

aAll strains carry the same malK-lacZ fusion and lack the same malImalXmalY gene cluster as strain REI7.

b ,-Galactosidase activity is given in micromoles of ONPGal hydrolyzedper minute per milligram of protein.

VOL. 175, 1993

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

5664 DECKER ET AL.

ing MalY. However, the induction of the maltose system byglucose is abolished (Table 2). The same result was obtainedwhen malK was overexpressed in this strain (chromatogra-phy not shown; induction data in Table 2). This demon-strated that contrary to our previous conclusion, MalK aswell as MalY does not control the expression of the maltosegenes by affecting the level of the inducer but by repressionvia a still unknown mechanism.

DISCUSSION

We have presented evidence that maltose and maltotriosecan be produced endogenously in the absence of exogenousmaltodextrins. We have shown that maltose and maltotriosecan be formed from glucose and glucose-i-phosphate in aphosphorylase-type reaction by an enzyme that is not underMalT control (scheme in Fig. 7). We propose that maltotri-ose produced in this way yields the necessary primer sub-strate for the utilization of maltose by amylomaltase as wellas internal inducer. A pgm mutant no longer producesinternal maltotriose and is reduced in the basal level ofexpression. Induction of the maltose system by trehalose hasbeen observed in the past (36, 39). It is clear that thisinduction is not due to trehalose itself, since metabolism oftrehalose is required for induction to occur (21). As in thecase of maltose metabolism, the final products of trehalosemetabolism are also glucose and glucose-l-phosphate (4).We feel that even the normal induction of the maltose

system by exogenous maltodextrins in the wild type ismediated by the elevated synthesis of maltotriose fromglucose and glucose-i-phosphate (Fig. 7), the final degrada-tion products of maltodextrin metabolism, rather than fromthe direct intermediary products in this degradative pathway(Fig. 1).

Previously, it had been observed thatpgm mutants exhibita galactose blue phenotype when grown in the presence ofgalactose. This was interpreted by the accumulation ofglucose-i-phosphate, a product in the galactose catabolicpathway of E. coli, and subsequent formation of maltodex-trin by the mass action-driven reversal of the maltodextrinphosphorylase (1). However, the smallest maltodextrin thatis recognized by maltodextrin phosphorylase is maltopen-taose (37). Thus, the reversal of maltodextrin phosphorylasealone cannot account for the formation of maltose andmaltotriose. Primer molecules with a length of at least 4glucose moieties would have to be synthesized first. Wepropose that the synthesis of these small maltodextrins is theproduct of the still unidentified maltose-maltotriose phos-phorylase that we have described here.The origin of free glucose in the endogenous formation of

maltotriose is less clear. The observation that galactose (viathe formation of glucose-l-phosphate) can induce the mal-tose system in apgm mutant would indicate that glucose canbe liberated from glucose-i-phosphate, either directly or viathe formation of nucleotide-activated glucose. The latter isthe explanation for the origin of free galactose in the endo-genous induction of the galactose system (43).The maltose-maltotriose phosphorylase that we propose in

the present publication cannot exhibit high activity; other-wise, malQ mutants would be able to grow on maltose by thephosphorylase-mediated degradation of maltose to glucoseand glucose-l-phosphate. One would expect that mutationsin the gene or genes encoding this enzyme would have beenisolated among maltose minus mutants, since they wouldlack the ability to synthesize the primer necessary for thedegradation of maltose by amylomaltase. Possibly, these

mutations have escaped detection, since the usual prepara-tions of maltose used as a carbon source contain smallamounts of maltotriose that would be sufficient for primerfunction (26).One of the remaining problems in the understanding of

maltose regulation is the function of MalK in this process.malK mutants are constitutive (7, 13). In the past, weexplained this phenomenon through endogenous inductionand through the hypothesis that MalK would eliminateendogenous inducer. However, this model can no longer becorrect. As we have demonstrated, MalK does not eliminatemaltotriose, and endogenous synthesis of maltotriose is notrequired for the constitutive expression of the maltose genesin the absence of MalK. Thus, it follows that in the absenceof MalK, MalT, the central regulator ofmal gene expression,does not require an inducer. Our explanation for this at firstsurprising conclusion is that MalT occurs in an equilibriumof two states; one is active in stimulating transcription,whereas the other is not. We propose that the active state ofMalT is stabilized by binding the inducer maltotriose, whilethe inactive state is stabilized by an interaction with MalK.In the wild-type situation, the inducer maltotriose competeswith MalK for MalT. Therefore, the induction processrequires the presence of MalK.

ACKNOWLEDGMENTS

We obtained bacterial strains and plasmids from Bernhard Erni,Regine Hengge-Aronis, Michael Island, Wolfgang Klein, Bob Mac-nab, Maxime Schwartz, and Howard Shuman.

This study was supported by grants from the Deutsche For-schungsgemeinschaft (SFB156) and the Fond der Chemischen In-dustrie.

REFERENCES1. Adbya, S., and M. Schwartz. 1971. Phosphoglucomutase mu-

tants of Escherichia coli K-12. J. Bacteriol. 108:621-626.2. Amann, E., B. Ochs, and K Abel. 1988. Tightly regulated tac

promoter vectors useful for the expression of unfused and fusedproteins in Escherichia coli. Gene 69:301-315.

3. Bochner, B. R., H. Huang, G. L. Schieven, and B. N. Ames.1980. Positive selection for loss of tetracycline resistance. J.Bacteriol. 143:926-933.

4. Boos, W., U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P.Postma. 1990. Trehalose transport and metabolism in Esche-richia coli. J. Bacteriol. 172:3450-3461.

5. Brand, B., and W. Boos. 1991. Maltose transacetylase of Esch-erichia coli; mapping and cloning of its structural gene, mac,and characterization of the enzyme as a dimer of identicalpolypeptides with a molecular weight of 20,000. J. Biol. Chem.266:14113-14118.

6. Bremer, E., T. J. Silhavy, and J. M. Weinstock 1985. Transpos-able Aplac Mu bacteriophages for creating lacZ operon fusionsand kanamycin resistance insertions in Escherichia coli. J.Bacteriol. 162:1092-1099.

7. Bukan, B., M. Ehrmann, and W. Boos. 1986. Osmoregulation ofthe maltose regulon in Escherichia coli. J. Bacteriol. 166:884-891.

8. Chang, A. C., and S. N. Cohen. 1978. Construction and charac-terization of amplifiable multicopy DNA cloning vehicles de-rived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141-1156.

9. Curtis, S. J., and W. Epstein. 1975. Phosphorylation of D-glU-cose in Escherichia coli mutants defective in glucophos-photransferase, mannosephosphotransferase, and glucokinase.J. Bacteriol. 122:1189-1199.

10. Dassa, J., C. Marck, and L. Bouquet. 1990. The completenucleotide sequence of the Eschenichia coli gene appA revealssignificant homology between pH 2.5 acid phosphatase andglucose-1-phosphatase. J. Bacteriol. 172:5497-5500.

J. BACTERIOL.

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SYNTHESIS OF MALTOTRIOSE IN E. COLI 5665

11. Davidson, A. L., and H. Nikaido. 1991. Purification and charac-terization of the membrane-associated components of the mal-tose transport system from Escherichia coli. J. Biol. Chem.266:8946-8951.

12. Davidson, A. L., H. A. Shuman, and H. Nikaido. 1992. Mecha-nism of maltose transport in Escherichia coli; transmembranesignaling by periplasmic binding proteins. Proc. Natl. Acad. Sci.USA 89:2360-2364.

13. Ehrmann, M., and W. Boos. 1987. Identification of endogenousinducers of the mal system in Escherichia coli. J. Bacteriol.169:3539-3545.

14. Emr, S. D., and T. J. Silhavy. 1980. Mutations affecting local-ization of an Escherichia coli outer membrane protein, thebacteriophage lambda receptor. J. Mol. Biol. 141:63-90.

15. Ferenci, T. 1980. The recognition of maltodextrins by Esche-richia coli. Eur. J. Biochem. 108:631-636.

16. Freundlieb, S., and W. Boos. 1986. a-Amylase of Eschenchiacoli, mapping and cloning of the structural gene, malS, andidentification of its product as a periplasmic protein. J. Biol.Chem. 261:2946-2953.

17. Freundlieb, S., U. Ehmann, and W. Boos. 1988. Facilitateddiffusion of p-nitrophenyl-a-D-maltohexaoside through theouter membrane of Escherichia coli. J. Biol. Chem. 263:314-320.

18. Hofnung, M., M. Schwartz, and D. Hatfield. 1971. Complemen-tation studies in the maltose-A region of Escherichia coli K12genetic map. J. Mol. Biol. 61:681-694.

18a.Island, M. D. Personal communication.19. Island, M. D., B. Y. Wei, and R. J. Kadner. 1992. Structure and

function of the uhp genes for the sugar phosphate transportsystem in Escherichia coli and Salmonella typhimurium. J.Bacteriol. 174:2754-2762.

20. Kelierman, O., and S. Szmelcman. 1974. Active transport ofmaltose in Escherichia coli K-12. Involvement of a periplasmicmaltose-binding protein. Eur. J. Biochem. 47:139-149.

21. Klein, W., and W. Boos. 1993. Induction of the lambda receptoris essential for the effective uptake of trehalose in Escherichiacoli. J. Bacteriol. 175:1682-1686.

22. Kuhnau, S., M. Reyes, A. Sievertsen, H. A. Shuman, and W.Boos. 1991. The activities of the Escherichia coli MalK proteinin maltose transport, regulation and inducer exclusion can beseparated by mutations. J. Bacteriol. 173:2180-2186.

23. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

24. Manson, M. D., W. Boos, P. J. Bassford, and B. A. Rasmussen.1985. Dependence of maltose transport and chemotaxis on theamount of maltose-binding protein. J. Biol. Chem. 260:9727-9733.

25. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

26. Palmer, N. T., B. E. Ryman, and W. J. Whelan. 1976. The actionpattern of amylomaltase from Escherichia coli. Eur. J. Bio-chem. 69:105-115.

27. Pugsley, A. P., and C. Dubreuil. 1988. Molecular characteriza-tion of malQ, the structural gene for the Escherichia colienzyme amylomaltase. Mol. Microbiol. 2:473-479.

28. Raha, M., I. Kawagishi, V. Muller, M. Kihara, and R. M.Macnab. 1992. Eschenchia coli produces a cytoplasmic a-amy-

lase, AmyA. J. Bacteriol. 174:6644-6652.29. Raibaud, O., and E. Richet. 1987. Maltotriose is the inducer of

the maltose regulon. J. Bacteriol. 169:3059-3061.30. Randall-Hazelbauer, L., and M. Schwartz. 1973. Isolation of the

bacteriophage lambda receptor from Escherichia coli. J. Bacte-riol. 116:1436-1446.

31. Reidl, J., and W. Boos. 1991. The malX malY operon ofEschenchia coli encodes a novel enzyme II of the phos-photransferase system recognizing glucose and maltose and anenzyme abolishing the endogenous induction of the maltosesystem. J. Bacteriol. 173:4862-4876.

32. Reidi, J., K. Romisch, M. Ehrnann, and W. Boos. 1989. MalI, anovel protein involved in regulation of the maltose system ofEscherichia coli, is highly homologous to the repressor proteinsGalR, CytR, and Lacd. J. Bacteriol. 171:4888-4899.

33. Richet, E., and 0. Raibaud. 1989. MalT, the regulatory proteinof the Escherichia coli maltose system, is an ATP-dependenttranscriptional activator. EMBO J. 8:981-987.

34. Schinzel, R., and D. Palm. 1990. Escherichia coli maltodextrinphosphorylase; contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans. Biochemistry 29:9956-9962.

35. Schneider, E., S. Freundlieb, S. Tapio, and W. Boos. 1992.Molecular characterization of the MalT-dependent periplasmicalpha-amylase of Escherichia coli encoded by malS. J. Biol.Chem. 267:5148-5154.

36. Schwartz, M. 1967. Sur l'existance chez Escherichia coli K12d'une regulation commune a la biosynthese des recepteur dubacteriophage X et au metabolism du maltose. Ann. Inst.Pasteur 113:687-704.

37. Schwartz, M., and M. Hofnung. 1967. La maltodextrin phos-phorylase d'Eschenichia coli. Eur. J. Biochem. 2:132-145.

38. Shuman, H. A. 1982. Active transport of maltose in Escherichiacoli: role of the periplasmic maltose-binding protein and evi-dence for a substrate recognition site in the cytoplasmic mem-brane. J. Biol. Chem. 257:5455-5461.

39. Silhavy, T. H., I. Hartig-Beecken, and W. Boos. 1976. Periplas-mic protein related to the sn-glycerol-3-phosphate transportsystem of Eschenichia coli. J. Bacteriol. 126:951-958.

40. Szmelcman, S., M. Schwartz, T. J. Silhavy, and W. Boos. 1976.Maltose tr4nsport in Eschenchia coli K12. A comparison oftransport kinetics in wild-type and X-resistant mutants with thedissociation constants of the maltose binding protein as mea-sured by fluorescence quenching. Eur. J. Biochem. 65:13-19.

41. Tapio, S., F. Yeh, H. A. Shuman, and W. Boos. 1991. The malZgene of Escherichia coli, a member of the maltose regulon,encodes a maltodextrin glucosidase. J. Biol. Chem. 266:19450-19458.

42. Wiesmeyer, H., and M. Cohn. 1960. The characterization of thepathway of maltose utilization by Eschenchia coli. II. Generalproperties and mechanism of action of amylomaltase. Biochim.Biophys. Acta 39:427-439.

43. Wu, H. C. P. 1967. Role of the galactose transport system in theestablishment of endogenous induction of the galactose operonin Escherichia coli. J. Mol. Biol. 24:213-223.

44. Yu, F., J. Jen, E. Takeuchi, M. Inouye, H. Nakayama, M.Tagaya, and T. Fukui. 1988. a-Glucan phosphorylase fromEscherichia coli: cloning of the gene and purification andcharacterization of the protein. J. Biol. Chem. 263:13706-13711.

VOL. 175, 1993

on March 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from