Vol. 172, No. 6 JOURNAL OF BACTERIOLOGY, June 1990, p. 3450-3461 0021-9193/90/063450-12$02.00/0 Trehalose Transport and Metabolism in Escherichia coli WINFRIED BOOS,'* ULRIKE EHMANN,1 HUBERT FORKL,1 WOLFGANG KLEIN,' MARTINA RIMMELE,1 AND PIETER POSTMA2 Department of Biology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany,' and The E. C. Slater Institute for Biochemical Research, University of Amsterdam, 1018 TV Amsterdam, The Netherlands2 Received 2 January 1990/Accepted 27 March 1990 Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITreHIlI c)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EfiTre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate- and HTre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system. The synthesis of internal trehalose in Escherichia coli in response to high osmolarity has been studied in detail on a genetic and biochemical level (12, 30), yet little is known about trehalose transport and metabolism. Early reports have described E. coli mutants that were partially defective in the utilization of trehalose. The mutations mapped at 26 min on the linkage map (3; for Salmonella typhimurium, see reference 29). Marechal (20) later reported the existence of a specific enzyme II of the phosphoenolpyruvate (PEP):car- bohydrate phosphotransferase system (PTS) by demonstrat- ing the PEP-dependent phosphorylation of trehalose. He also claimed, on the basis of biochemical studies, the exist- ence of an enzyme able to hydrolyze trehalose-6-phosphate to glucose-6-phosphate and glucose (20). Different results were obtained by Postma et al. (23), who reported that trehalose is transported in S. typhimurium via the mannose- PTS without phosphorylation. Both studies reported the existence of a trehalose-inducible trehalase in crude ex- tracts. A periplasmic trehalase was subsequently discovered and purified from E. coli, and mutants, termed treA, were isolated that lacked this enzyme. treA was mapped at 26 min (5), and the treA gene was cloned, sequenced, and found to be the only gene in the operon (14). Periplasmic trehalase synthesis is not induced by trehalose but rather by growth in the presence of 250 mM NaCl (5). Apparently, the function of the periplasmic trehalase is to ensure the utilization of trehalose under conditions of high osmolarity by hydrolysis to glucose and subsequent uptake via PTS, when, as we will report here, transport and internal hydrolysis of trehalose are turned off. This repression of trehalose transport and trehalose hydrolysis at high osmolarity is physiologically necessary, since under these conditions synthesis of large amounts of internal trehalose occurs. Under these condi- * Corresponding author. tions, external trehalose can be utilized as a carbon source but it does not contribute to osmoprotection (16, 30). The steps leading to internal trehalose synthesis involve transfer of glucose from UDP-glucose to glucose-6-phos- phate, followed by the hydrolysis of the resulting trehalose- 6-phosphate to trehalose (13). Thus, to prevent futile cycles, the pathways of trehalose utilization and synthesis have to be under separate regulation. As a step to elucidate this complicated regulatory net- work, we report the characterization of the PTS-mediated and osmorepressible uptake of trehalose. We demonstrate that the utilization of trehalose is mediated by the formation of trehalose-6-phosphate during transport, its hydrolysis in the cytoplasm to trehalose by a specific trehalose-6-phos- phate phosphatase followed by the degradation of free trehalose. These studies on trehalose transport were made possible by the development of a convenient and fast method to synthesize ['4C]trehalose from ['4C]glucose by using intact bacteria (6). MATERIALS AND METHODS Bacterial strains. Bacterial strains are described in Table 1. They were grown under aeration in Luria broth (LB) or in minimal medium A (MMA) (21) with 0.2% carbon source. High-osmolarity minimal medium contained in addition 250 mM NaCl. Strains containing either the cya or the crp mutation had to be grown in MMA containing 1% Casamino Acids. Strain construction and cotransductional analysis were done by P1 vir-mediated transductions by the method of Miller (21). Mapping of treBC and treD was done by P1 vir transduction by using the collection of mapped TnJO inser- tions (28) as donors. First, P1 vir lysates of TnJO insertions comprising 10 min on the linkage map (2) were pooled and used to transduce KRIM3 (treD) and KRIM4 (treB) to Tetr and screened for the Tre phenotype on minimal trehalose plates. The single lysates of the responding pooled lysate 3450 on May 3, 2018 by guest http://jb.asm.org/ Downloaded from
Transcript
Vol. 172, No. 6JOURNAL OF BACTERIOLOGY, June 1990, p. 3450-34610021-9193/90/063450-12$02.00/0
Trehalose Transport and Metabolism in Escherichia coliWINFRIED BOOS,'* ULRIKE EHMANN,1 HUBERT FORKL,1 WOLFGANG KLEIN,'
MARTINA RIMMELE,1 AND PIETER POSTMA2
Department of Biology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany,' and The E. C. SlaterInstitute for Biochemical Research, University ofAmsterdam, 1018 TV Amsterdam, The Netherlands2
Received 2 January 1990/Accepted 27 March 1990
Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolaritysynthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can
serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalosemetabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins anddegradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by theenzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehaloseutilization begins with phosphotransferase (IITreHIlI c)-mediated uptake delivering trehalose-6-phosphate tothe cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one
glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzingthis reaction was named amylotrehalase. Amylotrehalase and EfiTre were induced by trehalose in the mediumbut not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkagemap but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase alloweddemonstration of the phosphoenolpyruvate- and HTre-dependent in vitro phosphorylation of trehalose. Thephenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.
The synthesis of internal trehalose in Escherichia coli inresponse to high osmolarity has been studied in detail on agenetic and biochemical level (12, 30), yet little is knownabout trehalose transport and metabolism. Early reportshave described E. coli mutants that were partially defectivein the utilization of trehalose. The mutations mapped at 26min on the linkage map (3; for Salmonella typhimurium, seereference 29). Marechal (20) later reported the existence of aspecific enzyme II of the phosphoenolpyruvate (PEP):car-bohydrate phosphotransferase system (PTS) by demonstrat-ing the PEP-dependent phosphorylation of trehalose. Healso claimed, on the basis of biochemical studies, the exist-ence of an enzyme able to hydrolyze trehalose-6-phosphateto glucose-6-phosphate and glucose (20). Different resultswere obtained by Postma et al. (23), who reported thattrehalose is transported in S. typhimurium via the mannose-PTS without phosphorylation. Both studies reported theexistence of a trehalose-inducible trehalase in crude ex-tracts.A periplasmic trehalase was subsequently discovered and
purified from E. coli, and mutants, termed treA, wereisolated that lacked this enzyme. treA was mapped at 26 min(5), and the treA gene was cloned, sequenced, and found tobe the only gene in the operon (14). Periplasmic trehalasesynthesis is not induced by trehalose but rather by growth inthe presence of 250 mM NaCl (5). Apparently, the functionof the periplasmic trehalase is to ensure the utilization oftrehalose under conditions of high osmolarity by hydrolysisto glucose and subsequent uptake via PTS, when, as we willreport here, transport and internal hydrolysis of trehaloseare turned off. This repression of trehalose transport andtrehalose hydrolysis at high osmolarity is physiologicallynecessary, since under these conditions synthesis of largeamounts of internal trehalose occurs. Under these condi-
* Corresponding author.
tions, external trehalose can be utilized as a carbon sourcebut it does not contribute to osmoprotection (16, 30).The steps leading to internal trehalose synthesis involve
transfer of glucose from UDP-glucose to glucose-6-phos-phate, followed by the hydrolysis of the resulting trehalose-6-phosphate to trehalose (13). Thus, to prevent futile cycles,the pathways of trehalose utilization and synthesis have tobe under separate regulation.As a step to elucidate this complicated regulatory net-
work, we report the characterization of the PTS-mediatedand osmorepressible uptake of trehalose. We demonstratethat the utilization of trehalose is mediated by the formationof trehalose-6-phosphate during transport, its hydrolysis inthe cytoplasm to trehalose by a specific trehalose-6-phos-phate phosphatase followed by the degradation of freetrehalose. These studies on trehalose transport were madepossible by the development of a convenient and fast methodto synthesize ['4C]trehalose from ['4C]glucose by usingintact bacteria (6).
MATERIALS AND METHODS
Bacterial strains. Bacterial strains are described in Table 1.They were grown under aeration in Luria broth (LB) or inminimal medium A (MMA) (21) with 0.2% carbon source.High-osmolarity minimal medium contained in addition 250mM NaCl. Strains containing either the cya or the crpmutation had to be grown in MMA containing 1% CasaminoAcids.
Strain construction and cotransductional analysis weredone by P1 vir-mediated transductions by the method ofMiller (21). Mapping of treBC and treD was done by P1 virtransduction by using the collection of mapped TnJO inser-tions (28) as donors. First, P1 vir lysates of TnJO insertionscomprising 10 min on the linkage map (2) were pooled andused to transduce KRIM3 (treD) and KRIM4 (treB) to Tetrand screened for the Tre phenotype on minimal trehaloseplates. The single lysates of the responding pooled lysate
Strain*Known genotypeSource of strain orStrain Known genotype relevant alleles
ZSC112L treA::TnlOUE49 Lac' at 250 mM NaClHF1 treA TetsUE49 Lac' at 250 mM NaCl lacking the biosynthetic trehalose-6-phosphate
phosphataseHF50 treA Tets lacking the biosynthetic trehalose-6-phosphate phosphataseHF57 A(treB-treC), loss of 4'(treC::lacZ) A placMuSS, zjh-920::TnJO, Kans, lacking
were then used for fine mapping (Table 2, crosses 7 to 17).TnJO insertions zjg-713 and zfc-765, located next to treB andtreD, were isolated from a pooled lysate of random TnlOinsertions into MC4100 (15) by transducing KRIM3 andKRIM4 to Tetr and screening for Tre+. The zfc-706::TnJOinsertion was isolated from the same pooled lysate bytransducing PPA169, a Apts strain (5), to Pts+.
Strain HF80 arose during the transduction ofzjh-920: :TnJO into HF57 (Table 2, cross 26). It is most likelythe result of a deletion (caused by the excision of the4'(treC::lacZ+) X placMuS5 bacteriophage) covering at leasttreC and treB and possibly other yet unknown tre genes. Thedeletion is cotransducible with zjh-920: :TnJO. Strains RIM30and RIM31 were obtained by transduction of treA::TnJO (5)into ME484 (galU) and subsequently into MC4100, selectingfor tetracycline resistance (Tet) and screening for Gal-.
Selection for Tetr was done without phenotypic expres-sion plating on DYT (21) containing 5 ,ug of tetracycline per
ml. N-Methyl-N'-nitro-N-nitrosoguanidine mutagenesis was
performed by the method of Adelberg et al. (1), and selectionfor loss of Tetr was by the method of Maloy and Nunn (19).
The lactose (Lac) phenotype of the treC: :lacZ fusion was
tested on plates containing 50 ,ug of X-Gal (5-bromo-4-chloro-3-indolyl-,-D-galactoside) per ml. Quantitative mea-
surements of ,-galactosidase activity in liquid cultures weredone by the method of Miller (21). The specific activity isgiven in units of micromoles of o-nitrophenyl-p-D-galacto-pyranoside hydrolyzed per minute per milligram of protein.Uptake of [14C]trehalose. Uptake of ['4C]trehalose in dif-
ferent strains was measured after growing the strains over-night in MMA with 0.2% glycerol or 0.2% trehalose or bothin the presence or absence of 250 mM NaCl. The cultureswere washed three times with MMA with or without 250 mMNaCl and resuspended in the same medium to an opticaldensity of 1.0 (at 578 nm) at room temperature.[14C]trehalose (540 mCi/mmol) (6) was added to a finalconcentration of 45 nM, and 0.5-ml samples were filtered atdifferent time intervals through filters (0.45-,um pore size;Millipore Corp., Bedford, Mass.) and washed with 10 ml ofMMA, with or without 250 mM NaCl, and their radioactiv-ities were counted. For measuring the Km of uptake, thesame procedure was repeated in the presence of increasing
Selection was always for Tetr.b Screening plates contained Tet. The fusion was constructed with XplacMu55, which carries Kanr.c The reason for the appearance of this type of transductant is unclear. Gene duplication may account for it.
concentration of unlabeled trehalose. Care was taken to mixlabeled and unlabeled trehalose before the addition of thecells. At substrate concentrations higher than 20 ,uM, theoptical density of the culture was increased to balance thereduction in the amount of radioactivity taken up.For analyzing the chemical alterations of [14C]trehalose
taken up, 0.5-ml samples of culture were incubated with 0.1,uM [14C]trehalose and washed once with MMA by centrif-ugation in an Eppendorf centrifuge. The cell pellet wassuspended in 20 IlI of 7% trichloroacetic acid (TCA), andafter centrifugation, the supernatant was transferred ontosilica-coated thin-layer chromatography (TLC) plates anddeveloped with butanol-ethanol-water (5:3:2) followed byautoradiography (see Fig. 4).To observe the secretion of glucose from strain CB17 after
incubation with trehalose, we grew CB17 overnight in MMAwith glycerol as the carbon source and trehalose as theinducer. The cells were washed and resuspended in MMAcontaining 25 mM trehalose at an optical density (578 nm) of40. Samples of 20 IlI were withdrawn at different times andcentrifuged, and the supernatant was chromatographed asdescribed above. For detection of glucose, the TLC plateswere sprayed with 20% sulfuric acid and charred.Enzymatic assays. Enzymatic assays of amylotrehalase
and trehalose-6-phosphate phosphatase activities were de-termined in crude cellular extracts. Stationary-phase cellswere harvested, washed, and suspended in 10 mM Trishydrochloride (pH 7.2) containing 0.1% mercaptoethanoland broken by passage through a French pressure cell at16,000 lb/in2. Cell debris were removed by centrifugation at
40,000 x g for 30 min at 4°C. Cellular extracts were usedeither directly or after dialysis against the same buffer for 5h. The incubation mixture (20 plA) with or without 10 mMMgCl2 contained about 5 mg of protein per ml and 0.02 p,Ciof ['4C]trehalose (about 2 ,uM). After different time intervals,20 pl was subjected to TLC as described above and autora-diographed.
Trehalose-6-phosphate phosphatase was tested in a similarway, using in the incubation mixture 10 mM unlabeledtrehalose-6-phosphate (18). After chromatography, the TLCplate was sprayed with 20o sulfuric acid and charred.
In vitro PTS activity in cell extracts was measured aspreviously described (26).
Accumulation of internal trehalose. Accumulation of inter-nal trehalose was tested by growing strains in 20 ml ofMMAwith glucose as the carbon source in the presence of 250 mMNaCl overnight. The culture was harvested and washed oncein the same medium without the carbon source, and thepellet was suspended in 0.2 ml of 5% TCA, incubated for 20min at 0°C, and centrifuged. Ion-exchange resin was added,and 50-,u samples were spotted onto silica-coated TLCplates, chromatographed as described above, and charredafter spraying with 20% sulfuric acid.
RESULTS
Transport of trehalose is inducible by trehalose and re-pressed at high osmolarity. Strain UE15 (MC4100 treA)lacking the periplasmic trehalase is still able to grow ontrehalose (5). Figure 1 shows the ability of UE15 to take up
time (sec)FIG. 1. Uptake of [(4C]trehalose in strains lacking the periplas-
mic trehalase. UE15 (wild type) after growth on glycerol (0),trehalose (0), and glycerol plus trehalose in the presence of 250 mMNaCl (l). A, KRIM3 (treD) after growth on glycerol and trehalose;A, KRIM4 (treB) after growth on glycerol and trehalose. All strainsincluding UE15 lacked the periplasmic trehalase. ["4C]trehaloseconcentration was 45 nM. The cell density was 1.0 optical densityunit (at 578 nm).
[14C]trehalose after growth on either glycerol or trehalose.Uptake after growth on trehalose was induced about 100-fold. However, the strain did not grow on trehalose in thepresence of 250 mM NaCl, and when grown in glycerol andtrehalose together in the presence of 250 mM NaCl, uptakeof [14C]trehalose was strongly reduced (Fig. 1). Uptake of[14C]trehalose was not inhibited by 250 mM NaCl aftergrowth on trehalose at low osmolarity (data not shown).Thus, it is the synthesis and not the activity of the trehaloseuptake system that is sensitive to high osmolarity. Measur-ing uptake in this strain (after growth on trehalose at lowosmolarity) at different substrate concentrations allowed thedetermination of the apparent Km as 16 ,uM and of the Vmaxas 9 nmol/min per 109 cells for the uptake of trehalose atroom temperature.
Isolation and characterization of mutants defective in utili-zation of trehalose. With strain UE15, N-methyl-N'-nitroso-N-nitrosoguanidine mutagenesis was performed and mutantswere isolated that exhibited a Tre- phenotype on MacCon-key-trehalose plates. Strains KRIM3 and KRIM4 were cho-sen for further studies. Both strains were unable to grow ontrehalose, but only KRIM4 exhibited strongly reduced trans-port activity (Fig. 1). Using a P1 lysate of pooled randomTnJO insertions into strain MC4100 (15), we isolated TnWOinsertions that were cotransducible with the tre mutation inKRIM3 (zfc-765: :TnJO) (Table 2, cross 1) and KRIM4(zjg-713::TnJO) (Table 2, cross 2). Using these insertions as
selective markers in phage P1-mediated transduction, we
found that the two tre mutations were not linked (Table 2,crosses 3 and 6). We named the gene carrying the mutationin KRIM4 treB and the gene carrying the mutation in KRIM3treD.
TABLE 3. Expression of 4D(treC::1acZ) after growth on glycerolin the presence and absence of 250 mM NaCl
250 mM 13-Galactosidase activityStrain 250 of the treC::lacZ fusion
NaCi (U/mg of protein)a
UE49 0.08+ 0.03
WK111 (treD) - 0.01+ 0.01
HF1 0.75+ 0.79
HF57 0.16+ 0.37
a Units are defined as micromoles of o-nitrophenyl-3-D-galactopyranosidehydrolyzed per minute.
In addition, a lacZ operon fusion was isolated by the XplacMu technique (7) in strain MC4100 (treA+), yieldingUE48, which exhibited a weak Tre- phenotype on MacCon-key-trehalose plates. After transduction to treA::TnJO(yielding strain UE49), it no longer grew on trehalose as asole carbon source. UE49 is still able to transport[14C]trehalose after growth on glycerol (data not shown).Therefore, the fusion must have occurred in a gene codingfor an enzyme involved in the metabolism of trehalose ratherthan in a gene necessary for transport. After growth onglycerol, transport of trehalose appeared somewhat higherthan the uninduced level observed in UE15. The presence oftrehalose in the growth medium did not induce transportactivity reproducibly but resulted in a fivefold reduction ofthe growth rate (compared with glycerol as the carbonsource). The ,B-galactosidase activity of the lacZ fusion aswell as the trehalose transport activity in strain UE49 wererepressed when the cells were grown on glycerol in thepresence of 250 mM NaCl. The results on the expression ofthe lacZ fusion are summarized in Table 3. We termed thegene carrying the lacZ fusion treC.Using TnJO insertions next to treB (zjg-713::TnlO) and
next to treD (zfc-765::TnJO) as selective markers in P1-mediated transduction, we found that treB and treC werecotransducible with zjg-713::TnJO (Table 2, cross 4), whiletreD mapped elsewhere (Table 2, crosses 5 and 6). However,when treD was introduced with the nearby TnJO into a straincarrying the treC: :lacZ fusion, yielding WK111, the activityof the fusion was strongly reduced (Table 2, crosses 5 and 6;Table 3), indicating a regulatory function for treD. treD hadonly a small effect on the trehalose transport system (encod-ed by treB) (Fig. 1).The collection of mapped chromosomal TnJO insertions
(28) was used to define the map position of treB (treC) andtreD. Table 2, crosses 7 to 17, and Fig. 2 show the result ofthe genetic analysis. Accordingly, treB and treC are locatedat about 96.5 min, and treD is located at about 51.5 min. Thelatter is close to the PTS genes ptsl, ptsH, and crr as well asglk; the gene encoding glucokinase (11). Further Pl-medi-ated transductional analysis involving glk and treD (Table 2,crosses 18 to 20) demonstrated that these genes are notidentical. However, several lines of evidence indicated thattreD is an allele of crr. A crr mutation from strain PPA168when transduced into WK101[V(treC::lacZ)] resulted in therepression of the fusion (data not shown), and introducingthe same crr mutation into UE15 resulted in a Tre- pheno-type (Table 2, cross 22). When the crr allele of PPA168 was
FIG. 2. Phage P1 cotransductional analysis of treBC and treD. Cotransduction frequencies are given in fractions of 1 (equal to 100o). Thearrows point to the selected markers, which were in all cases TnWO insertions. The TnlO insertions were obtained from a collection of mappedTnlO insertions (28). (A) Map position of treD; (B) map position of treBC. The solid lines refer to crosses with KRIM4 (treB) as the recipient;the dashed lines refer to crosses with UE48 [4(treC::lacZ)] as the recipient. Markers are not drawn to scale.
introduced into KRIM3, only 1.2% of the transductantsbecame Tre+ (Table 2, cross 21), compatible with thefrequency of crossover within the same gene. Furthermore,introducing a crp* mutation that renders the catabolite geneactivator protein independent of cyclic AMP (cAMP) (4) intoKRIM3 again allowed growth on trehalose (Table 2, cross
23). This is expected when a defective IIIC encoded by thecrr gene of KRIM3 no longer stimulates adenylate cyclasefor the synthesis ofcAMP. Thus, we conclude that treD is anallele of crr whose product still permits transport of glucoseand trehalose but that no longer stimulates adenylate cyclasefor sufficient production of cAMP needed for the expressionof treC. In line with this conclusion was the observation thatderivatives of UE15 lacking crp or cya (coding for adenylatecyclase) were no longer Tre+ (data not shown).Even though not causing a Tre- phenotype by itself, a glk
mutation can affect trehalose metabolism. A mutant (CB17)that is defective in the periplasmic trehalase (treA), themannose-PTS (ptsM) and glucose-PTS (ptsG), and in glu-cokinase (glk) still exhibited trehalose-inducible uptake of
trehalose (Fig. 3) but was unable to grow on trehalose. WhenCB17 was given 25 mM trehalose, glucose was excreted intothe medium (as detected by TLC of the medium supernatant[data not shown]). Growth of CB17 on trehalose was re-stored by introducing either ptsG+ or glk+ (Table 2, cross24). A mutation in glk alone had no effect on the utilizationof trehalose (Table 2, cross 20). This indicates that phos-phorylation of internal glucose by glucokinase or of incom-ing glucose via the PTS (after exit of free glucose) is part ofthe metabolic pathway of trehalose.Another mutation that affects the utilization of trehalose is
galU. galU codes for the enzyme forming UDP-glucose fromUTP and glucose-i-phosphate. The introduction ofgalU intoMC4100 via cotransduction with treA::TnlO, yielding strainRIM31, resulted in strongly reduced growth on trehalose. Asdiscussed below, both glk and galU affect most likely theutilization of trehalose at the level of the internal degradationof trehalose mediated by the enzyme encoded by treC.
Reduction of growth rate in presence of trehalose. StrainUE49 [F(treC::lacZ) treA] did not grow on trehalose as the
time (sec)FIG. 3. Uptake of ['4C]trehalose in the presence of glucose.
Uptake was measured as described in the legend to Fig. 1. Beforethe uptake, glucose was mixed with ["4C]trehalose to the followingfinal concentrations: 0 M (0); 1i-0 M (A); 10-4 M (O); 10-3 M (0).The upper set of curves represent the uptake in strain UE15 (wildtype); the lower two curves represent the uptake of trehalose inCB17 (ptsM ptsG glk) after growth in glycerol (-) and glycerol plustrehalose (0). Both strains lack the periplasmic trehalase.
sole source of carbon and, in addition, showed a fivefoldreduction in growth rate on glycerol when trehalose waspresent. The major product that accumulated after exposureto external trehalose was free internal trehalose (Fig. 4B).Similarly, KRIM3 carrying the treD mutation that stronglyreduces the expression of the treC::lacZ fusion and thereforealso the intact treC gene was sensitive to trehalose whengrown on glycerol. In both UE49 and KRIM3, the sensitivityto trehalose was abolished at high osmolarity, at whichtrehalose is no longer taken up but internal trehalose issynthesized and accumulated (13, 16, 30). Apparently, theaccumulation of trehalose that is beneficial at high osmolar-ity is inhibitory for growth at low osmolarity.
Isolation of mutants that express 4(treC::lacZ) constitu-tively at high osmolarity. By plating UE49 on lactose minimalplates containing 250 mM NaCl, we selected mutants thatexpressed the treC::lacZ fusion constitutively (Table 3).Two types of mutants were obtained. The first, representedby strain HF1, was, in contrast to its parent, no longersensitive to trehalose when grown on glycerol. The mutationwas not separable by P1 transduction from treC::1acZ, andthe introduction of treD into HF4, the Tets derivative ofHF1, did not result in a repression of the treC::lacZ fusion(Table 2, cross 25). The second type of constitutive mutant,represented by HF50, was still sensitive to trehalose whengrown on glycerol. This strain, as well as its Tets derivative,HF57, still transported trehalose but accumulated exclu-
,A~
b a 1 5 10 15 20
^
4^ o ,4
a b c 1 2 3 5 10
FIG. 4. Thin-layer chromatographic analysis of radiolabeledcompounds after uptake of [14C]trehalose. The cell density was 4optical density units (at 578 nm) with strain UE49 [4.(treC::lacZ)]and HF57. The optical density was 1 with strain UE15. Substrateconcentration was 0.1 ,uM [14C]trehalose. After the times indicated,0.5-ml samples were rapidly centrifuged, washed once with MMA,resuspended in 20 ,ul of MMA, and precipitated with 7% TCA. Thesoluble extract was chromatographed. (A) UE15 grown in trehalose;(B) UE49 [4(treC-IacZ)] grown in glycerol; (C) HF57 grown inglycerol. Standards: lanes a, glucose; lanes b, trehalose; lanes c,trehalose-6-phosphate. This compound is unstable and slowlybreaks down to trehalose. Times of incubation (minutes) are indi-cated in the figure.
a b c d e f 9 h i Tre GicFIG. 5. Accumulation of internal trehalose after growth in the presence of 250 mM NaCl. Strains were grown in MMA containing 250 mM
NaCl and with 0.2% glucose as the carbon source. TCA-soluble extracts were chromatographed on TLC plates, and carbohydrates were
visualized by charring with 20% sulfuric acid. Lanes: a, MC4100; b, UE15; c, UE60; d, UE49; e, HF1; f, HF57; g, UE61; h, UE62; i, HF80.Controls: Tre, trehalose; Glc, glucose.
sively trehalose-6-phosphate (Fig. 4C). Therefore, in thiscase, and in contrast to its parent, strain UE49, the trehalosesensitivity of strain HF57 must be caused by the accumula-tion of the phosphorylated sugar, a phenomenon also ob-served in other systems (31). We conclude that the loss of atrehalose-6-phosphate phosphatase activity is the cause forthe constitutive expression of the treC: :lacZ fusion in HF50and HF57. It follows that trehalose-6-phosphate is the in-ducer of treC. Trehalose-6-phosphate synthase is present atlow osmolarity, even though with low activity (13).
In addition to the trehalose sensitivity seen with HF50 andHF57, both strains also exhibited a pronounced sensitivitytoward 250 mM NaCl when growing on glucose. The reasonfor this salt sensitivity must be caused by the inability tosynthesize internal trehalose at high osmolarity. Thus, thetrehalose-6-phosphate phosphatase lacking in HF50 andHF57 must be part of the biosynthetic pathway for internaltrehalose.
treD, when transduced into HF57, was still able to repressthe constitutive expression of 't(treC: :IacZ) (data notshown). When the fusion was transduced into UE14 select-ing for kanamycin resistance (the gene of which is part of thefusion phage) (7), the constitutivity of eI(treC: :lacZ) was notcotransduced among 200 transductants. This indicates thatthe mutation resulting in the constitutive expression is notlocated next to F(treC::lacZ).There is strong though circumstantial evidence for the
existence of a second catabolic trehalose-6-phosphate phos-phatase. When HF57 was transduced to Tetr with a Pl lysateof a strain harboring zjh-920: :TnJO, located next to treC+, alltransductants that had lost the treC::lacZ fusion becameTre+ (Table 2, cross 26). The salt sensitivity (and theinability to synthesize internal trehalose at high osmolarity)was not crossed out in the above transduction. Since PTS-mediated growth on trehalose must include a trehalose-6-phosphate phosphatase activity (Fig. 4B and C), and sinceboth types of transductants are still lacking the biosynthetictrehalose-6-phosphate phosphatase, one must conclude thatthe transduction to Tre+ introduced a phosphatase activity.
Thus, E. coli contains two specific trehalose-6-phosphatephosphatases. One is responsible for salt resistance and ispart of the trehalose biosynthetic pathway at high osmolar-ity. This enzyme is defective in HF50 and HF57. The otherhas to be part of the catabolic pathway of trehalose. SinceHF57 did not show any trehalose-6-phosphate phosphataseactivity (Fig. 4C) and since the transductants that obtainedtreC+ can grow on trehalose, we must conclude that thegene coding for the second (catabolic) trehalose-6-phosphatephosphatase is located distal to treC in the same operon andthat its expression must therefore be lacking in strainscarrying the treC::lacZ fusion. We propose the name treEfor the gene encoding the catabolic trehalose-6-phosphatephosphatase.Both mutations that lead to constitutive expression of
'D(treC::lacZ) at high osmolarity also expressedD(treC::IacZ) constitutively at low osmolarity, but they didnot render the expression of the transport gene(s) insensitiveto salt. Transport of trehalose in both mutants remainedrepressible by growth at high osmolarity. Therefore, treBand treC, even though closely linked, do not seem to belocated in the same operon. This conclusion is corroboratedby the finding that treD repressed the expression of1D(treC::1acZ) (Table 3) but not the transport gene treB (Fig.1).
Salt sensitivity is accompanied by reduced ability to synthe-size internal trehalose. The lack of biosynthetic trehalose-6-phosphate phosphatase in strain HF57 [constitutive'D(treC::lacZ)] suggested that it no longer was able to syn-thesize internal trehalose at high osmolarity (250 mM NaCl).Figure 5 shows the TLC analysis of the supernatants ofcellular extracts after TCA precipitation of strains grown onglucose in the presence of 250 mM NaCl. The mutation thatleads to the constitutive expression of the treC::lacZ fusion(strain HF57) was accompanied by the reduced ability toaccumulate trehalose. Instead, a compound migrating onTLC as trehalose-6-phosphate could be recognized in theseextracts. The appearance of the small amounts of trehaloseeither might be due to the leakiness of the mutation in the
gene encoding the biosynthetic trehalose-6-phosphate phos-phatase or more likely could be a consequence of theinstability of trehalose-6-phosphate during the long growthperiod (overnight).The inability to synthesize and accumulate trehalose dur-
ing growth on glucose at 250 mM NaCl was not connected toa Tre- phenotype. Neither UE61 and UE62, representativesof Tre+ and Tre- transductants from cross 26 in Table 2(treC+ into HF57), accumulated trehalose (Fig. 5). Interest-ingly, the Tre+ transductant did not even accumulatetrehalose-6-phosphate. This is consistent with the presenceof a second trehalose-6-phosphate phosphatase in UE61,encoded by treE, distal to treC which had been activated byrestoration of treC+.HF1, the other mutant that expressed '1(treC::lacZ) con-
stitutively and whose mutation was closely linked to thefusion, still produced large amounts of internal trehalose athigh osmolarity (Fig. 5).Nature of trehalose transport system. Mutants deleted for
ptsI, ptsH, and crr and lacking in addition the periplasmictrehalase (treA) are unable to grow on or to transporttrehalose (5, 14). This pointed to a PTS involvement in thetrehalose transport system. Since earlier reports (23) ontrehalose transport in S. typhimurium implicated participa-tion of the ptsM-encoded mannose-PTS, we tested E. coliptsM ptsG mutants for growth and transport of trehalose.Strain UE50, a treA::TnJO derivative of the ptsM ptsGmutant LR2-168 (17), did not grow on glucose or mannosebut grew normally on trehalose. In addition; strain CB17, atreA::TnJO derivative of ZSC112L (11) carrying ptsM ptsGglk, exhibited trehalose-inducible uptake of [14C]trehalose(Fig. 3), even though it did not grow on trehalose. Thus, it isclear that trehalose transport in E. coli is not mediated byptsM or ptsG.A crr mutation coding for IIIG1c was introduced into strain
UE15 by P1 transduction with the help of a nearby TnWOinsertion (Table 2, cross 22), yielding strain UE42. Thisstrain did not transport trehalose nor did it grow ontrehalose, even in the presence of cAMP, pointing to a directinvolvement of IlIGIc in a PTS-mediated transport oftrehalose. Indeed, uptake of [14C]trehalose in UE15 (ptsG+)in the presence of increasing concentrations of glucoseshowed that glucose was able to interfere to a certain extentwith trehalose uptake (Fig. 3). This is consistent with a
competition for phosphorylated IIIGlc in the uptake processof both sugars.Observing the fate of [14C]trehalose after uptake into
UE15, a strain able to metabolize trehalose, the formation ofsecondary metabolic products is too fast for detecting theinitial product of accumulation (Fig. 4A). Strain UE49,carrying the treC: :lacZ fusion, still transported trehalose butcould not use it as a carbon source. Figure 4B shows a TLCanalysis of the intracellular radioactive material present afterincreasing time intervals of exposure to [14C]trehalose. Sur-prisingly, the major component was free trehalose and nottrehalose phosphate as expected for a PTS-mediated trans-port system. Therefore, the cytoplasm must contain a highlyactive trehalose-6-phosphate phosphatase preventing theaccumulation of trehalose-6-phosphate. Indeed, the test fortrehalose-6-phosphate-hydrolyzing activity in cellular ex-tracts, with a 10 mM concentration of unlabeled substrateand TLC anaIy`is, revealed high activity in extracts of strainUE15 yielding as a product trehalose. This activity wasincreased by a factor of two to three when the cells weregrown in the presence of 250 mM NaCl (data not shown).
Considering the mutation in HF57 which renders the
TABLE 4. PEP-dependent in vitro phosphorylation of trehalose
a Specific activity expressed as nanomoles per minute per milligram ofprotein.
b Membranes of strain KRIM4 were used in combination with 100,000 x gsupernatants of strain HF50.
treC::lacZ fusion constitutive, we argued that the loss of atrehalose-6-phosphate phosphatase might lead to an accumu-lation of trehalose phosphate, which in turn might be aninternal inducer of treC. Therefore, we also analyzed theradioactive products accumulated in HF57 after exposureto [14C]trehalose. This strain accumulated exclusively tre-halose phosphate (Fig. 4C). This indicated that trehalosetransport occurs with simultaneous phosphorylation, and itdemonstrated that hydrolysis by a trehalose-6-phosphatephosphatase must be the first step in the trehalose degrada-tion pathway.PEP-dependent phosphorylation of trehalose by cell ex-
tracts. Table 4 shows the rate of PEP-dependent phosphor-ylation of [14C]trehalose with extracts of strain HF5O(trehalose transport positive but lacking the trehalose-6-phosphate phosphatase). After separating membranes fromsoluble extracts by centrifugation, it became clear thatmembranes were required for phosphorylation. In addition,membranes of strain KRIM4 carrying the treB mutationwere unable to phosphorylate, even though they were stillable to phosphorylate methyl-a-glucoside. This demon-strates that E. coli contains a trehalose-specific enzyme IIthat is defective in treB mutants.The enzyme that degrades internal trehalose is not a simple
trehalase. As shown above, UE49 carrying the treC::lacZfusion accumulated free trehalose after incubation withexternal trehalose (Fig. 4B). Therefore, it must lack anenzyme degrading free trehalose and not trehalose-6-phos-phate. Accordingly, we assayed cell extracts of strain UE15lacking periplasmic trehalase for their ability to break downtrehalose. We found an activity acting on trace amounts of[14C]trehalose producing mostly larger glucose polymers;the activity was induced after growth on trehalose (Fig. 6a),was dependent on Mg2", and was inhibited by 1 mM glucose(data not shown). The activity was not present in extracts ofstrain UE49 (Fig. 6c).The hydrolyzing-polymerizing activity could only be de-
tected when low concentrations of labeled trehalose wereused. Attempts with 10 mM unlabeled trehalose were unsuc-cessful. We therefore conclude that the enzymatic reactionneeds a cosubstrate present in limiting amounts in theextract that acts as an acceptor in a glucosyl transferreaction. Thus, the product of treC most likely represents anenzyme analogous in its action to amylomaltase, the keyenzyme in the degradation of maltose and maltodextrins: onemolecule of glucose is released and the other is transferredonto a suitable acceptor. The acceptor is possibly a glucosepolymer as indicated by the requirement for UDP-glucose(product of GalU) for growth on trehalose. We named theenzyme amylotrehalase.The picture of degradation of internal trehalose is further
complicated by the observation that cytoplasmic extracts ofstrains that lack the periplasmic trehalase (treA mutants)
FIG. 6. Metabolism of trehalose by cellular extracts. Extracts were dialyzed against Tris hydrochloride (pH 7.2) followed by incubationwith 2 FM [14C]trehalose. At 0.5, 5, 20, 40, 60, and 120 min, 10-u.l samples were spotted on TLC plates, chromatographed, andautoradiographed. The times of incubation were equal. The extracts of the following strains were used: lanes a, UE15 grown on trehalose;lanes b, UE15 grown on glycerol; lanes c, UE49 grown on glycerol. Standards: lane d, trehalose; lane e, glucose.
contained low activity of an enzyme that degrades trehaloseto glucose. This low activity was observed in extracts of allstrains tested so far, including UE49 and HF50. It wasindependent of induction by trehalose but could not be seenafter the strains were grown at low osmolarity (data notshown). Thus, it is not part of the pathway degradingexternal trehalose after PTS-mediated transport. The signif-icance of this cytoplasmic trehalase is unclear at present. Itmay represent a means of preventing the increase to toxiclevels of internal trehalose at high osmolarity. The origin ofthis activity was not further investigated.
DISCUSSION
Trehalose can serve different functions in enteric bacteria.E. coli can both utilize trehalose as carbon source for growth(3, 5, 20) and synthesize trehalose intracellularly to protectitself from high osmotic strength in the growth medium (13,16, 30). This dual function of trehalose implies that bothpathways have to be separately regulated.
In a previous paper, we reported that E. coli contains aperiplasmic trehalase that produces glucose from trehalosein the periplasmic space. The glucose is subsequently takenup by PTS (5). It was found that treA mutants, defective inthis trehalase, still grow on trehalose, however. Theseresults suggested that still another pathway for trehaloseuptake and metabolism exists. The nature of this system wasstudied.Our present view of trehalose metabolism is summarized
in Fig. 7. The first step involves the transport and concom-itant phosphorylation catalyzed by the PEP:carbohydratePNS. We identified via mutation an enzyme 11Tre, specific fortrehalose, which together with the glucose-specific enzymeIII"'c delivers trehalose-6-phosphate into the cell. Enzyme11Trc is the third enzyme II that is phosphorylated via IlIc.Apart from enzyme lIGIc, it was shown previously that aplasmid-encoded enzyme IIScr, specific for sucrose, alsorequired IIIG'c (17, 25). Whereas most PNS carbohydrates,once phosphorylated, are channelled directly into glycolysis,trehalose phosphate is subsequently converted intotrehalose by a trehalose phosphate phosphatase. In practice,
this means that one PEP molecule is spent to transporttrehalose unchanged into the cell.We presented evidence for the existence of two enzymes
that can hydrolyze trehalose-6-phosphate to trehalose. One,whose absence causes constitutivity of the treC::lacZ fu-sion, is part of the biosynthetic pathway for the productionof internal trehalose at high osmolarity. The gene locus forthis enzyme has not been mapped. The other, less-well-defined enzyme must be encoded in the tre gene cluster at96.5 min, distal to but on the same transcriptional unit astreC.The degradation of free trehalose is mediated by an
enzyme that we termed amylotrehalase. It most likely liber-ates one glucose molecule of trehalose, whereas the secondglucose moiety is added to a glucose polymer, analogous tothe formation of maltodextrins in the case of maltose metab-olism (27). Glucose is converted into glucose-6-phosphatevia glucokinase.The necessity of the glucose polymer in trehalose metab-
olism is indicated by the effect of a galU mutation ontrehalose utilization. We expect the existence of a trehalose-specific transferase that is needed for the synthesis of thispolymer. According to our model, the elongated polymerwill be recycled most likely by a phosphorylase. At present,it is unclear whether the glucose polymer is of the maltodex-trin type and whether the enzymes of glycogen metabolismare part of this metabolic scheme. It is clear, however, thatmaltodextrin phosphorylase (27) is not essential, since malTmutants that no longer express any mal genes still grownormally on trehalose (5). At present, there is no directbiochemical or genetic evidence for either the transferase orthe phosphorylase.
It is clear that metabolism of trehalose involves the releaseof one of its two glucose moieties as free internal glucose.One would assume that growth on trehalose should still bepossible even if one prevented the utilization of this glucosemolecule. However, CB17, a strain defective in ptsM, ptsG,and glk, was unable to grow on trehalose even though it stilltransported trehalose (and released glucose into the medi-um). Possibly, the in vitro-observed feedback inhibition of
FIG. 7. Proposed pathways for trehalose synthesis and degradation at low and high osmolarities. The different steps are explained in theDiscussion. The straight black arrows indicate the reactions at high osmolarity; the open arrows indicate the reactions at low osmolarity. Thephosphorylase is purely speculative. P, Phosphate.
the amylotrehalase by glucose inhibits the metabolic flow toan extent that no longer permits growth.A second (cytoplasmic) trehalase was found in all extracts
of strains grown at 250 mM NaCl. This activity releasedglucose from trehalose, and it was low, only present at highosmolarity; from preliminary data, we estimate a Km of theenzyme in excess of millimolars. This activity was over-looked in our previous studies on the periplasmic trehalase.At present, we do not know what the origin and the functionof this enzyme are. Possibly, this enzyme limits the concen-tration of internal trehalose during growth at high osmolar-ity.
It is clear that the pathway described here for trehalosemetabolism is different from that proposed by Marechal (20)in E. coli and S. typhimurium. Marechal described a
trehalose phosphate hydrolase that splits trehalose phos-phate into glucose and glucose-6-phosphate. We have beenunable to detect such an enzymatic activity. Results ob-tained with ptsM mutants of S. typhimurium (23) alsopointed to a different pathway. Although an inducibletrehalase was detected in S. typhimurium, results with ptsHIand ptsM mutants suggested that trehalose was taken up by
the mannose-PTS in the absence of phosphorylation (23). S.typhimurium LT2 differs from E. coli in that it lacks theactivity of the periplasmic trehalase (W. Boos, unpublishedobservations).The pathway for trehalose catabolism depends on the
osmotic strength of the medium. Whereas wild-type treA+strains can grow on trehalose in both the absence andpresence of 250 mM NaCl, treA mutants grow only in thelow-osmotic-strength media (14). Regulation of the synthesis(or activity) of the various trehalose-metabolizing enzymesby osmotic strength is to be expected since the synthesis ofintracellular trehalose from UDP-glucose and glucose-6-phosphate (13, 16, 30) and hydrolysis of the resultingtrehalose-6-phosphate at high osmolarity should not becounteracted by further breakdown via amylotrehalase.Thus, at least amylotrehalase should be turned off at highosmolarity. This was, in fact, observed in the treC::lacZfusion strain.We then isolated mutants that expressed the treC::lacZ
fusion at high osmolarity, allowing growth on lactose in thepresence of 250 mM NaCl. One (HF1) carried a tightly linkedmutation that most likely occurred in the promoter region.
The other, HF50, and its Tets derivative, HF57, lacked thebiosynthetic trehalose-6-phosphate phosphatase. Thus, weconclude that trehalose-6-phosphate is the inducer for treCand that the biosynthetic trehalose-6-phosphate synthase(13) exhibits enough basal-level activity for the production ofinducer even at low osmolarity. Consistent with this conclu-sion is the observation that the expression of the treC::lacZfusion in HF57 is increased at high osmolarity and not instrain HF1 (Table 3). Trehalose-6-phosphate synthase activ-ity would be elevated in its activity at high osmolarity (13).This could mean that the repression of trehalose metabolismobserved at high osmolarity is primarily due to inducerdegradation rather than to a separate osmorepression sys-tem.The regulation of the expression of the different tre genes
is not yet understood. treB is induced when trehalose ispresent in the medium, again provided that the mediumosmolarity is low. From the pattern of trehalose hydrolysis(Fig. 6) in cellular extracts, one can conclude that treC isalso induced when trehalose is present in the medium.However, treB and treC are not in one operon, since mutantsthat express the treC::lacZ fusion constitutively at 250 mMNaCl do not express I1Tre (treB) under these conditions.Also, treC appears to be more sensitive to catabolite repres-sion than treB, since treB-mediated transport of trehalose ismuch less affected by treD than is expression of thetreC::lacZ fusion.The analysis of the treD mutation revealed the dual
function of IIIcGc in trehalose transport and utilization. Amutation in crr that no longer allows the transport of glucose(strain PPA168) also does not allow the transport oftrehalose. This implicated a role for IIGIc in the I1Tre_mediated uptake of trehalose. On the other hand, the IIGlIcof KRIM3 allows transport of glucose and trehalose butapparently is no longer able to stimulate adenylate cyclaseenough for a sufficient production of cAMP needed toexpress treC. Consistent with the conclusion that treD isallelic with crr is the observation that a defined crr mutation(from strain PPA168) prevents the expression of thetreC::lacZ fusion as efficiently as treD. In addition, theintroduction of a cya or a crp mutation, lacking entirely bothcAMP-CAP-dependent transcription, into UE15 (treA) abol-ished growth on trehalose. In contrast, the introduction of acrp* mutation into KRIM3 again allowed growth ontrehalose.We showed in this study that metabolism of trehalose is
regulated by the osmolarity of the medium. Whereas thesynthesis of the specific PTS-mediated transport system aswell as the enzyme degrading internal trehalose are turnedoff at high osmolarity, synthesis of internal trehalose isturned on. Each pathway relies on the function of a specifictrehalose-6-phosphate phosphatase, oppositely regulated byosmolarity. The genes for the catabolic enzymes are locatedin a cluster on the E. coli chromosome. Their regulationmust be rather intriguing. There are elements of substrate-dependent induction, dependence on the cAMP-CAP sys-tem, and osmoregulatory signals. The study of this intricateregulatory circuit will add to the understanding of osmoti-cally regulated systems in bacteria (10).The next step in the elucidation of trehalose metabolism
will be the analysis of the cloned genes already at hand.
ACKNOWLEDGMENTSThe following individuals contributed to this study: E. Bremer
isolated the treC::lacZ fusion; the P1 lysate of pooled and randomTnlO insertions was prepared by M. Ehrmann; B. Overhoff provided
us with authentic trehalose-6-phosphate; the first preliminary exper-iments in respect to the trehalose utilization in UE49 were done byE. Schneider. We are grateful to Carol Gross and her co-workers forproviding us with the collection of mapped TnlO insertions thatallowed us to easily map the isolated mutations. Mutants wereobtained from J. Beckwith, B. Brand, W. Epstein, B. Erni, J.Lengeler, and A. Str0m. Major improvements in the readability ofthis complicated manuscript were provided by G. Sweet.
Financial support was obtained from the Deutsche Forschungs-gemeinschaft (SFB 156) and the Fond der Chemischen Industrie.
LITERATURE CITED1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965 Optimal
conditions for the mutagenesis by N-methyl-N'-nitro-N-nitroso-guanidine in Escherichia coli K12. Biochem. Biophys. Res.Commun. 18:788-795.
2. Bachmann, B. J. 1987. Linkage map of Escherichia coli K-12,edition 7, p. 807-876. In F. C. Neidhardt, J. L. Ingraham, K. B.Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.),Escherichia coli and Salmonella typhimurium: cellular andmolecular biology. American Society for Microbiology, Wash-ington, D.C.
3. Becerra de Lares, L., J. Ratouchniak, and F. Casse. 1977.Chromosomal location of genes governing the trehalose utiliza-tion in Escherichia coli K-12. Mol. Gen. Genet. 152:105-108.
4. Blazy, B., and A. Ulhmann. 1986. Properties of cyclic AMP-independent gene activator proteins of Escherichia coli. J. Biol.Chem. 261:11645-11649.
5. Boos, W., U. Ehmann, E. Bremer, A. Middendorf, and P.Postma. 1987. Trehalase of Escherichia coli. Mapping andcloning of its structural gene and identification of the enzyme asa periplasmic protein induced under high osmolarity growthconditions. J. Biol. Chem. 262:13212-13218.
6. Brand, B., and W. Boos. 1989. Convenient preparative synthesisof ["C]trehalose from ['4C]glucose by intact Escherichia colicells. Appl. Environ. Microbiol. 55:2414-2415.
7. Bremer, E., T. J. Silhavy, and G. M. Weinstock. 1988. Trans-position of lambda placMu is mediated by the A protein and itscarboxy-terminal end. Gene 71:177-186.
8. Brickman, E., L. Soll, and J. Beckwith. 1973. Genetic charac-terization of mutations which affect catabolite-sensitive operonsin Escherichia coli including deletions of the gene for adenylatecyclase. J. Bacteriol. 116:582-587.
9. Casadaban, M. J. 1976. Transposition and fusion of the lac-genes to selected promotors in Escherichia coli using bacterio-phage lambda and Mu. J. Mol. Biol. 104:541-555.
10. Csonka, L. N. 1989. Physiological and genetic responses ofEscherichia coli to osmotic stress. Microbiol. Rev. 53:122-147.
11. Curtis, S. J., and W. Epstein. 1975. Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephos-photransferase, mannosephosphotransferase, and glucokinase.J. Bacteriol. 122:1189-1199.
12. Dinnbier, U., E. Limpinsel, R. Schmid, and E. P. Bakker. 1988.Transient accumulation of potassium glutamate and its replace-ment by trehalose during adaptation of growing cells of Esche-richia coli K-12 to elevated sodium chloride concentrations.Arch. Microbiol. 150:348-357.
13. Giaever, H. M., 0. B. Styrvold, I. Kaasen, and A. R. Str0m.1988. Biochemical and genetic characterization of osmoregula-tory trehalose synthesis in Escherichia coli. J. Bacteriol. 170:2841-2849.
14. Gutierrez, C., M. Ardourel, E. Bremer, A. Middendorf, W.Boos, and U. Ehmnan. 1989. Analysis and DNA sequence of theosmoregulated treA gene encoding the periplasmic trehalase ofEscherichia coli K-12. Mol. Gen. Genet. 217:347-354.
15. Kleckner, N., J. R. Roth, and D. Botstein. 1977. Geneticengineering in vivo using translocatable drug resistance ele-ments. J. Mol. Biol. 116:125-159.
16. Larsen, P. I., L. K. Sydnes, B. Landfald, and A. R. Strom. 1987.Osmoregulation in Escherichia coli by accumulation of organicosmolytes: betaines, glutamic acid, and trehalose. Arch. Micro-biol. 147:1-7.
17. Lengeler, J., R. J. Mayer, and K. Schmid. 1982. Phosphoenol
pyruvate-dependent phosphotransferase system enzyme III andplasmid-encoded sucrose transport in Escherichia coli K-12. J.Bacteriol. 151:468-471.
18. MacDonald, D. L., and R. Y. K. Wong. 1964. A chemicalsynthesis of trehalose 6-phosphate. Biochim. Biophys. Acta86:390-392.
19. Maloy, S., and W. D. Nunn. 1981. Selection for loss of tetracy-cline resistance by Escherichia coli. J. Bacteriol. 145:1110-1112.
20. Markchal, L. R. 1984. Transport and metabolism of trehalose inEscherichia coli and Salmonella typhimurium. Arch. Microbiol.137:70-73.
21. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.
22. Postma, P. 1987. Phosphotransferase system for glucose andother sugars, p. 127-141. In F. C. Neidhardt, J. L. Ingraham,K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger(ed.), Escherichia coli and Salmonella typhimurium: cellularand molecular biology. American Society for Microbiology,Washington, D.C.
23. Postma, P. W., H. G. Keizer, and P. Koolwijk. 1986. Transportof trehalose in Salmonella typhimurium. J. Bacteriol. 168:1107-1111.
24. Sabourin, D., and J. Beckwith. 1975. Deletions of the Esche-richia coli crp gene. J. Bacteriol. 122:338-340.
25. Schmid, K., M. Schupfner, and R. Schmitt. 1982. Plasmid-
mediated uptake and metabolism of sucrose by Escherichia coliK-12. J. Bacteriol. 151:68-76.
26. Scholte, B. J., A. R. J. Schuitema, and P. W. Postma. 1982.Characterization pf factor Ill"c in catabolite repression-resis-tant (crr) mutants of Salmonella typhimurium. J. Bacteriol.149:576-586.
27. Schwartz, M. 1987. The maltose regulon, p. 1482-1502. In F. C.Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M.Schaechter, and H. E. Umbarger (ed.), Escherichia coli andSalmonella typhimurium: cellular and molecular biology. Amer-ican Society for Microbiology, Washington, D.C.
28. Singer, M., T. A. Baker, G. Schnitzler, S. M. Deischel, M. Goel,W. Dove, K. J. Jaaks, A. D. Grossman, J. W. Erickson, andC. A. Gross. 1989. A collection of strains containing geneticallylinked alternating antibiotic resistance elements for geneticmapping of Escherichia coli. Microbiol. Rev. 53:1-24.
29. St. Pierre, M. L. 1968. Isolation and mapping of Salmonellatyphimurium mutants defective in the utilization of trehalose. J.Bacteriol. 95:1185-1186.
30. Str0m, A. R., P. Falkenberg, and B. Landfald. 1986. Genetics ofosmoregulation in Escherichia coli: uptake and biosynthesis oforganic osmolytes. FEMS Microbiol. Rev. 39:79-86.
31. Yarmolinsky, M. B., H. Wiesmeyer, H. M. Kalckar, and E.Jordan. 1959. Hereditary defects in galactose metabolism inEscherichia coli mutants. II. Galactose-induced sensitivity.Proc. Natl. Acad. Sci. USA 45:1786-1791.
![The Role of Trehalose 6-Phosphate in Crop Yield and … · 2020. 5. 18. · Update on Trehalose 6-Phosphate Signaling The Role of Trehalose 6-Phosphate in Crop Yield and Resilience1[OPEN]](https://static.documents.pub/doc/80x56/60a94aac2e9d0b10d12c4d11/the-role-of-trehalose-6-phosphate-in-crop-yield-and-2020-5-18-update-on-trehalose.jpg)
