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):carbohydrate phosphotransferase system (PTS) by demonstrating the PEP-dependent phosphorylation of trehalose. He also claimed, on the basis of biochemical studies, the existence 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 mannosePTS without phosphorylation. Both studies reported the existence of a trehalose-inducible trehalase in crude extracts. 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-

*

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-phosphate, followed by the hydrolysis of the resulting trehalose6-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 network, 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-phosphate 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 insertions (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

Corresponding author. 3450

TREHALOSE METABOLISM IN E. COLI

VOL. 172, 1990

3451

TABLE 1. Bacterial strains Known genotype Strain*Known Strain

CB17 HF1 HF4 HF50

HF57 HF80 KRIM3 KRIM4 LR2-168 MC4100 ME484 PPA168 RIM30 RIM31 UE14 UE15 UE36 UE37 UE42, WK138 UE43 UE48 UE49 UE50 UE60 UE61 UE62 WK100 WK101

WK111 WK114 WK139 WK140 ZSC112L 18483 18467 18468 18470 18469 18427 12073 12019 18429

ZSC112L treA::TnlO UE49 Lac' at 250 mM NaCl HF1 treA Tets UE49 Lac' at 250 mM NaCl lacking the biosynthetic trehalose-6-phosphate phosphatase HF50 treA Tets lacking the biosynthetic trehalose-6-phosphate phosphatase HF57 A(treB-treC), loss of 4'(treC::lacZ) A placMuSS, zjh-920::TnJO, Kans, lacking the biosynthetic trehalose-6-phosphate phosphatase UE15 treD UE15 treB F- argG6 galT hisl manI metB thi nagE phoA ptsG ptsM F- araD139 A(argF-lac)UJ69flbB5301 ptsF25 rbsR relAl rpsLISO MC4100 4(malK::lacZ)347 X placMuSS (Kan) galU crr hsd lac leu met ptsL ptsM ptsP thr zfc::TnlO MC4100 treA::TnJO galU+ MC4100 treA::TnlO galU MC4100 treA::TnlO UE14 treA Tets KRIM4 treB+ zjg-713::TnlO KRIM4 zjg-713::TnlO UE15 crr zfc::TnlO ZSC112L zfc-706::TnlO MC4100 CF(treC::lacZ) X placMuSS (Kanr) treA+ UE48 treA::TnlO LR2-168 treA::TnlO MC4100 otsA::TnlO HF57 treC+ zjh-920::TnlO Kans HF57 zjh-920::TnlO UE15 treD zfc-765::TnJO UE15 4(treC::lacZ) X placMuS5 (Kan) WK101 treD zfc-765::TnlO MC4100 zfc-765::TnJO UE15 Acya ilv::TnJO UE15 Acrp zhd-732::TnlO ptsG ptsM glk

fadL-771::TnlO

zjb-J::TnJO nupCS0::TnlO purC80::TnlO

gua-26::TnlO zje-2241: :TnJO cycA30: :TnlO zjh-920::TnlO zji-6: :TnJO

were then used for fine mapping (Table 2, crosses 7 to 17). TnJO insertions zjg-713 and zfc-765, located next to treB and treD, were isolated from a pooled lysate of random TnlO insertions into MC4100 (15) by transducing KRIM3 and KRIM4 to Tetr and screening for Tre+. The zfc-706::TnJO insertion was isolated from the same pooled lysate by transducing PPA169, a Apts strain (5), to Pts+. Strain HF80 arose during the transduction of zjh-920: :TnJO into HF57 (Table 2, cross 26). It is most likely the result of a deletion (caused by the excision of the 4'(treC::lacZ+) X placMuS5 bacteriophage) covering at least treC and treB and possibly other yet unknown tre genes. The deletion is cotransducible with zjh-920: :TnJO. Strains RIM30 and RIM31 were obtained by transduction of treA::TnJO (5) into ME484 (galU) and subsequently into MC4100, selecting for tetracycline resistance (Tet) and screening for Gal-. Selection for Tetr was done without phenotypic expression 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 selection for loss of Tetr was by the method of Maloy and Nunn (19).

Source of strain or

relevant alleles

This This This This

study study study study

This study This study This study This study J. Lengeler (17) 9 M. Ehrmann P. Postma (5) This study This study 5 This study This study This study This study This study This study This study This study A. Str0m (13) This study This study This study This study This study This study J. Beckwith (8) J. Beckwith (24) W. Epstein (11) C. Gross (28) C. Gross (28) C. Gross (28) C. Gross (28) C. Gross (28) C. Gross (28) C. Gross (28) C. Gross (28) C. Gross (28)

The lactose (Lac) phenotype of the treC: :lacZ fusion was tested on plates containing 50 ,ug of X-Gal (5-bromo-4chloro-3-indolyl-,-D-galactoside) per ml. Quantitative measurements of ,-galactosidase activity in liquid cultures were done by the method of Miller (21). The specific activity is given in units of micromoles of o-nitrophenyl-p-D-galactopyranoside hydrolyzed per minute per milligram of protein. Uptake of [14C]trehalose. Uptake of ['4C]trehalose in different strains was measured after growing the strains overnight in MMA with 0.2% glycerol or 0.2% trehalose or both in the presence or absence of 250 mM NaCl. The cultures were washed three times with MMA with or without 250 mM NaCl and resuspended in the same medium to an optical density of 1.0 (at 578 nm) at room temperature. [14C]trehalose (540 mCi/mmol) (6) was added to a final concentration of 45 nM, and 0.5-ml samples were filtered at different time intervals through filters (0.45-,um pore size; Millipore Corp., Bedford, Mass.) and washed with 10 ml of MMA, with or without 250 mM NaCl, and their radioactivities were counted. For measuring the Km of uptake, the same procedure was repeated in the presence of increasing

3452

BOOS ET AL.

J. BACTERIOL. TABLE 2. Pl-mediated transductional analysisa

Cross

Donor

Recipient

1 2 3 4 5

WK100 treD zfc-765::TnlO UE36 zjg-713::TnlO UE36 zjg-713::TnlO UE36 zjg-713::TnJO WK100 treD zfc-765::TnlO

UE15 KRIM4 treB KRIM3 treD UE48 1'(treC::1acZ) UE48 4 (treC::1acZ)

6

WK100 treD zfc-765::TnJO

WK101 treA F(treC::1acZ)

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

18483 fadL-771::TnlO 18467 zfb-l::TnlO 18468 nupC50::TnlO 18470 purC80::TnJO 18469 gua-26::TnlO 18427 zje-2241::TnlO 12073 cycA30::TnJO 12073 cycA30::TnJO 12019 zjh::TnlO 12019 zjh-920::TnlO 18429 ji::TnlO UE43 glk zfc-706::TnlO WK100 treD glk+ zfc-765::TnJO UE43 glk zfc-706::TnlO PPA168 crr zfc::TnlO PPA168 crr zfc::TnlO RH83 crp* zhd-732::TnlO WK114 glk+ zfc-76S::TnJO WK100 treD zfc-765::TnlO

KRIM3 treD KRIM3 treD KRIM3 treD KRIM3 treD KRIM3 treD KRIM4 treB KRIM4 treB UE48 4D(treC::IacZ) KRIM4 treB UE48 CF(treC::1acZ) KRIM4 treB KRIM3 treD ZSCII2L ptsG ptsM glk UE15 treA KRIM3 treD UE15 treA KRIM3 treD ZSCII2L ptsG ptsM glk HF4 treA 4)(treC::1acZ[Con])

26

12019 zjh-920::TnJO

HF57 4(treC::lacZ[Con])

Screenb TreTre+ Tre+ Kans Kans Lac- Kanr Kans Lac- Kanr Tre+ Tre+ Tre+ Tre+ Tre+ Tre+ Tre+ Kans Tre+ Kans Tre+ Tre+ Tre+ TreTre+ TreTre+ Tre+ Kan' Lac- Kanr Tre- Kan' Tre+ Kan' Tre+ Kanr

% Linkage

96 (96/100) 86 (172/200) 0 (0/200) 68 (136/200) 0 (0/190) 76 (143/190) 0 (0/100) 71 (71/100) 0 (0/100) 24 (72/300) 62 (168/300) 0 (0/300) 0 (0/300) 0 (0/40) 19 (11/58) 5 (8/150) 39 (39/100) 17 (94/550) 0 (0/58)

22 (22/100) 8 (8/100) 0 (0/143) 1.2 (4/340) 17 (9/53) 30 (34/116) 60 (89/148) 0 (0/100) 0 (0/100) 83 (413/497) 12.4 (62/497) 4.4 (22/497)C

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 mix labeled and unlabeled trehalose before the addition of the cells. At substrate concentrations higher than 20 ,uM, the optical density of the culture was increased to balance the reduction 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 centrifugation in an Eppendorf centrifuge. The cell pellet was suspended in 20 IlI of 7% trichloroacetic acid (TCA), and after centrifugation, the supernatant was transferred onto silica-coated thin-layer chromatography (TLC) plates and developed with butanol-ethanol-water (5:3:2) followed by autoradiography (see Fig. 4). To observe the secretion of glucose from strain CB17 after incubation with trehalose, we grew CB17 overnight in MMA with glycerol as the carbon source and trehalose as the inducer. The cells were washed and resuspended in MMA containing 25 mM trehalose at an optical density (578 nm) of 40. Samples of 20 IlI were withdrawn at different times and centrifuged, and the supernatant was chromatographed as described above. For detection of glucose, the TLC plates were sprayed with 20% sulfuric acid and charred. Enzymatic assays. Enzymatic assays of amylotrehalase and trehalose-6-phosphate phosphatase activities were determined in crude cellular extracts. Stationary-phase cells were harvested, washed, and suspended in 10 mM Tris hydrochloride (pH 7.2) containing 0.1% mercaptoethanol and broken by passage through a French pressure cell at 16,000 lb/in2. Cell debris were removed by centrifugation at

x g for 30 min at 4°C. Cellular extracts were used either directly or after dialysis against the same buffer for 5 h. The incubation mixture (20 plA) with or without 10 mM MgCl2 contained about 5 mg of protein per ml and 0.02 p,Ci of ['4C]trehalose (about 2 ,uM). After different time intervals, 20 pl was subjected to TLC as described above and autora-

40,000

diographed. Trehalose-6-phosphate phosphatase was tested in a similar way, using in the incubation mixture 10 mM unlabeled trehalose-6-phosphate (18). After chromatography, the TLC plate was sprayed with 20o sulfuric acid and charred. In vitro PTS activity in cell extracts was measured as previously described (26).

Accumulation of internal trehalose. Accumulation of internal trehalose was tested by growing strains in 20 ml of MMA with glucose as the carbon source in the presence of 250 mM NaCl overnight. The culture was harvested and washed once in the same medium without the carbon source, and the pellet was suspended in 0.2 ml of 5% TCA, incubated for 20 min at 0°C, and centrifuged. Ion-exchange resin was added, and 50-,u samples were spotted onto silica-coated TLC plates, chromatographed as described above, and charred after spraying with 20% sulfuric acid. RESULTS Transport of trehalose is inducible by trehalose and repressed at high osmolarity. Strain UE15 (MC4100 treA) lacking the periplasmic trehalase is still able to grow on trehalose (5). Figure 1 shows the ability of UE15 to take up

TREHALOSE METABOLISM IN E. COLI

VOL. 172, 1990

TABLE 3. Expression of 4D(treC::1acZ) after growth on glycerol in the presence and absence of 250 mM NaCl

70 [ S

U)

Strain

u

QJ

0

0 oJ E

WK111 (treD)

0~

A

30

C3

A

0

ll~ z

A

o'A 0

30

-

--

I

A

01----I-0 A,

90

time (sec) FIG. 1. Uptake of [(4C]trehalose in strains lacking the periplasmic trehalase. UE15 (wild type) after growth on glycerol (0), trehalose (0), and glycerol plus trehalose in the presence of 250 mM NaCl (l). A, KRIM3 (treD) after growth on glycerol and trehalose; A, KRIM4 (treB) after growth on glycerol and trehalose. All strains including UE15 lacked the periplasmic trehalase. ["4C]trehalose concentration was 45 nM. The cell density was 1.0 optical density unit (at 578 nm).

[14C]trehalose after growth on either glycerol or trehalose. Uptake after growth on trehalose was induced about 100fold. However, the strain did not grow on trehalose in the presence of 250 mM NaCl, and when grown in glycerol and trehalose together in the presence of 250 mM NaCl, uptake of [14C]trehalose was strongly reduced (Fig. 1). Uptake of [14C]trehalose was not inhibited by 250 mM NaCl after growth on trehalose at low osmolarity (data not shown). Thus, it is the synthesis and not the activity of the trehalose uptake system that is sensitive to high osmolarity. Measuring uptake in this strain (after growth on trehalose at low osmolarity) at different substrate concentrations allowed the determination of the apparent Km as 16 ,uM and of the Vmax as 9 nmol/min per 109 cells for the uptake of trehalose at room

+

0.08 0.03

+

0.01 0.01

+

0.75 0.79

+

0.16 0.37

HF57

A

10

activity 13-Galactosidase of the treC::lacZ fusion

NaCi

HF1

0~~~~~

0. -I.

C-

250 250 mM

UE49

50

4-

3453

temperature.

Isolation and characterization of mutants defective in utilization of trehalose. With strain UE15, N-methyl-N'-nitrosoN-nitrosoguanidine mutagenesis was performed and mutants were isolated that exhibited a Tre- phenotype on MacConkey-trehalose plates. Strains KRIM3 and KRIM4 were chosen for further studies. Both strains were unable to grow on trehalose, but only KRIM4 exhibited strongly reduced transport activity (Fig. 1). Using a P1 lysate of pooled random TnJO insertions into strain MC4100 (15), we isolated TnWO insertions that were cotransducible with the tre mutation in KRIM3 (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 mutation in KRIM4 treB and the gene carrying the mutation in KRIM3 treD.

(U/mg of protein)a

a Units are defined as micromoles of o-nitrophenyl-3-D-galactopyranoside hydrolyzed per minute.

In addition, a lacZ operon fusion was isolated by the X placMu technique (7) in strain MC4100 (treA+), yielding UE48, which exhibited a weak Tre- phenotype on MacConkey-trehalose plates. After transduction to treA::TnJO (yielding strain UE49), it no longer grew on trehalose as a sole 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 coding for an enzyme involved in the metabolism of trehalose rather than in a gene necessary for transport. After growth on glycerol, transport of trehalose appeared somewhat higher than the uninduced level observed in UE15. The presence of trehalose in the growth medium did not induce transport activity reproducibly but resulted in a fivefold reduction of the growth rate (compared with glycerol as the carbon source). The ,B-galactosidase activity of the lacZ fusion as well as the trehalose transport activity in strain UE49 were repressed when the cells were grown on glycerol in the presence of 250 mM NaCl. The results on the expression of the lacZ fusion are summarized in Table 3. We termed the gene 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 P1mediated transduction, we found that treB and treC were cotransducible with zjg-713::TnJO (Table 2, cross 4), while treD mapped elsewhere (Table 2, crosses 5 and 6). However, when treD was introduced with the nearby TnJO into a strain carrying the treC: :lacZ fusion, yielding WK111, the activity of the fusion was strongly reduced (Table 2, crosses 5 and 6; Table 3), indicating a regulatory function for treD. treD had only a small effect on the trehalose transport system (encoded by treB) (Fig. 1). The collection of mapped chromosomal TnJO insertions (28) was used to define the map position of treB (treC) and treD. Table 2, crosses 7 to 17, and Fig. 2 show the result of the genetic analysis. Accordingly, treB and treC are located at about 96.5 min, and treD is located at about 51.5 min. The latter is close to the PTS genes ptsl, ptsH, and crr as well as glk; the gene encoding glucokinase (11). Further Pl-mediated transductional analysis involving glk and treD (Table 2, crosses 18 to 20) demonstrated that these genes are not identical. However, several lines of evidence indicated that treD is an allele of crr. A crr mutation from strain PPA168 when transduced into WK101[V(treC::lacZ)] resulted in the repression of the fusion (data not shown), and introducing the same crr mutation into UE15 resulted in a Tre- phenotype (Table 2, cross 22). When the crr allele of PPA168 was

3454

J. BACTERIOL.

BOOS ET AL. treD

A

guua-26--TnlO

glk

zfb- I..Tn 10

zfc-765::Tn 10

nupC::Tn I 0 -

50 min I

1 pts 0.6

54 mvin

0.96 0.62 0.24

0.0

B

treB/C

zjg-7 1 3::Tn 10

zjh-920::Tn 10

cgcA-30::Tn 10 95 mlin

I

fdp 0.9 0.19

I

I

purfi urgl

97

mlin

4

0.06 0.68

0.39 0.17

FIG. 2. Phage P1 cotransductional analysis of treBC and treD. Cotransduction frequencies are given in fractions of 1 (equal to 100o). The arrows point to the selected markers, which were in all cases TnWO insertions. The TnlO insertions were obtained from a collection of mapped TnlO 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 transductants became Tre+ (Table 2, cross 21), compatible with the frequency of crossover within the same gene. Furthermore, introducing a crp* mutation that renders the catabolite gene activator protein independent of cyclic AMP (cAMP) (4) into KRIM3 again allowed growth on trehalose (Table 2, cross 23). This is expected when a defective IIIC encoded by the crr gene of KRIM3 no longer stimulates adenylate cyclase for the synthesis of cAMP. Thus, we conclude that treD is an allele of crr whose product still permits transport of glucose and trehalose but that no longer stimulates adenylate cyclase for sufficient production of cAMP needed for the expression of treC. In line with this conclusion was the observation that derivatives of UE15 lacking crp or cya (coding for adenylate cyclase) 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), the mannose-PTS (ptsM) and glucose-PTS (ptsG), and in glucokinase (glk) still exhibited trehalose-inducible uptake of

trehalose (Fig. 3) but was unable to grow on trehalose. When CB17 was given 25 mM trehalose, glucose was excreted into the medium (as detected by TLC of the medium supernatant [data not shown]). Growth of CB17 on trehalose was restored by introducing either ptsG+ or glk+ (Table 2, cross 24). A mutation in glk alone had no effect on the utilization of trehalose (Table 2, cross 20). This indicates that phosphorylation of internal glucose by glucokinase or of incoming glucose via the PTS (after exit of free glucose) is part of the metabolic pathway of trehalose. Another mutation that affects the utilization of trehalose is galU. galU codes for the enzyme forming UDP-glucose from UTP and glucose-i-phosphate. The introduction of galU into MC4100 via cotransduction with treA::TnlO, yielding strain RIM31, resulted in strongly reduced growth on trehalose. As discussed below, both glk and galU affect most likely the utilization of trehalose at the level of the internal degradation of trehalose mediated by the enzyme encoded by treC. Reduction of growth rate in presence of trehalose. Strain UE49 [F(treC::lacZ) treA] did not grow on trehalose as the

TREHALOSE METABOLISM IN E. COLI

VOL. 172, 1990

3455

A

I_

30 ol cL 0

E 0.

20

ci~0-

,..,,.., *-O0ft~

-4.-

00

d 0~

cii au d4 oJ

10

b

a

c

0.L S

9~~~

0 I. I----

0

60

120

---

1

2

3

5

15

20

*1P.

U~

180

time (sec)

,A~

FIG. 3. Uptake of ['4C]trehalose in the presence of glucose. Uptake was measured as described in the legend to Fig. 1. Before the uptake, glucose was mixed with ["4C]trehalose to the following final 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 (wild type); the lower two curves represent the uptake of trehalose in CB17 (ptsM ptsG glk) after growth in glycerol (-) and glycerol plus trehalose (0). Both strains lack the periplasmic trehalase.

sole source of carbon and, in addition, showed a fivefold reduction in growth rate on glycerol when trehalose was present. The major product that accumulated after exposure to external trehalose was free internal trehalose (Fig. 4B). Similarly, KRIM3 carrying the treD mutation that strongly reduces the expression of the treC::lacZ fusion and therefore also the intact treC gene was sensitive to trehalose when grown on glycerol. In both UE49 and KRIM3, the sensitivity to trehalose was abolished at high osmolarity, at which trehalose is no longer taken up but internal trehalose is synthesized and accumulated (13, 16, 30). Apparently, the accumulation of trehalose that is beneficial at high osmolarity is inhibitory for growth at low osmolarity. Isolation of mutants that express 4(treC::lacZ) constitutively at high osmolarity. By plating UE49 on lactose minimal plates containing 250 mM NaCl, we selected mutants that expressed the treC::lacZ fusion constitutively (Table 3). Two types of mutants were obtained. The first, represented by strain HF1, was, in contrast to its parent, no longer sensitive to trehalose when grown on glycerol. The mutation was not separable by P1 transduction from treC::1acZ, and the introduction of treD into HF4, the Tets derivative of HF1, 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 when grown on glycerol. This strain, as well as its Tets derivative, HF57, still transported trehalose but accumulated exclu-

b

a

1

5

10

^

4^

o

,4

2 b c 3 5 1 10 FIG. 4. Thin-layer chromatographic analysis of radiolabeled compounds after uptake of [14C]trehalose. The cell density was 4 optical density units (at 578 nm) with strain UE49 [4.(treC::lacZ)] and HF57. The optical density was 1 with strain UE15. Substrate concentration 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. The soluble extract was chromatographed. (A) UE15 grown in trehalose; (B) UE49 [4(treC-IacZ)] grown in glycerol; (C) HF57 grown in glycerol. Standards: lanes a, glucose; lanes b, trehalose; lanes c, trehalose-6-phosphate. This compound is unstable and slowly breaks down to trehalose. Times of incubation (minutes) are indicated in the figure. a

3456

BOOS ET AL.

J. BACTERIOL.

S dlW

iS

a

b

c

d

e

f

9

h

i Tre Gic

FIG. 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 this case, and in contrast to its parent, strain UE49, the trehalose sensitivity of strain HF57 must be caused by the accumulation of the phosphorylated sugar, a phenomenon also observed in other systems (31). We conclude that the loss of a trehalose-6-phosphate phosphatase activity is the cause for the constitutive expression of the treC: :lacZ fusion in HF50 and HF57. It follows that trehalose-6-phosphate is the inducer of treC. Trehalose-6-phosphate synthase is present at low osmolarity, even though with low activity (13). In addition to the trehalose sensitivity seen with HF50 and HF57, both strains also exhibited a pronounced sensitivity toward 250 mM NaCl when growing on glucose. The reason for this salt sensitivity must be caused by the inability to synthesize internal trehalose at high osmolarity. Thus, the trehalose-6-phosphate phosphatase lacking in HF50 and HF57 must be part of the biosynthetic pathway for internal trehalose. treD, when transduced into HF57, was still able to repress the constitutive expression of 't(treC: :IacZ) (data not shown). When the fusion was transduced into UE14 selecting for kanamycin resistance (the gene of which is part of the fusion phage) (7), the constitutivity of eI(treC: :lacZ) was not cotransduced among 200 transductants. This indicates that the mutation resulting in the constitutive expression is not located next to F(treC::lacZ). There is strong though circumstantial evidence for the existence of a second catabolic trehalose-6-phosphate phosphatase. When HF57 was transduced to Tetr with a Pl lysate of a strain harboring zjh-920: :TnJO, located next to treC+, all transductants that had lost the treC::lacZ fusion became Tre+ (Table 2, cross 26). The salt sensitivity (and the inability to synthesize internal trehalose at high osmolarity) was not crossed out in the above transduction. Since PTSmediated growth on trehalose must include a trehalose6-phosphate phosphatase activity (Fig. 4B and C), and since both types of transductants are still lacking the biosynthetic trehalose-6-phosphate phosphatase, one must conclude that the transduction to Tre+ introduced a phosphatase activity.

Thus, E. coli contains two specific trehalose-6-phosphate phosphatases. One is responsible for salt resistance and is part of the trehalose biosynthetic pathway at high osmolarity. This enzyme is defective in HF50 and HF57. The other has to be part of the catabolic pathway of trehalose. Since HF57 did not show any trehalose-6-phosphate phosphatase activity (Fig. 4C) and since the transductants that obtained treC+ can grow on trehalose, we must conclude that the gene coding for the second (catabolic) trehalose-6-phosphate phosphatase is located distal to treC in the same operon and that its expression must therefore be lacking in strains carrying the treC::lacZ fusion. We propose the name treE for the gene encoding the catabolic trehalose-6-phosphate phosphatase. Both mutations that lead to constitutive expression of 'D(treC::lacZ) at high osmolarity also expressed D(treC::IacZ) constitutively at low osmolarity, but they did not render the expression of the transport gene(s) insensitive to salt. Transport of trehalose in both mutants remained repressible by growth at high osmolarity. Therefore, treB and treC, even though closely linked, do not seem to be located in the same operon. This conclusion is corroborated by the finding that treD repressed the expression of 1D(treC::1acZ) (Table 3) but not the transport gene treB (Fig. 1). Salt sensitivity is accompanied by reduced ability to synthesize internal trehalose. The lack of biosynthetic trehalose6-phosphate phosphatase in strain HF57 [constitutive 'D(treC::lacZ)] suggested that it no longer was able to synthesize internal trehalose at high osmolarity (250 mM NaCl). Figure 5 shows the TLC analysis of the supernatants of cellular extracts after TCA precipitation of strains grown on glucose in the presence of 250 mM NaCl. The mutation that leads to the constitutive expression of the treC::lacZ fusion (strain HF57) was accompanied by the reduced ability to accumulate trehalose. Instead, a compound migrating on TLC as trehalose-6-phosphate could be recognized in these extracts. The appearance of the small amounts of trehalose either might be due to the leakiness of the mutation in the

VOL. 172,

TREHALOSE METABOLISM IN E. COLI

1990

encoding the biosynthetic trehalose-6-phosphate phosphatase or more likely could be a consequence of the instability of trehalose-6-phosphate during the long growth period (overnight). The inability to synthesize and accumulate trehalose during growth on glucose at 250 mM NaCl was not connected to a Tre- phenotype. Neither UE61 and UE62, representatives of Tre+ and Tre- transductants from cross 26 in Table 2 (treC+ into HF57), accumulated trehalose (Fig. 5). Interestingly, the Tre+ transductant did not even accumulate trehalose-6-phosphate. This is consistent with the presence of a second trehalose-6-phosphate phosphatase in UE61, encoded by treE, distal to treC which had been activated by restoration of treC+. HF1, the other mutant that expressed '1(treC::lacZ) constitutively and whose mutation was closely linked to the fusion, still produced large amounts of internal trehalose at high osmolarity (Fig. 5). Nature of trehalose transport system. Mutants deleted for ptsI, ptsH, and crr and lacking in addition the periplasmic trehalase (treA) are unable to grow on or to transport trehalose (5, 14). This pointed to a PTS involvement in the trehalose transport system. Since earlier reports (23) on trehalose transport in S. typhimurium implicated participation of the ptsM-encoded mannose-PTS, we tested E. coli ptsM ptsG mutants for growth and transport of trehalose. Strain UE50, a treA::TnJO derivative of the ptsM ptsG mutant LR2-168 (17), did not grow on glucose or mannose but grew normally on trehalose. In addition; strain CB17, a treA::TnJO derivative of ZSC112L (11) carrying ptsM ptsG gene

glk, exhibited trehalose-inducible uptake

of

[14C]trehalose

(Fig. 3), even though it did not grow on trehalose. Thus, it is clear that trehalose transport in E. coli is not mediated by ptsM or ptsG. A crr mutation coding for IIIG1c was introduced into strain UE15 by P1 transduction with the help of a nearby TnWO insertion (Table 2, cross 22), yielding strain UE42. This strain did not transport trehalose nor did it grow on

trehalose, even in the presence of cAMP, pointing to a direct involvement of IlIGIc in a PTS-mediated transport of trehalose. Indeed, uptake of [14C]trehalose in UE15 (ptsG+) in the presence of increasing concentrations of glucose showed that glucose was able to interfere to a certain extent with trehalose uptake (Fig. 3). This is consistent with a competition for phosphorylated IIIGlc in the uptake process of both sugars. Observing the fate of [14C]trehalose after uptake into UE15, a strain able to metabolize trehalose, the formation of secondary metabolic products is too fast for detecting the initial product of accumulation (Fig. 4A). Strain UE49, carrying the treC: :lacZ fusion, still transported trehalose but could not use it as a carbon source. Figure 4B shows a TLC analysis of the intracellular radioactive material present after increasing time intervals of exposure to [14C]trehalose. Surprisingly, the major component was free trehalose and not trehalose phosphate as expected for a PTS-mediated transport system. Therefore, the cytoplasm must contain a highly active trehalose-6-phosphate phosphatase preventing the accumulation of trehalose-6-phosphate. Indeed, the test for trehalose-6-phosphate-hydrolyzing activity in cellular extracts, with a 10 mM concentration of unlabeled substrate and TLC anaIy`is, revealed high activity in extracts of strain UE15 yielding as a product trehalose. This activity was increased by a factor of two to three when the cells were grown in the presence of 250 mM NaCl (data not shown). Considering the mutation in HF57 which renders the

3457

TABLE 4. PEP-dependent in vitro phosphorylation of trehalose Sp acta

Strain

HF50 KRIM4b

iiTre

+

Trehalose Trhls

Methyla-glucoside

a-lcsd

phosphorylation

phosphorylation

2.3

Trehalose transport and metabolism in Escherichia coli.

Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotec...
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