JOURNAL OF BACTERIOLOGY, OCt. 1993, p. 6150-61570021-9193/93/196150-08$02.00/0Copyright © 1993, American Society for Microbiology

Vol. 175, No. 19

Identification of the glmU Gene Encoding N-Acetylglucosamine-1-Phosphate Uridyltransferase in Escherichia coli

DOMINIQUE MENGIN LECREULX* AND JEAN vAN HEIJENOORTLaboratoire des Enveloppes Bacteriennes et des Peptides, Unite6 de Recherche Associee 1131 du Centre

National de la Recherche Scientifique, Universite Paris-Sud, Baitiment 432 91405 Orsay, FranceReceived 20 May 1993/Accepted 20 July 1993

The physiological properties of the EcoURF-1 open reading frame, which precedes the gbnS gene at 84 minon the Escherichia coli chromosome (J. E. Walker, N. J. Gay, M. Saraste, and A. N. Eberle, Biochem. J.224:799-815, 1984), were investigated. A thermosensitive conditional mutant in which the synthesis of the geneproduct was impaired at 43°C was constructed. The inactivation of the gene in exponentially growing cellsrapidly inhibited peptidoglycan synthesis. As a result, various alterations of cell shape were observed, and celllysis finally occurred when the peptidoglycan content was 37% lower than that of normally growing cells.Analysis of the pools of peptidoglycan precursors revealed a large accumulation of N-acetylglucosamine-l-phosphate and the concomitant depletion of the pools of the seven peptidoglycan nucleotide precursors locateddownstream in the pathway, a result indicating that the mutational block was in the step leading fromN-acetylglucosamine-l-phosphate and UTP to the formation of UDP-N-acetylglucosamine. In vitro assaysshowed that the overexpression of this gene in E. col cells, directed by appropriate plasmids, led to a highoverproduction (from 25- to 410-fold) of N-acetylglucosamine-l-phosphate uridyltransferase activity. Thisallowed us to purify this enzyme to homogeneity in only two chromatographic steps. The gene for this enzyme,which is essential for peptidoglycan and lipolysaccharide biosyntheses, was designated glnmU.

In bacteria, UDP-N-acetylglucosamine (UDP-GlcNAc) isone of the main cytoplasmic precursors of cell wall pepti-doglycan (14, 28). In Escherichia coli and most other relatedgram-negative bacteria, it is also the precursor for thedisaccharide (glucosamine) moiety of lipid A, which is anessential component of outer membrane lipopolysaccharide,as well as for the synthesis of the enterobacterial commonantigen (16, 30). It is tempting to postulate that this branch-point (shown schematically in Fig. 1) is a site of regulation,considering that most of the glucosamine in the E. colienvelope is found in peptidoglycan and lipopolysaccharide(28, 30). Genes and enzymes involved in the steps locateddownstream from UDP-GlcNAc in these different pathwayshave in most cases been identified and studied in detail (8, 9,16, 20-23, 26, 28, 30).However, the synthesis of UDP-GlcNAc has been only

poorly characterized. UDP-GlcNAc is believed to be synthe-sized from fructose-6-phosphate by four enzyme-catalyzedreactions (7, 11, 36). Glucosamine-6-phosphate synthase (L-glutamine:D-fructose-6-phosphate amidotransferase) cata-lyzes the first reaction (10). The glmS gene encoding thisenzyme in E. coli has been identified at 84 min on thephysical map, and mutants defective in glmS (which requireglucosamine for growth) have been characterized (2, 33, 35,37). Subsequent steps from glucosamine-6-phosphate to UDP-GlcNAc presumably are via glucosamine-1-phosphate (Fig. 1)(7, 36). The enzymes for the first two reactions, phosphoglu-cosamine mutase and glucosamine-1-phosphate acetyltrans-ferase, as well as their genes, remain to be characterized. TheN-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltrans-ferase activity (also named UDP-GlcNAc pyrophosphorylase),which synthesizes UDP-GlcNAc from GlcNAc-1-P and UTP,has previously been partially purified and characterized forBacillus lichenifonnis and Staphylococcus aureus (1, 34),

* Corresponding author.

but no data were available on the E. coli enzyme and itsgene.DNA sequencing around the E. coli unc operon has

previously shown that theglmS gene sequence was precededby an open reading frame of unknown function, namedEcoURF-1, which theoretically encodes a polypeptide of 456amino acids with a molecular weight of 49,130 (35). Theshort intergenic distance between EcoURF-1 and glmS andthe absence of an obvious promoter consensus sequenceupstream ofglmS suggested that these genes were cotrans-cribed (29, 35). We inferred that the function of EcoURF-1might be also related to cell wall synthesis and that a likelycandidate for its product was one of the enzymes involved inthe steps leading from glucosamine-6-phosphate to UDP-GlcNAc. We here demonstrate that the EcoURF-1 gene is anessential gene encoding the GlcNAc-1-P uridyltransferaseactivity. We propose glmU (for glucosamine uridyltrans-ferase) as the name for this E. coli gene, according to thenomenclature previously adopted for the glmS gene encod-ing glucosamine-6-phosphate synthase (35, 37).

MATERIALS AND METHODS

Bacterial strains, phages, and plasmids. E. coli JM83 [araA(lac-proAB) rpsL thi 480 dlacZAM15] (38) was used as thehost strain for plasmids and for the large-scale purification ofthe overproduced glmU gene product. Plasmid pJP900,constructed by insertion of the 2.4-kb XhoII fragment ofbacteriophage lambda carrying the PR promoter into theBamHI site of the pUC9 vector, was obtained from J. Pla(CSIC, Universidad Aut6noma de Madrid, Madrid, Spain).Cloning vectors pUC18 and pUC19 and the Kanr GenBlockoriginating from the pUC4K plasmid were purchased fromPharmacia. Plasmid pMAK705 bearing a thermosensitivereplicon was obtained from S. R. Kushner via Y. Mechulam(Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau,France) (13). The pGM7 plasmid, which was used as the

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BIOSYNTHESIS OF UDP-N-ACETYLGLUCOSAMINE IN E. COLI 6151

Fructose-6-P

nagB gimS

glucosamine-6-P

glucosamine-1-P

N-acetylglucosamine-1-P

UTP glmU

UDP-N-acetylglucosamine

lipopolysaccharide peptidoglycan

FIG. 1. Biosynthesis and cellular utilization of UDP-GlcNAc inE. coli.

DNA source for the cloning of the E. coliglmU gene, was agenerous gift from B. Badet (Institut de la Chimie desSubstances Naturelles, CNRS, Gif sur Yvette, France) (10).Growth conditions. Unless otherwise noted, 2YT rich

medium was used for growing cells (25). Growth was mon-itored at 600 nm with a spectrophotometer (model 240;Gilford Instrument Laboratories, Inc., Oberlin, Ohio). Forstrains carrying drug resistance genes, antibiotics were usedat the following concentrations: ampicillin, 100 ug- ml-l;kanamycin, 30 p.g ml-1; and chloramphenicol, 25 pg ml-l.Broth for plates was solidified with 1.5% agar, and whenplasmid inserts were screened for the absence of a-comple-mentation, 5-bromo-4-chloro-3-indolyl-3-D-galactopyrano-side (X-Gal) was added at 40 ,g. ml-'.

General DNA techniques and E. cofi cell transformation.Small- and large-scale plasmid isolations were carried out bythe alkaline lysis method, and plasmids were further purifiedby using cesium chloride-ethidium bromide gradients (32).Standard procedures for endonuclease digestions, ligation,filling in of 5' protruding ends by using the Klenow fragmentof DNA polymerase I, and agarose electrophoresis wereused (6, 32). E. coli cells were made competent for transfor-mation with plasmid DNA by the method of Dagert andEhrlich (5).

Construction of plasmids. The pGM7 plasmid (10) carryingthe 4.6-kb NcoI-ClaI fragment which contains theglmU andglmS genes from the E. coli chromosome was used asstarting material for the construction of the different plas-mids described in this study (Fig. 2). Plasmid pMLD71 wasconstructed by inserting the 2.7-kb PstI-EcoRI fragment intothe corresponding sites of the pUC19 vector. The pMLD72plasmid was constructed by inserting the 2.5-kb Dral-EcoRIfragment into the pUC19 vector cut by SmaI and EcoRI. Aninternal NaeI-EcoRI deletion in pMLD71 provided plasmidpMLD86 containing only the glmU gene (Fig. 2). For theexpression of glmU under the control of the lambda PRpromoter, plasmid pMLD78 was constructed by insertingthe 2.4-kb XhoII lambda fragment (carrying the structuralgene cI857 encoding a thermosensitive form of the lambda cIrepressor, the strongpR promoter, and the ribosome-binding

NC P DI . [

InC_- _ _~c_ E

A H NAI a I

V E P P DCI .II

glmU gimS J [hoSpMLD71 +r* INIIINNM

pMLD72 +* 'N"x N "i

pMLDS6 +l

ka I

P EpMLD76 +1*FZ;M 500 bp

FIG. 2. Localization of the glmU gene at 84 min on the E. colichromosome. Locations of some other genes are indicated at the top(2). Bacterial DNA present in plasmid inserts is shoWn below. TheglmU gene is represented as a hatched region, and the position ofthe lac promoter relative to the insert in each pUC19-derivedplasmid is indicated by an arrow. Positions of cleavage sites areshown for AccI (A), DraI (D), EcoRI (E), EcoRV (V), HpaI (H),NaeI (NA), NcoI (Ne), and PstI (P).

site of cro) into the unique SalI site of pMLD72. Disruptionof the glmU gene was done by inserting the 1.28-kb HincIIkan cartridge originating from pUC4K into the unique AccIsite of plasmid pMLD71 within the glmU gene codingsequence, generating plasmid pMLD76 (Fig. 2). The pGMU-kan plasmid was constructed by inserting the 3.8-kb DraI-EcoRV fragment from pMLD76 (harboring the disruptedglmU gene) into the HincII site of pMAK705 (13). ThepGMU plasmid was isolated as described below during theexcision process that followed integration of the pGMUkanplasmid at the chromosomal glmU locus.

Disruption of the chromosomal glnU gene. The wild-typechromosomal copy ofglmUwas replaced by a disrupted oneby following the procedure of Hamilton et al. (13), whichused pMAK705, a plasmid bearing a thermosensitive repli-con. pGMUkan, a pMAK705 derivative carrying the dis-rupted glmU gene, was transformed into JM83. Integrationof the plasmid into the chromosome was selected by platingthe cells at 44°C on 2YT-chloramphenicol plates. Severalclones were picked up, and the integration of pGMUkan atthe glmU locus was verified by Southern analysis (data notshown). Excision of the plasmid from the chromosome wasthen done as follows. Cointegrants were grown at 30°C in2YT-chloramphenicol for at least 30 doubling times andsubsequently plated at 30°C. Individual clones were thenscreened for sensitivity to chloramphenicol at 44°C, which isindicative of plasmid excision. The structures of the excisedplasmids from several clones were determined by DNArestriction analysis. One clone containing a plasmid bearingthe wild-type glmU gene (called pGMU) was chosen. Fi-nally, the replacement in that strain (named UGS83) of thechromosomalglmU copy by the inactivated one was verifiedby Southern analysis (data not shown).

Pool levels of peptidoglycan precursors. Cells of UGS83(1-liter cultures) were grown exponentially at 30°C in 2YTmedium. At the appropriate cell concentration (3 x106. ml-1), the temperature of the culture was either main-tained at 30°C or increased to 43°C. Incubation was contin-ued until the optical density of the culture at 43°C reached aplateau value of 0.5 to 0.7 about 4 h later (see Fig. 3). At thistime, cells were rapidly chilled to 0°C and harvested in thecold. Control cultures were made with strain JM83(pGMU),

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6152 MENGIN-LECREULX AND vAN HEIJENOORT

which carries an intact chromosomal copy of glmU. Theextraction of peptidoglycan nucleodide precursors as well asthe analytical procedure used for their quantitation were aspreviously described (20, 21). GlcNAc-1-P was purified fromcell extracts by first using the same two-step chromato-graphic procedure that is commonly used to purify thepeptidoglycan nucleotide precursors: a gel filtration onSephadex G-25, where GlcNAc-i-P is eluted just after UDP-GlcNAc with a coefficient of distribution (Kd) of 0.35,followed by high-performance liquid chromatography on acolumn of ,uBondapak C18, where it is eluted in mixture withUDP-GlcNAc (20, 21). A high-voltage electrophoresis per-formed under the same conditions as those described belowfor the enzymatic assays was used to completely purifyGlcNAc-1-P. To ensure its detection and to evaluate itsrecovery at each step, a given amount (2,000 Bq) of[14CJGlcNAc-l-P was initially added to the extracts. Theglucosamine content of the GlcNAc-1-P was determinedafter acid hydrolysis. Glucosamine-6-phosphate and glu-cosamine-1-phosphate were extracted and purified as de-scribed above for GlcNAc-1-P, except that the electro-phoretic step was unnecessary and was replaced by thedirect separation and estimation of these compounds with anamino acid analyzer.

Isolation of sacculi and quantitation of peptidoglycan. Ex-ponential-phase cells of JM83(pGMU) and UGS83 weregrown at 30°C or first at 30°C and then 43°C as describedabove. Harvested cells were washed with a cold 0.85% NaClsolution and centrifuged again. Bacteria were then rapidlysuspended with vigorous stirring in 40 ml of a hot (95 to100°C) aqueous 4% sodium dodecyl sulfate (SDS) solutionfor 30 min. After standing overnight at room temperature,the suspensions were centrifuged for 30 min at 200,000 x g,and the pellets were washed several times with water. Finalsuspensions made in 5 ml of water were homogenized bybrief sonication. Aliquots were hydrolyzed and analyzed aspreviously described, and the peptidoglycan content of thesacculi was expressed in terms of its diaminopimelic acidcontent (20, 24).

Preparation of crude enzyme. Cells of JM83 harboring theplasmids listed in Table 2 (except pMLD78) were grownexponentially at 37°C in 2YT medium supplemented withampicillin (1-liter cultures). Cells were harvested in the coldwhen the optical density of the cultures reached 0.7. Adifferent protocol was used with strain JM83(pMLD78): cellswere grown first at 30°C, and at an absorbance of 0.2 (108cells. ml-') cultures were shifted to 42°C for 3 h, duringwhich time the absorbance reached a plateau value of 0.7. Inall cases, cells were washed with 40 ml of cold 0.02 Mpotassium phosphate buffer (pH 7) containing 1 mM 3-mer-captoethanol. The wet cell pellet was suspended in 12 ml ofthe same buffer and disrupted by sonication (sonicator 150;T.S. Ultrasons, Annemasse, France) for 10 min with cool-ing. The resulting suspension was centrifuged at 4°C for 30min at 200,000 x g with a Beckman TL100 centrifuge. Thesupernatant was dialyzed overnight at 4°C against 100 vol-umes of the same phosphate buffer, and the resulting solu-tion (11 ml; 5.5 mg of protein. ml-1), designated crudeenzyme, was stored at -20°C. SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis of proteins was per-formed as previously described (17), using 13% polyacryl-amide gels. Protein concentrations were determined by themethod of Lowry et al., using bovine serum albumin as astandard (19).Assay for GlcNAc-1-P uridyltransferase activity. The stan-

dard assay mixture contained 50 mM Tris-hydrochloride

buffer (pH 8.6), 1 mM UTP, 0.1 mM [14C]GlcNac-1-P (700Bq), 5 mM MgCl2, and enzyme (10 to 700 pg of protein,depending on overexpression or purification factors) in afinal volume of 100 pl. Mixtures were incubated at 37°C for1 h, and reactions were terminated by the addition of 20 ,ul ofacetic acid. Reaction products were separated by high-voltage electrophoresis on Whatman 3MM filter paper inpyridine-acetic acid-water (6/23/971) (pH 4.0) for 100 min at40 V/cm with an LT36 apparatus (Savant Instruments,Hicksville, N.Y.), and the radioactive spots were located byovernight autoradiography using type R2 films (3M, St. Paul,Minn.) or with a radioactivity scanner (Multi-TracermasterLB285; Berthold France, Elancourt, France). The only tworadioactive spots (GlcNAc-1-P and UDP-GlcNAc) were cutout, and the radioactivity was counted in an IntertechniqueSL 30 liquid scintillation spectrophotometer with a solventsystem consisting of 2 ml of water and 13 ml of Aqualytemixture (J. T. Baker Chemicals, Deventer, Netherlands).One unit of enzyme activity was defined as the amountwhich catalyzed the synthesis of 1 ,umol of UDP-GlcNAc in1 min.

Purification of GlmU from overproducing strain JM83(pMLD78). The total extract (67 mg of protein, 552 U ofuridyltransferase activity) secured from strain JM83(pMLD78) as described above was loaded onto a column(12.5 by 2.5 cm) of DEAE-Trisacryl-M (IBF, Vileneuve-la-Garenne, France) that had been preequilibrated with bufferA (20 mM potassium phosphate buffer, pH 7.4, containing 1mM ,B-mercaptoethanol and 10% [vol/vol) glycerol). Theelution was run at a flow rate of 1 ml. min -, first with 50 mlof buffer A and then with a linear gradient (400 ml) of NaCl(0 to 500 mM) in buffer A. Fractions (10 ml) were collectedand assayed for GlcNAc-1-P uridyltransferase activity. Themost active fractions were pooled (317 U, 25 mg of protein),concentrated to 12 ml by ultrafiltration through PM10 mem-branes (Amicon, Beverly, Mass.), and stored at -20°C. Halfof the protein solution was thawed, and the buffer wasexchanged with buffer B (2 mM potassium phosphate buffer,pH 7.4, containing the same additives as in buffer A) byrepeated concentration and dilution using PM10 membranes.The protein solution was loaded onto a column (10 by 2.6cm) of hydroxylapatite-Ultrogel (IBF) that had beenpreequilibrated with buffer B. The elution was run at a flowrate of 1 ml min-1, first with 60 ml of buffer B and then witha linear gradient (400 ml) of potassium phosphate (2 to 500mM). Fractions (10 ml) containing GlcNAc-1-P uridyltrans-ferase were pooled (121 U, 8 mg of protein), concentrated asdescribed above to about 12 ml, and dialyzed against bufferA.Amino acid composition. Quantitative amino acid analyses

were performed with an analyzer (model LC 2000;Biotronik, Frankfurt, Germany) after hydrolysis of samplesin 6 M HCl at 95°C for 16 h.

RESULTS

Inactivation of the chromosomal gbnU gene and its effect onbacterial growth. The pGM7 plasmid (10) carrying the 4.6-kbNcoI-ClaI fragment that encompasses both the glmU andglmS genes (Fig. 2) was used as the starting DNA material.A series of plasmid derivatives carrying only the glmU genewere constructed as described in Materials and Methods andshown in Fig. 2. To inactivate the glmU gene on the E. colichromosome, we used the procedure described by Hamiltonet al. (13), which is particularly well suited for the disruptionof essential genes, as recently shown for the construction of

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0.5

0.1

0.01 /

0 2 4 6 8 0 2 4 6 8

hoursFIG. 3. Effect of inactivation of the glmU gene on the growth of

E. coli K-12. UGS83 (A) and JM83(pGMU) (B) cells were grownexponentially at 30'C in 2YT medium. At the time indicated by thearrows, the temperature of the culture was either maintained at 30'C(open symbols) or shifted to 43'C (closed symbols).

mutants altered in the murI gene encoding the glutamateracemase (8).

First, the glmU coding sequence carried by the pMLD71plasmid was disrupted by inserting at the unique AccI sitethe 1.28-kb kanamycin resistance gene from pUC4K, gener-ating plasmid pMLD76 (Fig. 2). Most of the insert from thepMLD76 plasmid was then inserted into the pMAK705vector, which bears a thermosensitive replicon. The result-ing plasmid, pGMUkan, was used as detailed in Materialsand Methods in the construction of a strain derived fromJM83, UGS83 [JM83 glmU::kan(pGMU)], having the inac-tivated glmU gene on the chromosome and the wild-typeallele on the pMAK705 vector. At the restrictive tempera-ture for plasmid replication (43 to 440C), UGS83 failed togrow on 2YT plates, indicating that this strain with adisrupted copy of the glmU gene on the chromosome wasviable only in the presence of a plasmid carrying the wild-type glmU gene. The impossibility of transducing by phageP1 the glmU::kan marker from UGS83 to other E. coli K-12strains further confirmed that glmU was an essential gene.

Since the pGMU plasmid bears a thermosensitive repli-con, the effects of the inactivation of the glmU gene wereobserved by shifting cultures of UGS83 and JM83(pGMU)cells growing at 30'C in 2YT medium to 43°C. The strainsshowed identical growth rates and cell morphologies whengrown at 30°C. However, after a 4-h incubation at 43'C, thegrowth rate of UGS83 rapidly slowed, and cells apparentlyentered stationary phase at a lower cell mass (Fig. 3).Furthermore, UGS83 cells exhibited clear morphologicaldifferences when observed by phase-contrast microscopy,whereas the morphology of the parental strain was unaltered(data not shown). During this period, the UGS83 cellsprogressively changed from rods to greatly enlarged ovoids.Some rare short filaments with partial constrictions at posi-tions expected for septal sites were also observed. As judgedby the presence of many ghosts within the cell population,cell lysis finally occurred after prolonged incubation at therestrictive temperature.

It was noteworthy that these different morphological

TABLE 1. Peptidoglycan content and pool levels of mainpeptidoglycan precursors in the parental and glmU

mutant strainsa

Strain and Amt (nmol/g [dry weight] of bacteria)growth temp GlcNAc- UDP- UDP-MurNAc- Pepti-

(OC) 1-P GlcNAc pentapeptide doglycan

JM83(pGMU)30 120 975 605 8,60043 170 740 700 9,050

UGS8330 110 840 650 9,20043 2,150 210 95 5,800

a Cells were grown exponentially in 2YT medium at 30'C or first at 300C andthen for 4 h (the time at which the growth rate of the mutant strain begins todecrease) at 43'C. Cells were harvested, and the peptidoglycan and itsprecursors were extracted and quantified as detailed in Materials and Meth-ods. The peptidoglycan content of sacculi is expressed in terms of itsdiaminopimelic acid content.

changes, and in particular the filamentous phenotype, wereexacerbated when a 2YT medium deprived of NaCl was usedfor growing cells. Furthermore, the early stationary phasewhich characterized UGS83 cells grown at 430C was nolonger observed when either 2% NaCl or 20% sucrose wasadded to the growth medium. All these different results wereconsistent with the involvement of theglmU gene product inthe biosynthesis of a cell envelope component. It should bementioned that the above-described phenomena were alsoobserved in a medium supplemented with 0.2% glucosamine,indicating that the phenotype caused by the insertionallyinactivatedglmU allele was not due to polarity effects on theglmS gene (whose inactivation results in glucosamine aux-otrophy [33, 37]) located downstream on the chromosome.

Involvement of the gbnU gene product in peptidoglycansynthesis. Since the effects on bacterial shape and cellintegrity provoked by the defect of glmU resembled thephenotypes of previously characterized mutants defective inpeptidoglycan synthesis (4, 8, 22, 23, 26, 33, 37), the effect ofa temperature upshift on peptidoglycan synthesis in bothstrains JM83(pGMU) and UGS83 was determined. In orderto estimate the rate of synthesis of this macromolecule, wedirectly quantitated the cell peptidoglycan content by aminoacid and amino sugar analyses of isolated sacculi. After 4 hof growth at 43'C, the peptidoglycan content of UGS83 cellsappeared to be 37% lower (when expressed per bacterial dryweight) than that of parental cells grown in similar conditions(Table 1). It could be assumed that the highly reducedpeptidoglycan content observed in the mutant cells probablyrepresents the lowest physiological value compatible withcell integrity in this case. Since inactivation of the glmUgene product function had the effect of decreasing the rate ofpeptidoglycan synthesis, we also investigated the effect of itsoverproduction in exponentially growing cells. In particular,the peptidoglycan content of cells harboring plasmidpMLD71, in which theglmU gene is under the control of thelac promoter (which overproduced the glmU gene product23-fold [see Table 2]), was identical to that of control cells(data not shown), a result indicating that the glmU geneproduct was probably not a limiting factor in the peptidogly-can synthesis pathway.

Site of the mutational block. Other experiments werecarried out to determine which step in peptidoglycan syn-thesis was affected by theglmU mutation. We first examinedthe possible function of theglmU gene product in some early

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TABLE 2. Levels of GlcNAc-l-P uridyltransferase inE. coli cells

Strain Sp act Amplification(growth temp, °C) (U. mg of protein-1) (fold)

JM83(pUC19) (37) 0.02 1JM83(pMLD71) (37) 0.46 23JM83(pMLD72) (37) 0.50 25JM83(pMLD86) (37) 0.60 30JM83(pMLD76) (37) 0.02 1JM83(pMLD78) (42) 8.20 410

cytoplasmic step by determining the effect of its inactivationon the pool levels of the different cytoplasmic nucleotideprecursors characteristic of this metabolic pathway. Asshown in Table 1, an arrest in the de novo synthesis offunctional GlmU protein molecules led to the depletion of thepools of the two main precursors UDP-GlcNAc and UDP-N-acetylmuramoyl-L-Ala-,y-D-Glu-meso-diaminopimilic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide), which are the firstand last nucleotidic precursors in the pathway, respectively(20, 28). The pool levels of the other intermediate precursorsfrom UDP-GlcNAc-enolpyruvate to UDP-MurNAc-tripep-tide, which are always detected at much lower intracellularconcentrations (20, 21), were also significantly decreased(data not shown). This result suggested that the mutationalblock was located upstream from UDP-GlcNAc and conse-quently that the glmU gene product might be involved insome early cytoplasmic step also required for lipopolysac-charide synthesis (Fig. 1).To further localize this step, new chromatographic proce-

dures were developed for a rapid quantification of the poolsof the early precursors from glucosamine-6-phosphate toGlcNAc-l-P (see Materials and Methods). As shown inTable 1, the amount of the pool of GlcNAc-1-P determinedfor the mutant strain growing at 30°C was low (100 nmol/g[dry weight] of bacteria, corresponding to a 0.05 mM cellularconcentration) and roughly equivalent to that measured inthe parental strain at either 30 or 43°C. However, growth ofthe mutant strain at the restrictive temperature led to aconsiderable (18-fold) increase of the pool of this compound.Pools of glucosamine-6-phosphate and glucosamine-1-phos-phate, which are located upstream in the pathway, were notsignificantly enhanced (data not shown). All the data pre-sented in Table 1, and in particular the opposite variations ofthe pools of GlcNAc-1-P and UDP-GlcNAc, clearly indi-cated that the activity of the GlcNAc-1-P uridyltransferasewas most probably impaired in strain UGS83.

Levels ofGlcNAc-l-P uridyltransferase. To corroborate theprevious assumption, the GlcNAc-l-P uridyltransferase ac-tivities of strains overexpressing the glmU gene were esti-mated. For this purpose, an assay was developed whichconsisted in following the formation of UDP-[14CJGlcNAcfrom [14C]GlcNAc-1-P and UTP, with both labelled com-pounds being separated by high-voltage electrophoresis.This procedure allowed us to detect this activity in cellextracts of wild-type E. coli strains and to demonstrate itssignificant (up to 30-fold) overproduction in cells carryingplasmids expressing the gbnU gene under the control of thelac promoter (Table 2). Only a slight increase of a proteinspecies of about 50,000 molecular weight was seen bySDS-PAGE analysis of the corresponding crude extracts. Toobtain a more efficient overproducing plasmid, the strategyoriginally described by Leplatois and Danchin (18), whichconsists of expressing the considered gene under the control

x xi

C1867 *7pM 2

/Sall

zq { pMLD78.0

Cro :>

ATGGAACAA --- TACAGTCAGGACGCGTATITGAATAA----

GlmU :>FIG. 4. Construction of plasmid pMLD78. This plasmid was

constructed by inserting the 2.4-kb XhoII lambda fragment thatcarries the PR promoter into the SalI site of pMLD72. Restrictionenzymes: B, BamHI; E, EcoRI; H, HindIII; S, SalI; X, XhoII. Thejunction between lambda and E. coliDNA sequences is shown at thebottom, indicating the translational coupling of Cro (interrupted)and GlmU proteins. The ribosome-binding site and initiation codonof the glmU gene are underlined.

of the lambda PR promoter, was used. This was done byinserting upstream from theglnU gene coding sequence in apUC19-derived plasmid (pMLD72 [Fig. 2]) the 2.4-kb XhoIIlambda fragment carrying the structural gene cI857 encodinga thermosensitive form of the lambda cI repressor, the PRpromoter, and the lambda cro ribosome-binding site (Fig. 4).When cells of JM83 harboring the resulting plasmid,pMLD78, were grown exponentially at 30°C in 2YT mediumand then shifted (at an optical density of 0.2) to 42'C, aninitial increase in the growth rate was observed, but growthrapidly stopped 2 h later when the optical density of theculture reached a plateau value of 0.7. At different times ofgrowth, cells were recovered and their protein content wasfractionated and analyzed as described in Materials andMethods. At 1 h after the temperature shift, a large accumu-lation of the 50,000-molecular-weight protein was observed,which further increased to finaLly account for more than 40%of the cell proteins after a 3-h incubation period (Fig. 5). Itcomigrated with the radioactive protein band expressed inmaxicells from plasmids carrying the glmU gene, such aspMLD71 (data not shown). The molecular weight of theoverproduced protein was consistent with the value of49,130 calculated from the DNA sequence (35). A typicalfractionation of cell extracts showed that the highly overpro-duced protein was found mainly in the soluble fraction butthat significant amounts (10 to 20%) remained associatedwith the particulate fraction, a finding which could be due tothe formation of aggregates at this high level of protein

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mw A B C D E F mw

94

67

43

30

20

FIG. 5. Overproduction of the GlmU protein and its purificationfrom crude extracts of strain JM83(pMLD78). StrainJM83(pMLD78) was grown exponentially at 30°C in 2YT-ampicillinmedium. At an optical density of the culture of 0.2, the temperaturewas either maintained at 30'C or shifted to 42°C. Cells wereharvested 1, 2, or 3 h later and were disrupted by sonication. Asdescribed in Materials and Methods, the protein contents of thesoluble and particulate fractions obtained after high-speed centrifu-gation of the corresponding crude extracts were analyzed by SDS-PAGE. Molecular weight (mw) standards (thousands) indicated onthe left are as follows: phosphorylase b (94), bovine serum albumin(67), ovalbumin (43), carbonic anhydrase (30), and soybean trypsininhibitor (20). Lanes A to D: analysis of the soluble fraction fromJM83(pMLD78) cells grown at 30°C (lane A) or first at 30°C and thenfor 1 h (lane B), 2 h (lane C), or 3 h (lane D) at 42°C. (E) Pool offractions containing the GlmU activity eluted from the DEAE-Trisacryl column. (F) Pool of fractions containing the GlmU activityeluted from the hydroxylapatite-Ultrogel column.

expression. Finally, we showed that after 3 h of growth at42°C, JM83(pMLD78) cells contained 410-fold more GlcNAc-1-P uridyltransferase activity than cells carrying the pUC19control vector (Table 2). All these different results takentogether supported the initial proposal that the ghnU geneencodes the GlcNAc-1-P uridyltransferase involved in thebiosynthesis of UDP-GlcNAc.

Purification of E. cofi GlcNAc-l-P uridyltransferase. Thehigh-level overproduction of theglmU gene product directedby the pMLD78 plasmid was subsequently used for itspurification, which was a quite straightforward procedurewith only two chromatographic steps on columns of DEAE-Trisacryl and hydroxylapatite-Ultrogel. The final enzymepreparation was judged essentially pure by denaturing gelelectrophoresis (Fig. 5, lane F), and its amino acid compo-sition after acid hydrolysis corresponded to that calculatedfrom the gene sequence (35). From only 1 liter of culture,approximately 16 mg of homogeneous GlcNAc-l-P uridyl-transferase was obtained in an overall unit yield of 44%, with

an overall purification factor of 1.8 (Table 3). Compared withthe specific activity of the crude extract from the nonover-producing strain (Table 2), a purification factor of 755 couldbe estimated for this final preparation.

DISCUSSION

The product of the EcoURF-1 open reading frame (35),which precedes the glmS gene on the E. coli chromosome,has now been clearly identified as the GlcNAc-1-P uridyl-transferase. This enzyme catalyzes the formation of UDP-GlcNAc, which is an essential precursor of cell wall pepti-doglycan and lipopolysaccharide. We were able to constructa strain with the corresponding chromosomal glmU genedisrupted that was viable only in the presence of a plasmidcarrying the wild-type gene. Since the plasmid bears athermosensitive replicon, the effects of this inactivationwere easily visualized by shifting exponentially growing cellsto the restrictive temperature. Cells were shown to accumu-late GlcNAc-1-P, while pools of the nucleotide peptidogly-can precursors located downstream in the pathway wererapidly depleted. As a result, peptidoglycan synthesis (andprobably also lipopolysaccharide synthesis) was inhibited,and cells progressively lost their rod shape before cell lysisfinally occurred. The fact that these different effects wereobserved only after a few hours is explained by the timerequired for curing cells (by progressive dilution or inactiva-tion) of the low-copy-number pGMU plasmid and of thefunctional GhmU protein molecules present at the time of thetemperature shift.A search of data bases for sequences homologous to

EcoURF-1 revealed the tms gene of Bacillus subtilis (27)(44% identical amino acids in the predicted protein sequenc-es). The tms gene was identified previously by the isolationof a mutant strain (tms-26 strain) that was temperaturesensitive for growth (4). While this work was in progress,this B. subtilis tms-26 mutant strain was characterized bio-chemically and also shown to be altered in GlcNAc-l-Puridyltransferase activity (15), a finding in agreement withour data and which further supported the correct assignmentof the function of the glmiU gene.The effects of inactivating GlcNAc-l-P uridyltransferase

on the growth and cell morphology of E. coli cells weresimilar to those previously observed by Copeland and Mar-mur with the Bacillus tms-26 mutant strain (4). Interestingly,in both bacterial species, this defect led to an earlier station-ary growth phase and a loss in cell viability, but these effectswere not immediately followed by a rapid decrease of theculture absorbance (cell lysis) as generally observed withmutants defective in peptidoglycan synthesis (8, 22, 23, 26,33, 37). Nevertheless, the increasing number of ghosts in theE. coli mutant cell population grown at the restrictivetemperature (more than 50% in overnight cultures) wasconsistent with a thermosensitive lytic phenotype. The ab-sence of an abrupt cell lysis could be related to the fact thatthe mutation affects not only peptidoglycan but also thesynthesis of the lipopolysaccharide. It could be effectivelyassumed that a simultaneous inhibition of the biosynthesesof both cell envelope components affects the functioning ofpeptidoglycan hydrolases in some way. Alternatively, thiseffect could be due to this particular mutation and to theaccumulation of GlcNAc-l-P in the pool content of E. colicells. This precursor, which is normally present at a lowcellular concentration, could possibly interfere with anotherstep of the general cell metabolism when it accumulates,leading to an overall growth inhibition (bacteriostatic effect).

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6156 MENGIN-LECREULX AND vAN HEIJENOORT

TABLE 3. Purification of GlcNAc-1-P uridyltransferase from E. coli

Step Total protein Total activity Sp act Purification Yield (%)(mg) (U) (U.- mg-') (fold)Crude extract 67 552 8.2 1 100DEAE-Trisacryl 25 317 12.7 1.5 65Hydroxylapatite-Ultrogela 16 241 15.1 1.8 44

a The values for hydroxylapatite-Ultrogel were calculated for the total protein recovered from the DEAE-Trisacryl. In fact, only 50% of the extract was loadedonto the hydroxylapatite column.

In the present paper the first purification to homogeneityof a bacterial GlcNAc-1-P uridyltransferase is described.The final preparation obtained from the highly overproduc-ing strain had a specific activity of 15.1 U. mg of protein-'(Table 3), whereas that of the crude extract from theplasmid-free strain was approximately 0.02 U. mg-1 (Table2). Assuming that there was no loss in activity in this crudeextract from 3.8 x 1011 cells (60 mg of protein) and that thepurified enzyme contained only active GlmU molecules, acopy number of at most 2,500 per cell could be estimated forthis enzyme in a plasmid-free parental strain. It was there-fore understandable that this protein was efficiently detectedby SDS-PAGE only with extracts from the very highlyoverproducing strain harboring the plasmid with the glmUgene under the control of the strongPR promoter.

In the enzymatic assay used throughout this work,GlcNAc-l-P and UTP were at saturating concentrations asjudged by the Km values of these substrates (0.062 and 0.12mM, respectively, determined with a crude enzyme prepa-ration [data not shown]). From the specific activity of 15.1U. mg of protein-' determined for the pure enzyme andassuming a molecular weight of 49,131 for the glmU geneproduct (35), a turnover number of 742 min-1 was calcu-lated. Under the in vitro conditions considered here, theuridyltransferase activity could catalyze the formation ofapproximately 50 x 106 molecules of UDP-GlcNAc in eachcell during a 30-min generation time (2,500 x 742 x 30).Even though a 50% turnover of peptidoglycan is taken intoaccount (12), this value is much higher than that (7 x 106)required for the formation of the average peptidoglycancontent of cells growing in rich medium, previously esti-mated at 3.5 x 106 U (20, 24). However, the specificrequirements for UDP-GlcNAc molecules of the lipopoly-saccharide pathway, which seem to be more or less equiva-lent, should be also taken into account (31). Furthermore, anumber of in vivo conditions (e.g., substrate concentrationand pH) can differ substantially from the optimal in vitroconditions we used. Considering that the pool level of UTPis about 1 mM in gram-negative bacteria (3) and that the poolof GlcNAc-1-P has been estimated here at approximately0.05 mM, the enzyme seems to work in vivo with saturatingconcentrations of UTP and with concentrations of GlcNAc-1-P close to its Km value. The in vitro assay conditions areprobably not far from the physiological ones, and we con-clude that the enzyme activity in the cells is not in greatexcess compared with that needed for the synthesis of thesedifferent cell envelope components.We mentioned above that the lack of an obvious promoter

consensus on the DNA sequence upstream of the gimS geneencoding glucosamine synthase suggested that theglmU andgimS genes were cotranscribed. Recently, Plumbridge et al.showed that the glmS gene of E. coli is subject to a controlmechanism which causes its expression to be reduced whenthe nag regulon of genes coding for amino sugar-degrading

enzymes is derepressed (29). The transcription in this par-ticular chromosomal region and the possible coregulation ofthe glmU gene expression now have to be examined. More-over, since no other open reading frame of unknown func-tion has been identified in the unc-phoS region, the genesinvolved in the two putative steps leading from glucosamine-6-phosphate to GlcNAc-l-P may belong to a separate chro-mosomal region. These genes, as well as their products,remain to be characterized.

ACKNOWLEDGMENTSWe thank J. Plumbridge and B. Badet for helpful discussions and

for communicating results before publication.This work was supported by grants from the Centre National de la

Recherche Scientifique (URA 1131) and the Institut National de laSante et de la Recherche M6dicale (900314).

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