Vol. 164, No. 2JOURNAL OF BACTERIOLOGY, Nov. 1985, P. 854-8600021-9193/85/110854-07$02.00/0Copyright © 1985, American Society for Microbiology

Regulation of Sialic Acid Metabolism in Escherichia coli: Role ofN-Acylneuraminate Pyruvate-Lyase

ERIC R. VIMRt* AND FREDERIC A. TROYDepartment ofBiological Chemistry, University of California School of Medicine, Davis, California 95616

Received 2 April 1985/Accepted 23 August 1985

In Escherichua coli, synthesis of sialic acid is not regulated by allosteric inhibition mediated by cytidine5'-nmonophospho-N-acetylneuraminic acid (CMP-NeuNAc). Evidence for the lack of metabolic control byfeedback inhibition was demonstrated by measuring the intracellular level of sialic acid and CMP-NeuNAc inmutants defectiv'e in sialic acid polymerization and in CMP-NeuNAc synthesis. Polymerization-defectivemutants could not synthesize the polysiplic acid capsule and accumulated ca. 25-fold more CMP-NeuNAc thanthe wild type. Mutants unable to activate sialic acid because of a defect in CMP-NeuNAc synthetaseaccumulated ca. sevenfold more sialic acid than the wild type. An additional threefold increase in sialic acidlevels occurred when a mutation resulting in loss of N-acylneuraminate pyruvate-lysase (sialic acid aldolase)was introduced into the CMP-NeuNAc synthetase-deficient mutant. The aldolase mutation could not beintroduced into tpe polymerization-defective mutant, suggesting that any further increase in the intracellularCMP-NeuNAc concentration was toxic. these results show that sialic acid aldolase can regulate theintracellular concentration of sialic acid and therefore the concentration of CMP-NeuNAc. We conclude thatregulation of aldolase, mediated by sialic acid induction, is necessary not only for dissimilating sialic acid (E. R.Vimr and F. A. Troy, J. Bacteriol. 164:845-853, 1985) but also for modulating the level of metabolicintermediates ip'the sialic acid pathway. In agreement with this conclusion, an increase in the intracellularsialic acid concentration was correlated with an increase in aldolase activity. Direct evidence for the central roleof aldolase in regulating the metabolic flux of sialic acid in E. coli was provided by the finding that exogenous,radiolabeled sialic"acid was specifically incorporated into sialyl polymer in an aldolase-negative strain but notin the wild type.

In procaryotic and eucaryotic cells, sialic acids such as

N-acetylneuraminic acid (NeuNAc) are activated for partic-ipation in sialoglycoconjugate synthesis by cytidine 5'-mhonophospho-N-acetylneuraniinic acid synthetase (CMP-NeuNAc synthetase, EC 2.7.7.43). This enzyme catalyzesthe reaction NeuNAc + CTP -, CAMP-NeuNAc t PPi. In

eucaryotes, CMP-NeuNAc is an allosteric inhibitor of thereaction UDP-N-acetylglucosainine (GlcNAc). N-acetylmannosasmine + UDP, which is catalyzed by UDP-GlcNAc 2-epimerase (EC 5.1.3.14). The epimerase is thefirst committed step in thre pathway to CMP.NeuiNAc (re-viewed in reference 2). Transfer of sialic acid to oligosac-charide chains in eucaryotic cells is often the terniinal eventin glucoconjugate synthesis. Therefore, the cellular concen-tration of CMP-NeuNAc could affect the rate at whichsialoglycoconjugates are, expressed. In bacteria that synthe-size sialic acid, there may be a less stringent requirement forregulation of CMP:NeuNAc levels because synthesis of thepolysialic 'acid capsule, catalyzed by a membranesialyltransferase,complex (16), is not essential for growth.

In the accompanying paper, we showed that the intracel-lular accumulation of sialic acid derived from exogenouslyadded sialic acid was toxic in Escherichia coli (20). It seemedpossible that'the accumulation of sialic acid derived from theendogenous biosynthetic pathway might also be toxic, andalternative mechanisms to self-reg,ulate the concentration ofsialic acid were studied. In this communication, we presentan analysis of E. coli mutants that have defects in sialic acid

* CQrresponding author.t Present address: Department of Veterinary Pathobiology, Col-

lege of Veterinary Medicine, University of Illinois, Urbana, IL61801.

degradation, activation, and polymerization. The data showthat, in contrast to eucaryotic cells, CMP-NeuNAc in E. colidoes not act as an allosteric inhibitor of its own biosynthesis.We now report that aldolas'e can effectively modulate theintracellular concentration of sialic acid and thereby theconcentration of CMP-NeuNAc. We also demonstrate thataccumulation of CMP-NeuNAc is potentially more toxicthan is accumulation of the free sugar, underscoring thephysiolofical need to regurate sialic acid metabolism.

(Portions of this work have been presented earlier [E. R.Vimr, R. I. Merker, and F. A. Troy, Fed. Proc.'42:2164,1983].)

MATERIALS AND METHODS

Bacterial strains. All bacterial strains except RS1085 wereE. coli K-12 or K-12-K1 hybrid derivatives (Table 1). Hybridstrains were constructed by conjugation of appropriate F-recipients with the Hfr kps+ strain' RS1085. This Hfr strainwas constructed by R. P. Silver and J. Foulds by selectingLac' colonies at 42°C after having introduced an F' tsJJ4lac+ plasmid into a Lac- derivative of the kps+ strain D699(R. P. Silver, personal communication). Our prototypicalK-12-K1 hybrid' strain, EV1, was obtained by crossingRS1085 with PA360, selecting for Ser+, and counterselectingwith 200 ,ug of streptomycin sulfate' per ml. Approximately70% of the Ser+ exconjugates were kps+ as judged by theirsensitivity to bacteriophage K1F. This phage only infectsKl-encapsulated strains of E. coli and has been describedpreviously (19, 27). Other kps' hybrid strains wereconstructed in auxotrophic recipients with mutations map-ping near serA. For example, strain EV36 (Table 1) wasisolated as an argA+ exconjugate after being crossed withstrain RS1085. All phage-sensitive recombinants produced

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TABLE 1. Bacterial strains and their origin

E. coli Relevant genotype Origin or referencestrain

PA360 thi-J leuB6 hisGi serAl argHl CGSC288athr-J lacYl gal-6 malAlxyl-7 mtl-2 rpsL9 tonA2supE44

JL3664 argA22 gaIP23 rha-200 20RS1085 Hfr kps+ R. P. SilverEV1 PA360 [kps+ serA+ malA +]b This studyEV4 EV1 kps-21 Spontaneous K1FlEV5 EV1 kps-22 Spontaneous K1FrEV11 EV1 kps-23 Spontaneous KlFlEV13 EV1 kps-24 Spontaneous KlFrEV24 EV1 kps-25 Spontaneous K1FrEV31 JL3664 rpsL9 P1/EV1 x JL3664EV36 EV32 [argA + kps+ rha +] This studyEV52 EV36 nanA4 zgj-791::TnJO uxa 20EV70 EV36 nanA4 zgj-791::TnlO 20EV72 EV36 nanA+ zgj-791::TnlO 20EV76 JL3664 nanA4 zgj-791::TnJO 20EV77 JL3664 nanA+ zgj-791::TnJO 20EV90 EV5 nanA4 zgj-791::TnlO Pl/EV52 x EV5

a E. coli Genetic Stock Center number (Yale University School of Medi-cine, New Haven, Conn.).

b Markers in brackets are known to be derived from the non-K-12 strainRS1085.

polysialic acid capsular antigen (Kl antigen) that wasimmunoreactive with anti-a-2,8-polysialic acid antiserum.The preparation and use of this antiserum has been described(13, 19). Using the methods of antibody stabilization andtransmission electron microscopic visualization (27), wedemonstrated that strains EV1 and EV36 expressed polysialicacid capsules that were morphologically indistinguishablefrom those produced by two independent clinical isolates ofE. coli Kl (C. Whitfield, E. R. Vimr, J. W. Costerton, and F.A. Troy, unpublished observations). These observationsindicated that the hybrid Kl strains described here receivedall the kps genetic information required for synthesizingcapsular polysialic acid. The success of our hybrid strainconstructions was predicated on the earlier observations of0rskov et al. (11), who showed that kps genes mapped nearserA in Kl strains.Mutant isolation. Overnight Luria broth cultures of strain

EV1 were diluted 100-fold into the same medium and platedon Luria broth agar seeded with ca. 3 x 109 KlF infectiveparticles. Spontaneous phage-resistant colonies arising afterovernight incubation at 37°C were picked and streaked forsingle colonies to dilute the phage. Colonies were purified bytwo additional single-colony isolations and then retested forKlF resistance. All KlFr mutants in this study were nega-tive for the presence of Ki antigen by a test using anti-a-2,8-polysialic acid antibodies as previously described (13).Growth conditions and cell fractionation procedures. Luria

broth and minimal E medium were described in the accom-panying paper (20). Unless otherwise indicated, cell extractswere prepared from stationary-phase cultures that wereharvested within 1 h after the cells had completed exponen-tial growth. Cells were sedimented by centrifugation andwashed once prior to disruption by sonication with four 30-spulses (Branson model 185 sonifier with microtip), with a30-s cooling period between each pulse. Samples weremaintained on ice during all procedures. The buffer used inpreparing extracts for sialyltransferase assays was 50 mMTris hydrochloride, pH 8.0, containing 2 mM dithiothreitoland 3.5 mM magnesium acetate. For CMP-NeuNAc synthe-

tase assays, 10 mM Tris hydrochloride (pH 7.6)-0.1 mMMgSO4 was used. After sonication, unbroken cells wereremoved by centrifugation for 10 min at 1,930 x g. Materialnot sedimenting after 1 h of centrifugation at 23,640 x g wasdesignated the soluble or supernatant fraction. As describedbelow, this fraction contained CMP-NeuNAc synthetase andsialic acid aldolase. The 23,640 x g pellet was washed bysuspension in buffer and sedimentation as above'. Thewashed pellet was designated the membrane fraction; itcontained the membrane sialyltransferase.

Assay for sialyltransferase. Sialyltransferase activity wasmeasured by a modification of previously published proce-dures (12). This assay measures the incorporation of[14C]NeuNAc from CMP-['4C]NeuNAc into polymeric prod-ucts, which are then separated from the substrate bychromatography. Membranes suspended in the Tris bufferdescribed above were incubated in a 40-,ul total volumecontaining 150 to 450 jig of membrane protein and 0.05 ,uCiof CMP-[14C]NeuNAc (7,000 dpm/nmol). Incubations werecarried out for 5 to 15 min at 33°C, after which 15-,ul sampleswere spotted onto Whatmann 3MM paper and chromato-graphed in ethanol-1 M ammonium acetate (7:3), pH 7.5, aspreviously described (12). Radioactivity remaining at theorigin represented incorporation of [14C]NeuNAc into poly-meric material and was quantitated by liquid scintillationspectrometry (12). Under these conditions, radioactivityremaining at the origin was proportional to protein, but wasnot linear with time.' Each assay was performed at twodifferent protein concentrations to verify'proportionality toprotein. One unit of sialyltransferase activity was defined as1 nmol of sialic acid incorporated into polymeric products in1 h at 33TC and was calculated by assuming linearity,Colominic acid (Sigma Chemical'Co., St. Louis, Mo.), anoligomeric form of sialic acid composed of 10 to 12 sialic acidresidues joined through a-2,8-ketosidic linkages, was in-cluded in some incubation mixtures as an exogenous accep-tor at a final concentration of 7.5 mg/ml. Under theseconditions, reaction rates were linear for up to 1 h. Theinclusion of colominic acid increased sialyl polymer synthe-sis in wild-type membranes three- to fivefold by serving as anexogenous acceptor for sialyltransferase, as described pre-viously (6, 18).

Assay for CMP-NeuNAc synthetase. CMP-NeuNAc syn-thetase converts CTP plus NeuNAc into CMP-NeuNAc andPP, in the presence of magnesium and a sulfhydryl reagent.Activity in the soluble fraction was measured as describedby Warren and Blacklow (23) except that L-cysteine wasused in place of reduced glutathione. This assay takesadvantage of the fact that ketosidically linked sialosyl resi-dues in CMP-NeuNAc are resistant to alkaline-borohydridereduction, whereas unreacted sialic acid substrate is con-verted to sialitol. Sialitol fails to give a color reaction in theperiodate-thiobarbituric acid (TBA) assay used here forcolorimetric determination of sialic acids (21). Sialic acidconverted to CMP-NeuNAc is released after borohydridereduction by the acidic conditions used in the TBA assay.Values reported are averages of duplicate determinationscorrected for background color production by subtraction,using duplicate assay tubes which lacked exogenous CTP.One unit of activity represented 1 nmol of sialic acid con-verted to CMP-NeuNAc in 1 h at 37°C.

Assay for sialic acid aldolase. Sialic acid aldolase wasmeasured as described in the accompanying paper (20).

Analytical procedures. The amount of membrane-boundsialic acid was determined by the TBA procedure after acidhydrolysis of the sialyl polymer in 0.1 N H2SO4 (800C, 2.5 h)

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TABLE 2. Phenotypes of acapsular mutants based on membrane-bound sialic acid and enzymatic activities relevant to Kl antigenbiosynthesis

Sialytransferase activity (U/mg of membrane Membrane-bound NeuNAc (kg/mg ofprotein) CMP-NeuNAc membrane protein) released by:

Strain synthetase activityWith exogenous Without exogenous (U/mg of soluble protein) Acidb Exo-neuraminidase

acceptor" acceptor

PA360 0.06 0.03 0.4 0 (30.6) <0.7EV1 3.75 1.14 24.4 52.1 (82.7) 63.9EV4 2.20 0.08 18.6 12.1 (42.7) 9.0EV5 2.97 2.37 3.9 4.5 (35.1) <0.7EV11 0.80 0.03 39.4 6.6 (37.2) <0.7EV13 0.19 0.05 3.2 5.6 (36.2) <0.7EV24 2.61 1.96 17.3 4.6 (35.2) <0.7

a Each standard incubation mixture contained colominic acid at a final cohcentration of 7.5 mg/ml as described in Materials and Methods.b 0.1 N H2SO4, 80°C, 2.5 h. Because much of the TBA-positive material released by acid hydrolysis of total membranes is not due to the presence of sialic acid,

the value obtained from the TBA assay of the control strain, PA360, was subtracted from each value shown in parentheses to give the indicated amounts of boundsialic acid.

or by exo-neuraminidase digestion (0.1 M sodium acetatebuffer, pH 5.5, 37°C, 4 h) with 1 U of Clostridium perfringensneuraminidase (Worthington Biochemical Corp., Freehold,

N.J.). Membranes (1.5 to 1.9 mg of protein in 0.5 ml) were

prepared in either acid or buffer and incubated as above.After treatment, insoluble material was removed by centrif-ugation in a microfuge, and TBA assays were carried out onthe supernatant.Methods for protein determination, paper chromatogra-

phy, and radiometric scanning of chromatograms and thesources of radiolabeled chemicals are described in the ac-

companying paper (20). Uniformly 14C-labeled glucose (210mCi/nimol) was purchased from ICN Pharmaceuticals, Inc.,Irvine, Calif. DEAE-cellulose chromatography on SephadexA-25 anion exchange has been described (19). Polyacryl-amide gel electrophoresis (PAGE) (10% resolving gel, 4%stacking gel) was carried out in the presence of sodiumdodecyl sulfate (SDS) as described by Laemmli (7).Fluorography with the fluorographic enhancer Amplify was

carried out as described by the manufacturer (AmershamCorp., Arlington Heights, Ill.).

Determination of intracellular concentrations of NeuNAcand CMP-NeuNAc. Appropriate strains were grown to mid-exponential phase in minimal medium containing all requiredsupplements and 0.3% glycerol. Cultures were sedimentedby low-speed centrifugation, and the cell pellets were gentlyrinsed with water. Pellets were immediately suspended in 10mM Tris hydrochloride, pH 7.2, containing 1 mM MgSO4and disrupted by six cycles of sonication as described above.Particulate material was removed by centrifugation as de-scribed for the sialyltransferase assay, and the protein con-tent in the soluble fraction was determined. The solublefraction was then extracted with 50% ethanol by adding 1volume of 100% ethanol to 1 volume of supernatant, After 1h at 0°C, precipitated material was removed by centrifuga-tion, and the supernatants were lyophilized and redissolvedin 1 volume of water. The concentration of NeuNAc andCMP-NeuNAc was determined by TBA assay before andafter alkaline borohydride reduction, as described above forthe CMP-NeuNAc synthetase assay. The sialic acid contentin extracts of strains EV5, EV11, and EV90 was estimatedfrom absorbance curves, using pure sialic acid as the stan-dard. This was possible because the amount of sialic acidrelative to interfering substances was high, as indicated byA549/A532 ratios of 2.0 to 2.3. These ratios were similar tothose obtained with pure sialic acid. The absorbance ratiowas 0.9 to 1.3 in extracts of strain EV1, requiring a correc-

tion to estimate sialic acid levels accurately (21). Cellularconcentrations of TBA-reactive intermediates were calcu-lated by assuming that the nanomoles of sialic acid permilligram of soluble protein measured represented completerecovery of NeuNAc or CMP-NeuNAc and that a singlebacterium contained 8 x 10-14 g of soluble protein in anintracellular volume of 6.2 x 10-13 ml (3, 15).

RESULTSIsolation of mutants with defects in sialic acid activation and

polymerization. Spontaneous mutants derived from strainEV1 that survived plating with bacteriophage KlF wereisolated at frequencies of ca. 10-5. To identify the likely sitesof mutational lesions in these phage-resistant mutants,subcellular fractions were prepared and assayed for mem-brane sialyltransferase activity, soluble CMP-NeuNAc syn-thetase activity, and the amount of membrane-boundpolysialic acid. Membranes prepared from these strainseither lacked or had low levels of polysialic acid, as deter-mined by the TBA assay after acid or enzymatic release offree sialic acid (Table 2). Compare, for example, the amountof membrane-associated sialic acid in the wild type, EV1,with that in the kps' strain PA360 (see reference 20 fordefinition of the kps-null [kps'] genotype) and the variousmutant strains (Table 2). This result indicated that thephage-resistant mutants would have identifiable defects inenzyme activities required for sialic acid synthesis, activa-tion, or polymerization.The potential biochemical basis for mutations that could

result in an acapsular phenotype may be inferred from Fig. 1.Several mutants analyzed in detail had defects in sialic acidactivation (EV5) or polymerization (EV4 and EV1l) (Table2). One mutant, EV13, had a pleiotropic defect resulting indecreased CMP-NeuNAc synthetase and sialyltransferaseactivities. EV24 had approximately wild-type levels of theseenzymes, suggesting a defect at a step prior to sialic acidactivation. At least 11 other independently isolatedacapsular mutants were analyzed and shown to have pheno-types similar to those shown in Table 2.

If the biosynthetic pathway (Fig. 1) is efficiently regulatedby end product inhibition, a mutant blocked in sialic acidpolymerization (sialyltransferase deficient) would not beexpected to accumulate intracellular concentrations ofCMP-NeuNAc much above the wild-type basal level. Conversely,if CMP-NeuNAc is not an allosteric inhibitor, then a poly-merization-deficient mutant might be expected to accumu-late CMP-NeuNAc and perhaps free sialic acid. To deter-

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UDP-Gic NAcEpimerose K UDP

G Epimerase MGIcAc* - ManNAc<-

ATPKinaseADP

GIcNAc-6-P

Epimerose P

Man NAc-6-P /

Pi

p

MEMBRANE

Pyrr

NeuNAc-AldoloseNeuNAc <

rCTP,

CMP- Neu NAcPi 8 tSynthetose

to

x

-I0

EC.

0

70

6Pyr

CMP-NeuNAcKLCMP

Sialyltransferose(s)

Sialyl Polymer

FIG. 1. Proposed metabolic interconversions of sialic acid in E.coli. See reviews by Warren (22), McGuire (9), and White (24).Epimerizations indicated by the dashed lines have not been identi-fied in bacteria. Pyr, Pyruvic acid; ManNAc, N-acetylman-nosamine; 6-P, 6-phosphate; PEP, phosphoenolpyruvate.

mine whether the pathway is self-regulated, the intracellularconcentrations of free sialic acid and CMP-NeuNAc inmutants defective in CMP-NeuNAc synthetase activity(EV5) and sialyltransferase activity (EV11) were comparedwith those in the wild type (EV1). This required that wemeasure CMP-NeuNAc in the presence of free sialic acid(Table 3).

Isolation and characterization of CMP-NeuNAc accumu-lated in EVil. During assays ofCMP-NeuNAc synthetase inextracts of strain EV11, we noted high endogenous levels ofTBA-reactive material. This material was resistant toborohydride reduction, but became sensitive after mild acid

0.40.30.20.1o Z

0 20 40 60 80 100 120 140 160FRACTION NUMBER

FIG. 2. Purification of accumulated CMP-NeuNAc. Strain EV11was grown to mid-exponential phase in 1 liter of minimal mediumcontaining 0.2% glucose. Cells were sedimented, washed, andsuspended in 300 ml of minimal medium containing 0.01% glucoseplus 200 ,uCi of [U-'4Clglucose and incubated for 3 h with shaking at37°C. Labeled cells were collected by centrifugation, washed, andextracted with ethanol as described in Materials and Methods.Ethanol-soluble material was fractionated on a column (1.5 by 30cm) of DEAE-Sephadex A-25 at 4°C. Fractions (2.5 ml) wereanalyzed for radioactivity ( ) and conductance (-- -). Arrows,Elution of [4-14C]NeuNAc and CMP-[9-3H]NeuNAc determinedfrom a separate column run with these standards. The bar indicatesfractions 83 to 92, which were pooled for further analysis.

hydrolysis. This indicated that the keto group at C-2 in sialicacid was in an acid-labile linkage (1, 23), the result expectedif the TBA reactivity were due to sialic acid released fromCMP-NeuNAc. To confirm this assessment, strain EV11was labeled by growth on [U-14C]glucose. The sugar nucle-otides were isolated by ethanol extraction and purified byfractionation on DEAE-Sephadex A-25. The major sugarnucleotide had chromatographic properties identical to thoseof authentic CMP-NeuNAc (Fig. 2). Fractions (indicated bythe bar in Fig. 2) were pooled, lyophilized, and analyzed bydescending paper chromatography (Fig. 3). The 14C-labeledmaterial migrated with an Rf (Fig. 3A) similar to that ofauthentic CMP-[4-14C]NeuNAc (Fig. 3D). Mild acid hydro-lysis of the isolated material resulted in two peaks ofradioactivity (Fig. 3B). The front peak migrated with an Rfnearly identical to that of sialic acid obtained by hydrolysisof CMP-[4-14C]NeuNAc (Fig. 3C). The slower peak migratedwith an Rf of ca. 0.2, as expected for free [14C]CMP (8). Weconclude from these results that the 14C-labeled material

TABLE 3. Concentrations of NeuNAc and CMP-NeuNAc in mutant and wild-type E. coli strains

Strain Relevant Concn (nmol/mg of soluble protein) Intracellular concn (mM)genotype CMP-NeuNAca Free NeuNAcb NeuNAc CMP-NeuNAc

PA360 kpsn nan+ <0.5 <0.5 0 0EV1 kps+ nan+ 1.3 4.4 0.6 0.2EV5 kps-22 nan+ <0.5 31.7 4.1 0EV90 kps-22 nanA4 <0.5 93.1 12.0 0EV11 kps-23 nan+ 39.4 8.3 1.1 5.1

a Sialic acid resistant to borohydride reduction.b Total sialic acid (by the TBA assay) minus the CMP-NeuNAc concentration.

z- 7-IY7Z.= .1 77777777777777

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isolated as shown in Fig. 2 was CMP-NeuNAc. Furtherconfirmation was provided by showing that the purifiedmaterial was resistant to borohydride reduction and onlybecame TBA reactive after mild acid hydrolysis. In addition,the biosynthetically derived, radiolabeled CMP-NeuNAcwas used as a substrate by the membrane sialyltransferaseprepared from each of the strains shown in Table 2. Thelarger amount ofCMP-NeuNAc accumulated by strain EV11than by the wild type is consistent with the idea that thismetabolite does not act as an end product inhibitor of its ownsynthesis.

Metabolic fate of sialic acid. In the accompanying paper(20), we showed that mutants defective in sialic acid aldolaseactivity were unable to use sialic acid as a carbon source.This result implied that a single aldolase functions in sialicacid degradation in E. coli and suggested that aldolase mightalso regulate the intracellular concentration of sialic acid.According to the pathway shown in Fig. 1, the only knownmetabolic fate for sialic acid in the absence of aldolaseshould be activation to CMP-NeuNAc and polymerization togive sialyl polymer, regenerating CMP. Therefore, the sialicacid accumulated by aldolase-negative mutants that are alsonull for sialyl polymer synthesis (kps') should not be de-graded or polymerized. Confirmation of these predictions isshown in Fig. 4. In the absence of aldolase, a strain with afunctional kps locus produced a single high-molecular-weight radiolabeled component in vivo after accumulatingexogenous '4C-labeled sialic acid (lane 2). A strain lackingaldolase and functional kps genes (EV76) did not, as ex-pected, produce labeled polymer (lane 4), nor did it catabo-lize sialic acid. In contrast, strains with aldolase, regardlessof their kps genotype, synthesized multiple radiola-

I

0.

I-w

0

RELATIVE MIGRATION (Rf)I.0

FIG. 3. Characterization ofCMP-NeuNAc accumulated in strainEV11. Samples of biosynthetic '4C-labeled CMP-NeuNAc (A),prepared as shown in Fig. 2, and authentic CMP-[4-'C]NeuNAc(D), at 35,000 dpm each, were incubated for 30 min at 37°C in water,fractionated by descending paper chromatography, and detected byradiometric scanning. The amount of contaminating NeuNAc (fast-er-migrating peak) was approximately equal in both preparations ofCMP-NeuNAc (slower-migrating peak). Prior to chromatography,biosynthetically labeled CMP-[U-14C]NeuNAc was incubated for 30min at 37°C in 0.1 N HCl (B). Note the quantitative loss ofCMP-NeuNAc and the appearance of a slowly migrating peak of14C-CMP at ca. Rf 0.2 that was missing from the chromatogram ofacid-hydrolyzed authentic CMP-[14C]NeuNAc (C). S T F, Solventfront.

1 2 3 4 5 6 7 8

FIG. 4. Specific radiolabeling of sialyl polymer in aldolase-negative E. coli. The metabolic fate of internalized sialic acid wasdetermined by SDS-PAGE. Exponentially growing cultures of E.coli (200 ml of minimal medium) were labeled with 0.05 mMNeuNAc containing 2.3 pCi of [4-14C]NeuNAc (1.6 mCilmmol).After 2.5 h, cells were sedimented, washed, and suspended to 2 ml.Samples were treated with an equal volume of double-concentratedLaemmli sample buffer (7) at 370C for 30 min (lanes 1 through 4) orat 900C for 10 min (lanes 5 through 8). Lanes: 1 and 5, EV72 (kps'nan'), 10,800 cpm loaded; 2 and 6, EV70 (kps' nanA4), 6,360 cpmloaded; 3 and 7, EV77 (kps' nan'), 17,175 cpm loaded; 4 and 8,EV76 (kpSn nanA4), 2,000 cpm loaded. For PAGE, 60-pJl portionswere fractionated in each lane on a 0.75-mm-thick polyacrylamidegel. Arrow, Top of the separating gel.

beled products that were derived from the exogenouslyadded sialic acid (lanes 1 and 3). The apparent molecularweight of the labeled polymer in the kps', aldolase-negativemutant EV70 (lane 2) was ca. 1.5 x i05, which is similar tothe size reported for authentic sialyl polymer (19). Thesepolymers are also characterized by polydispersity in SDS-PAGE (19; unpublished observations). Additional confirma-tion that these polymers were polysialic acid was obtainedby demonstrating their heat lability (Fig. 4, lane 6) andsensitivity to endo-a-2,8-polysialic acid depolymerase puri-fied from phage KlF (data not shown). Together theseresults provided evidence for the central role of aldolase insialic acid dissimilation and clearly indicated that this en-zyme modulated the pool of exogenously acquired sialic acidthat became avaiable for activation and polymerization.Because our hybrid E. coli mutant and wild-type strainswere sensitive to P1 infection, we were able to constructappropriate double mutants to determine directly the effectof loss of aldolase on the accumulation of intracellular sialicacid and CMP-NeuNAc.

Effects of aldolase mutation on intracellular concentrationof NeuNAc and CMP-NeuNAc. The suggestion that CMP-NeuNAc did not regulate the sialic acid biosynthetic path-way by feedback inhibition led to four predictions. (i) Effiuxof CMP-NeuNAc, if it occurs at all, must be slower than therate of CMP-NeuNAc synthesis; (ii) E. coli must lacksignificant CMP-NeuNAc hydrolase activity; (iii) in theabsence of CMP-NeuNAc synthetase, NeuNAc should ac-cumulate; and (iv) NeuNAc aldolase, which is induced byfree sialic acid, should be affected by fluctuations in the sialicacid pathway and in turn should affect the steady-stateconcentration of sialic acid. Experimental evidence consis-tent with these predictions follows.The efflux rate must be slow compared to the rate of

synthesis of CMP-NeuNAc (Table 3). For example, strainEV1i accumulated CMP-NeuNAc at least 25-fold above theconcentration measured in wild-type EVi (Table 3). Theaccumulated CMP-NeuNAc would not be removed byhydrolase, since E. coli lacks this enzyme (8, 17). We were

A.

D.* ORIGIN S#F

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TABLE 4. Induction of aldolase by sialic acid producedbiosynthetically

Relevant Sialic acid aldolase RelativeStrain genotype (U/mg of soluble protein) inductiona (fold)

PA360 kps' nan+ 3.8 1EV1 kps+ nan+ 2.1 1EV5 kps-22 ndn+ 20.6 7EV90 kps-22 nanA4 <0.3

a Aldolase specific activity measured in strains PA360 and EV1 wasaveraged (2.95 U/mg of soluble protein) and assigned an induction value of 1.By comparison, strain EV5 aldolase levels were induced sevenfold.

unable to transduce the nanA4 allele into strain EVil,suggesting that in the absence of aldolase, CMP-NeuNAclevels rose to toxic concentrations. This result providedevidence that aldolase can indirectly regulate the intracellu-lar concentration of CMP-NeuNAc by modulating the poolof free sialic acid.

In the absence of CMP-NeuNAc synthetase, strain EV5accumulated at least sevenfold more free sialic acid than thewild type (Table 3), indicating that sialic acid synthesis wasalso not inhibited by end product control exerted by the freesugar. Furthermore, this result implied that the intracellularconcentration of sialic acid in strain EV5 was suboptimal foraldolase induction. Evidence that aldolase was controllingthe sialic acid pool size was provided by introducing analdolase mutation into strain EV5 to give the double mutantEV90. The further threefold increase in sialic acid pool sizein strain EV90 (Table 3) was additional evidence for modu-lation of the biosynthetic pathway by aldolase. We concludethat the only direct control on sialic acid levels was beingexerted by aldolase.

If aldolase directly participated in controlling sialic acidaccumulation, then increased aldolase levels should be de-tected under conditions that raise the intracellular concen-tration of free sialic acid. To test this possibility, we mea-sured aldolase activity in an extract of EV5, a strain that waspreviously shown to have a sevenfold increase in sialic acidlevels. The aldolase level measured in strain EV5 (Table 4)paralleled the increased pool size of free sialic acid (Table 3).Apparently, the intracellular concentration of sialic acid inthe wild type, EV1 (0.6 mM, Table 3), was not sufficient toinduce aldolase because the specific activity in EV1 (2.1U/mg of protein) was essentially the same as in the kpsnstrain PA360 (3.8 U/mg of protein), which did not accumu-late detectable levels of sialic acid (Tables 3 and 4). Noaldolase activity was detected in strain EV90 (Table 4),demonstrating that all of the activity detected in strain EV5was due to a single aldolase that responded to an increasedpool of sialic acid. The level of aldolase detected in thepresence of a larger sialic acid pool (EV5, Table 4) wasapparently not sufficient to reduce sialic acid to a wild-typelevel, which is further evidence that full aldolase induction,as seen in the accompanying paper (20), may require arelatively high (>4 mM) intracellular concentration of thissugar.

DISCUSSIONThe major results of these studies support our conclusion

that free sialic acid and CMP-NeuNAc do not function asallosteric inhibitors of a committed step in the sialic acidpathway in E. coli. Rather, our results show that a sialic acidaldolase can function to regulate the intracellular concentra-tions of free sialic acid and modulate the level of CMP-NeuNAc. Thus, the data (Table 3) show that mutants with

defects in the terminal and penultimate steps of polysialicacid synthesis accumulated NeuNAc or CMP-NeuNAc toconcentrations 7- to 25-fold higher than those in the wildtype. The pool size of these metabolites in the mutants wasminimized by sialic acid aldolase. As for the sialic acidtoxicity described in the accompanying paper (20), we do notknow the mechanism by which high levels of CMP-NeuNAcapparently become toxic, preventing construction of akps-23 nanA4 double mutant. We would expect from theresults (Table 3) that the 25-fold increase in CMP-NeuNAcin the kps-23 background (polymerization-defective mutantEV11) might increase another threefold upon introduction ofnanA4, resulting in a 75-fold increase in the CMP-NeuNAcpool. Toxicity could therefore be due simply to exhaustionof the CTP pool. We do not know whether CMP-NeuNAcaccumulation occurs physiologically. Presumably, a situa-tion that limits sialyl polymer synthesis, for example growthat 15°C (18, 25), could result in a physiological condition thatmimics the mutational defect in strain EV11.The findings reported here and in the accompanying paper

(20) highlight the multiple functions of sialic acid aldolase.This enzyme (i) participates in dissimilating and detoxifyingsialic acid that accumulates intracellularly after being addedexogenously to the medium and (ii) regulates the intracellu-lar level of sialic acid produced biosynthetically in Klstrains, presumably to recycle sialic acid carbon and toprevent overproduction of CMP-NeuNAc. The multiplephysiological functions carried out by this aldolase may bereflected in the mechanism of its induction and in how thatinduction is controlled.The most straightforward interpretation of our results is

that none of the enzymes required for sialic acid biosynthesisidentified in Fig. 1 are allosterically regulated by CMP-NeuNAc or sialic acid. The apparent lack of control by endproduct inhibition in bacteria is in contrast to mammaliansystems, in which allosteric regulation of UDP-GlcNAc2-epimerase by CMP-NeuNAc is a major factor in control-ling biosynthesis of sialic acid in the liver (5).The lack of regulation by UDP-GlcNAc feedback inhibi-

tion in bacteria (4) led to the suggestion that accumulation ofUDP-GlcNAc, and perhaps other sugar nucleotides, in E.coli was controlled by a specific pyrophosphatase (10). Asimilar mechanism for regulating CMP-NeuNAc levels mayhave been identified in Neisseria meningitidis (8). N. men-ingitidis group B strains produce an a-2,8-ketosidicallylinked polysialic acid capsule that is structurally identical tothe Ki antigen of E. coli (see reference 16 for a review).Masson and Holbein (8) identified a membrane-associatedCMP-NeuNAc hydrolase that was inhibited by CMP, andthey postulated that this enzyme controlled the rate ofpolysialic acid synthesis by regulating the availability ofsugar nucleotide precursor. E. coli lacks this hydrolyticactivity (8, 17), and it is interesting that N. meningitidis lackssialic acid aldolase (8). Thus, N. meningitidis and E. colisynthesize structurally identical capsules, yet may regulatesialic acid and possibly sialyl polymer metabolism by dif-ferent mechanisms.We have recently identified a set of 5 to 11 membrane

polypeptides whose synthesis is temporally correlated withexpression of the polysialic acid capsule (28). Usingminicells and kps genes recombined into a plasmid vector,Silver et al. (14) identified 12 polypeptides correlated withcapsule synthesis or expression. In general, the function ofthe polypeptides identified by either approach was notdeduced. We assume that at least one membrane-associatedprotein other than sialyltransferase is required for polymer

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synthesis or transmembrane translocation, explaining therequirement for de novo protein synthesis observed incapsule expression during temperature shiftup experiments(27). Other experiments indicate that E. coli fails to expresscapsular polysaccharide at 15°C because of a defect in theassembly or synthesis of components required for a func-tional membrane-associated sialyltransferase complex ratherthan in the synthesis of sialyltransferase itself (25). Thiscomplex presumably functions in polymer initiation, elonga-tion, and termination and translocation of the finished prod-uct to the outer membrane (25, 26). The molecular compo-nents of this enzyme complex have not been identified.Appropriate aldolase-negative strains permit specific label-ing of polymeric material with properties identical to authen-tic polysialic acid (Fig. 4). The ability to specifically labelsialyl polymer in vivo may therefore assist in developing anexperimental system to investigate polymer translocationand perhaps to understand the function of membrane-associated polypeptides in this process. Further work di-rected to understanding how the complex series of solubleand membrane-associated reactions which are involved incapsular polysaccharide production are coordinated will berequired for any complete description of microbial mem-brane assembly.

ACKNOWLEDGMENTSWe gratefully acknowledge B. J. Bachmann and R. P. Silver's

contribution of bacterial strains and advice. We thank W. F. Vannand J. B. Robbins for the kind gift of anti-polysialic acid antiserum.We acknowledge many stimulating discussions concerning this workwith R. I. Merker and C. Whitfield.

This work was supported by Public Health Service research grantAI-09352 (to F.A.T.) from the National Institutes of Health.

LITERATURE CITED1. Comb, D. G., D. R. Watson, and S. Roseman. 1966. The sialic

acids. IX. Isolation of cytidine 5'-monophospho-N-acetyl-neuraminic acid from Escherichia coli K-23S. J. Biol. Chem.241:5637-5642.

2. Corfield, A. P., and R. Schauer. 1982. Metabolism of sialicacids, p. 195-261. In R. Schauer (ed.), Sialic acids: chemistry,metabolism, and function. Springer-Verlag, New York.

3. Ingraham, J. L., 0. Maale, and F. C. Neidhardt. 1983. Growthof the bacterial cell, p. 7. Sinauer Associates Inc., Sunderland,Mass.

4. Kornfeld, R. 1967. Studies on L-glutamine D-fructose 6-phosphate amidotransferase. I. Feedback inhibition by uridinediphosphate-N-acetylglucosamine. J. Biol. Chem. 242:3135-3141.

5. Kornfeld, S., R. Kormfeld, E. F. Neufeld, and P. J. O'Brien.1964. The feedback control of sugar nucleotide biosynthesis inliver. Proc. Natl. Acad. Sci. USA 52:371-379.

6. Kundig, F. D., D. Aminoff, and S. Roseman. 1971. The sialicacids. XII. Synthesis of colominic acid by a sialyltransferasefrom Escherichia coli K-235. J. Biol. Chem. 246:2543-2550.

7. Laemmli, U. K. 1970. Cleavage of structural proteins duringassembly of the head of bacteriophage T4. Nature (London)227:680-685.

8. Masson, L., and B. E. Holbein. 1983. Physiology of sialic acidcapsular polysaccharide synthesis in serogroup B Neisseriameningitidis. J. Bacteriol. 154:728-736.

9. McGuire, E. J. 1976. Anabolic reactions involving sialic acids,p. 123-158. In A. Rosenberg and C.-L. Schengrund (ed.),Biological roles of sialic acid. Plenum Publishing Corp., NewYork.

10. Melo, A., and L. Glaser. 1966. Nucleotide diphosphate hexosepyrophosphatase. Biochem. Biophys. Res. Commun. 22:524-531.

11. 0rskov, I., V. Sharma, and F. 0rskov. 1976. Genetic mapping ofthe Kl and K4 antigens (L) of Escherichia coli. Nonallelism ofK(L) antigens with K antigens of 08:K27(A), 08:K8(L) and09:K57(B). Acta Pathol. Microbial. Scand. B 84:125-131.

12. Rohr, T. E., and F. A. Troy. 1980. Structure and biosynthesis ofsurface polymers containing polysialic acid in Escherichia coli.J. Biol. Chem. 255:2332-2342.

13. Sarff, L. D., G. H. McCracken, Jr., M. S. Schiffer, M. P. Glode,J. B. Robbins, I. 0rskov, and F. 0rskov. 1975. Epidemiology ofEscherichia coli Kl in healthy and diseased newborns. Lanceti:1099-1104.

14. Silver, R. P., W. F. Vann, and W. Aaronson. 1984. Genetic andmolecular analyses of Escherichia coli Kl antigen genes. J.Bacteriol. 157:568-575.

15. Stock, J. B., B. Rauch, and S. Roseman. 1977. Periplasmic spacein Salmonella typhimurium and Escherichia coli. J. Biol. Chem.252:7850-7861.

16. Troy, F. A. 1979. The chemistry and biosynthesis of selectedbacterial capsular polymers. Annu. Rev. Microbiol. 33:519-560.

17. Troy, F. A., and M. A. McCloskey. 1979. Role of a membranoussialyltransferase complex in the synthesis of surface polymerscontaining polysialic acid in Escherichia coli. Temperature-induced alteration in the assembly process. J. Biol. Chem. 254:7377-7387.

18. Viay, I. K., and F. A. Troy. 1975. Properties of membrane-associated sialyltransferase of Escherichia coli. J. Biol. Chem.250:164-170.

19. Vimr, E. R., R. D. McCoy, H. F. Voliger, N. C. Wilkison, andF. A. Troy. 1984. Use of prokaryotic-derived probes to identifypoly(sialic) acid in neonatal neuronal membranes. Proc. Natl.Acad. Sci. USA 81:1971-1975.

20. Vimr, E. R., and F. A. Troy. 1985. Identification of an induciblecatabolic system for sialic acids (nan) in Escherichia coli. J.Bacteriol. 164:845-853.

21. Warren, L. 1959. The thiobarbituric assay of sialic acids. J.Biol. Chem. 234:1971-1975.

22. Warren, L. 1972. The biosynthesis and metabolism of aminosugars and amino sugar-containing heterosaccharides, p.1097-1126. In A. Gottschalk (ed.), Glycoproteins: their compo-sition, structure, and function, part B. Elsevier Publishing Co.,Amsterdam.

23. Warren, L., and R. S. Blacklow. 1962. The biosynthesis ofcytidine 5'-monophospho-N-acetylneuraminic acid by an en-zyme from Neisseria meningitidis. J. Biol. Chem. 237:3527-3534.

24. White, R. J. 1968. Control of amino sugar metabolism inEscherichia coli and isolation of mutants unable to degradeamino sugars. Biochem. J. 106:847-858.

25. Whitfield, C., D. A. Adams, and F. A. Troy. 1984. Biosynthesisand assembly of the polysialic acid capsule in Escherichia coliKl. Role of a low-density vesicle fraction in activation of theendogenous synthesis of sialyl polymer. J. Biol. Chem. 259:12769-12775.

26. Whitfield, C., and F. A. Troy. 1984. Biosynthesis and assemblyof the polysialic acid capsule in Escherichia coli Kl. Activationof sialyl polymer synthesis in inactive sialyltransferase com-plexes requires protein synthesis. J. Biol. Chem. 259:12776-12780.

27. Whitfield, C., E. R. Vimr, J. W. Costerton, and F. A. Troy. 1984.Protein synthesis is required for in vivo activation of polysialicacid capsule synthesis in Escherichia coli Kl. J. Bacteriol.159:321-328.

28. Whitfield, C., E. R. Vimr, J. W. Costerton, and F. A. Troy. 1985.Membrane proteins correlated with expression of the polysialicacid capsule in Escherichia coli Kl. J. Bacteriol. 161:743-749.

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