Erythritol feeds the pentose phosphate pathway viathree new isomerases leading to D-erythrose-4-phosphate in BrucellaThibault Barbiera,1, François Collardb,1, Amaia Zúñiga-Ripac, Ignacio Moriyónc, Thibault Godardd, Judith Beckere,Christoph Wittmanne, Emile Van Schaftingenb,2,3, and Jean-Jacques Letessona,2,3

aResearch Unit in Microorganisms Biology, University of Namur, B-5000 Namur, Belgium; bWelbio and de Duve Institute, Université Catholique de Louvain,B-1200 Brussels, Belgium; cDepartamento de Microbiología e Instituto de Salud Tropical, Universidad de Navarra, 31008 Pamplona, Spain; dInstitute ofBiochemical Engineering, Technische Universität Braunschweig, 38106 Braunschweig, Germany; and eInstitute of Systems Biotechnology, Saarland University,66123 Saarbrücken, Germany

Edited by Hans Leo Kornberg, Boston University, Boston, MA, and approved November 3, 2014 (received for review July 31, 2014)

Erythritol is an important nutrient for several α-2 Proteobacteria,including N2-fixing plant endosymbionts and Brucella, a worldwidepathogen that finds this four-carbon polyol in genital tissues.Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter toL-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is acceptedthat further steps involve oxidation by the putative dehydrogenaseEryC and subsequent decarboxylation to yield triose-phosphates. Ac-cordingly, growth on erythritol as the sole C source should requirealdolase and fructose-1,6-bisphosphatase to produce essentialhexose-6-monophosphate. However, we observed that a mutantdevoid of fructose-1,6-bisphosphatases grew normally on eryth-ritol and that EryC, which was assumed to be a dehydrogenase,actually belongs to the xylose isomerase superfamily. Moreover, wefound that TpiA2 and RpiB, distant homologs of triose phosphateisomerase and ribose 5-phosphate isomerase B, were necessary, aspreviously shown for Rhizobium. By using purified recombinantenzymes, we demonstrated that L-3-tetrulose-4-phosphate wasconverted to D-erythrose 4-phosphate through three previouslyunknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase;renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; re-named EryI), a pathway fully consistent with the isotopomer distribu-tion of the erythrose-4-phosphate-derived amino acids phenylalanineand tyrosine obtained from bacteria grown on 13C-labeled erythritol.D-Erythrose-4-phosphate is then converted by enzymes of the pentosephosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is thefirst description to our knowledge of a route feeding carbohydratemetabolism exclusively via D-erythrose 4-phosphate, a pathway thatmay provide clues to the preferential metabolism of erythritol byBrucella and its role in pathogenicity.

Brucella | erythritol | alphaproteobacteria | pentose phosphate cycle |tetrose metabolism

Brucellosis is a widespread bacterial zoonosis caused by thefacultative intracellular pathogens Brucella spp. Domestic

ruminants and swine, which altogether represent more than 4billion animals worldwide, are highly susceptible, and in thislivestock, the brucellae characteristically target the genital organsand cause abortion and infertility (1). Close to parturition, thesebacteria reach exceedingly high numbers (up to 1014 bacteria perconceptus) (2), thus making brucellosis abortion a highly conta-gious condition.It is accepted that this tropism and extensive multiplication relate

to the high erythritol concentration in the genital organs of thoseanimals (3, 4). In vitro, this four-carbon polyol is a preferential car-bon source of Brucella, promotes growth, and up-regulates the ex-pression of major virulence factors and key enzymes of centralmetabolism (5–9). In vivo, it has been observed that parenteral

injections of erythritol increase the numbers of Brucella in the spleensof intraperitoneally infected calves (3, 7). For these reasons, erythritolhas been considered a major factor in Brucella pathogenesis.Erythritol metabolism (Fig. 1A, accepted pathway) starts with

its phosphorylation by EryA to yield L-erythritol-4-P (10, 11),which is then oxidized by the dehydrogenase EryB to L-3-tetrulose-4-P (10). It is accepted that the next three steps imply the oxida-tion of the first carbon of L-3-tetrulose-4-P to an aldehyde by anNAD-dependent dehydrogenase, purportedly EryC (12), followedby dehydrogenation and decarboxylation reactions generating di-hydroxyacetone-P and carbon dioxide as end products (10).The use of this pathway to grow on erythritol is not restricted

to brucellae or to their intracellular pathogenic lifestyle but isshared by other members of the Rhizobiales, such as Ochrobactrumanthropi (an environmental bacteria), Rhizobium leguminosarum,and Sinorhizobium meliloti (both plant symbionts) (13, 14). In allthese bacteria, there is, downstream of the ery operon, a secondhighly conserved operon that contains three ORFs includingtpiA2 and rpiB, which are homologous to triose phosphate isom-erase and ribose 5-phosphate isomerase B, respectively (13). In-terestingly, rpiB and tpiA2 mutants of R. leguminosarum (15) areunable to grow on erythritol as a carbon source.

Significance

Erythritol is a preferential substrate for Brucella, a common zoo-notic bacterial pathogen. This four-carbon polyol is found in thereproductive organs of several affected species, a feature thatmay account for the characteristic viscerotropism of Brucella thatleads to sterility and abortion. Although described previously asfeeding glycolysis via dihydroxyacetone-phosphate, we showhere that erythritol is actually converted into D-erythrose-4-phosphate through a hitherto undescribed set of reactions thatinvolves three isomerases and that allows hexose-mono-phosphate synthesis and growth by feeding the pentose phos-phate shunt. Elucidation of this unique carbohydrate pathway,which also applies to the Rhizobiales plant endosymbionts, opensthe way for further research on the metabolic adaptation of animportant facultative intracellular pathogen to target organs.

Author contributions: T.B., F.C., E.V.S., and J.-J.L. designed research; T.B., F.C., A.Z.-R., T.G., J.B.,and C.W. performed research; T.B., F.C., A.Z.-R., I.M., T.G., J.B., C.W., E.V.S., and J.-J.L. analyzeddata; and T.B., F.C., A.Z.-R., I.M., T.G., J.B., C.W., E.V.S., and J.-J.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1T.B. and F.C. contributed equally to this work.2E.V.S. and J.-J.L. contributed equally to this work.3To whom correspondence may be addressed. Email: emile.vanschaftingen@uclouvain.beor jean-jacques.letesson@unamur.be.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1414622111/-/DCSupplemental.

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Because the involvement of two putative isomerases is notconsidered in the accepted pathway, where a dehydrogenase anda decarboxylase are predicted to catalyze the last two steps, wereassessed the erythritol pathway. A combination of genetic,biochemical, and isotopic evidence brought us to the conclusionthat erythritol metabolism involves three as-yet undescribed isom-erization reactions catalyzed by EryC, TpiA2, and RpiB, whichtogether lead to the formation of D-erythrose-4-P, rather thandihydroxyacetone-P. Erythrose-4-P can then be converted intoglyceraldehyde-3-P and fructose-6-P through reactions catalyzedby enzymes of the pentose phosphate pathway (Fig. 1B).

ResultsFructose-1,6-Bisphosphatases Are Dispensable for Growth on Erythritol.Because hexose-monophosphates are essential biomass precursors,the accepted pathway predicts that growth on erythritol as the

sole carbon source requires isomerization of dihydroxyacetone-Pto glyceraldehyde-3-P, triose-P condensation by aldolase, anddephosphorylation of fructose-1,6-P2 by fructose-1,6-bisphos-phatase (FBPase). Brucella spp. carry only two FBPase genes [fbpand glpX (16)], and surprisingly, a double FBPase mutant (Bru-cella abortus 2308ΔfbpΔglpx) showed no growth defect whenprovided with erythritol as the only carbon source (Fig. S3A),indicating that this polyol is converted not into trioses-P but intoa product that can generate fructose-6-P without involvement offructose-1,6-P2. Because D-erythrose-4-P can be converted to fruc-tose-6-P and glyceraldehyde-3-P via the pentose phosphate pathway(Fig. 1B), we hypothesized that L-3-tetrulose-4-P is converted toD-erythrose-4-P by a series of isomerization reactions (Fig. 1A).

Three Putative Isomerases/Epimerases Are Required Instead ofDehydrogenases. Our hypothesis was supported by sequencecomparisons showing that EryC was not a dehydrogenase

Fig. 1. Erythritol catabolism by Brucella. (A) The two first reactions of the “accepted” and the “revised” pathways are similar (highlighted in gray and furthercharacterized in Figs. S1 and S2 and Table S1). Although it was previously admitted that L-3-tetrulose-4-P is afterward oxidized to dihydroxyacetone-phos-phate and CO2, we now show that this intermediate is converted to D-erythrose-4-P via three isomerization reactions catalyzed by EryC, TpiA2, and RpiB. Therelevant enzyme-encoding genes are organized as illustrated in two operons also comprising two regulators (EryD and DeoR). (B) D-erythrose-4-P is furtherprocessed by enzymes of the pentose phosphate pathway to generate fructose-6-P and glyceraldehyde-3-P, two intermediates of glycolysis/gluconeogenesis(C). The proteins whose genes are induced by erythritol (9) are circled in red. The new proposed gene names are indicated in parentheses below the olddesignations. Fba, fructose bisphosphate aldolase; Fbp, fructose bisphosphatase; G6P, glucose-6-P; Gapdh, glyceraldehyde-3-P dehydrogenase; Pgk, phos-phoglycerate kinase; Pgm, phosphoglycerate mutase; R5P, ribose-5-P; Rpe, ribulose-5-P epimerase; Rpi, ribose-5-P isomerase; Ru5P, ribulose-5-P; S7P, sedo-heptulose-7-P; Tal, transaldolase; Tkt, transketolase; Tpi, triose-P isomerase; X5P, xylulose-5-P.

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homolog but, rather, a member of the metal-dependent xyloseisomerase-like superfamily (17). Two other hypothetical iso-merases, TpiA2 and RpiB, which are homologous to triose-Pisomerase and ribose-P isomerase, respectively, are requiredfor growth of R. leguminosarum on erythritol (15). Similarly,B. abortus mutants (ΔtpiA2 and ΔrpiB) in the orthologous genes,which are downstream of the ery operon, did not grow onerythritol (Fig. S3 B and C). These results, together with previousevidence on the indispensability of EryC (12), indicate that threeputative isomerases are in fact involved in erythritol metabolism.

EryC, TpiA2, and RpiB Convert L-3-Tetrulose-4-P to D-Erythrose-4-P. Toconfirm the role of EryC, TpiA2, and RpiB, L-3-tetrulose-4-Pwas produced from L-erythritol-4-P with EryB, purified, andtested for conversion to D-erythrose-4-P by the purified proteins.Using an assay in which D-erythrose-4-P formation was deter-mined spectrophotometrically, with purified Escherichia colierythrose-4-P dehydrogenase (18) as the coupling enzyme, wefound that D-erythrose-4-P was formed from the product of EryBwhen EryC, TpiA2, and RpiB were present together in the assaymixture (Fig. 2A). No reaction was observed if any of these threeproteins or the coupling enzyme was omitted or if EDTA waspresent, in all likelihood because of the metal-dependency ofEryC (Fig. S4). These findings confirmed that all three isomer-ases are required and D-erythrose-4-P is the end-product oferythritol catabolism.

Identification of the Sequence for the Three Isomerization Reactions.We took advantage of the metal-dependency of EryC to checkwhether this protein acted directly on L-3-tetrulose-4-P. Thiscompound was preincubated with EryC, the reaction was stop-ped with EDTA, and the formation of D-erythrose-4-P wasmeasured in the presence of TpiA2 and RpiB (Fig. 2B, secondcolumn). We found that under these conditions, about 50% ofthe L-3-tetrulose-4-P had been converted by EryC during thepreincubation step to a substrate for TpiA2 and RpiB, suggestingthat EryC catalyzed the racemization of L-3-tetrulose-4-P to itsD-isomer. Experiments in which L-3-tetrulose-4-P was preincubatedwith EryC and either TpiA2 or RpiB indicated that TpiA2, butnot RpiB, increased the amount of tetrose-4-phosphate that be-came available for conversion to erythrose-4-P and erythronate-4-P in the subsequent assay (Fig. 2B, columns 3 and 4; see SIResults for a more detailed interpretation of this experiment).Accordingly, the last specific step of the pathway should be theisomerization catalyzed by RpiB. This was confirmed by showingthat incubation of D-erythrose 4-phosphate with RpiB (althoughnot with EryC or TpiA2) caused about a 90% decrease in itsconcentration (Fig. 2C), a result consistent with the fact that theequilibrium of the D-erythrulose-4-P/erythrose-4-P isomerization ismuch in favor of D-erythrulose-4-P (19).

Identification of the Intermediates with Tritiated Borohydride andPeriodate Oxidation. To analyze further the reactions catalyzedby the three isomerases, we examined the nature of the succes-sively formed tetrose-4-Ps by taking advantage of the fact thatreduction of erythrose-4-P, erythrulose-4-P, and 3-tetrulose-4-Pwith tritiated borohydride leads to specific incorporation of tri-tium on their C1, C2, and C3, respectively. Oxidation withperiodate breaks down polyols according to well-known rules(20) predicting that the first carbon of tetritol-4-P will be con-verted to formaldehyde, the second one to formate, and the thirdand fourth ones to phosphoglycolaldehyde (see the expectedpatterns in Fig. S5). These breakdown products, which keep theoriginal hydrogen–carbon linkages, can be easily separated byanion-exchange chromatography. The nature of the radiolabeledbreakdown products thus informs directly on the position of theoxo group in the intermediates.

The experiments performed on the products resulting fromincubations of either D-erythrose-4-P or L-3-tetrulose-4-P withthe three isomerases alone or in combinations (Fig. 3) led to thefollowing conclusions: First, EryC acts on L-3-tetrulose-4-Pwithout moving the position of the oxo function (Fig. 3 B, C, E,and F), confirming therefore that it is a 3-tetrulose-4-P racemase(Fig. 1A). Second, TpiA2 moves reversibly the oxo group be-tween the second and the third carbons (Fig. 3 B and C), thusinterconverting a 3-tetrulose-4-P and an erythrulose-4-P. It isspecific for D-3-tetrulose-4-P because it does not act directly onthe product of EryB, but well after its racemization with EryC.As illustrated in Fig. 1A, it acts in the reverse direction on theisomer of erythrulose-4-P made from erythrose-4-P by RpiB (i.e.,the D-isomer; Fig. 3 E and F). Not unexpectedly, the stereo-chemistry of the TpiA2 reaction is similar to that of the ho-mologous enzyme triose-phosphate isomerase (TPI in Fig. S6).As shown in Fig. S7, the replacement of the highly conservedAsn10 in triose phosphate isomerase by a serine in TpiA2 pre-sumably allows the accommodation of a larger substrate; that is,a tetrose-P instead of a triose-P. Finally, RpiB reversibly movesthe oxo group between C1 and C2 (Fig. 3 D and E), thus cata-lyzing the isomerization of D-erythrose-4-P with D-erythrulose-4-P(Fig. 1A).

Isotopic Labeling of Phenylalanine and Tyrosine Supports the RevisedPathway. Because phenylalanine and tyrosine are formed fromone molecule of D-erythrose-4-P and two molecules of phos-phoenolpyruvate (Fig. 4A), analysis of the isotopomers of theseamino acids in cells grown with a mixture of 80% [12C] and 20%[U-13C] (uniformly labeled) erythritol should allow us to dis-criminate the “accepted” and the “revised” pathways. In theformer, the M+4 isotopomer of D-erythrose-4-P directly resultsfrom [U-13C] erythritol metabolism and amounts thereforeto ∼ 20% of total D-erythrose-4-P. In the latter, D-erythrose-4-Pwould result from recombination of [U-13C] triose-phosphatewith a 13C-labeled carbon from another intermediate of thepentose phosphate pathway, thus amounting to only about 4% oftotal D-erythrose-4-P (Table S2). The observed distribution ofthe M+1 to M+8 isotopomers in carbons C2–C9 of phenylalanineand tyrosine were in much better agreement with the predictionof the revised than of the accepted pathway (Fig. 4 B and C),particularly if we took into account the possibility that part ofD-[U-13C]erythrose-4-P may be converted to [2,3,4-13C] and [1-13

C] erythrose-4-P through exchange reactions catalyzed bytransaldolase and transketolase.

DiscussionThis work leads to a substantial revision of the erythritol path-way, most particularly of the steps beyond L-3-tetrulose-4-P.These steps involve three enzymes that have not been describedfor any other species. Because the role of TpiA2 and RpiB in themetabolism of erythritol is now established, we propose torename their genes eryH and eryI [the designations eryE, eryF,and eryG have already been proposed for the erythritol transportsystem of B. abortus (21)]. The gene encoding the putative reg-ulatory protein DeoR, which precedes tpiA2 on the chromosome,has been designated eryR by Geddes and colleagues (13). Ex-pression of these genes, including eryA,B,C,D, is induced in thepresence of erythritol (9, 12). This regulation is partly based onthe release by erythritol of the EryD-dependent repression oferyA,B,C,D expression (12), whereas the role of EryR remainsunknown. By analogy with other members of the DeoR family,EryR binds in all likelihood a phosphate ester, presumably anintermediate of the revised erythritol pathway (15). It would thusparticipate, together with EryD, in the regulation of the ex-pression of enzymes involved in erythritol catabolism.Of note, adjacent homologs of tpiA2 (with the typical Asn

to Ser substitution, Fig. S7) and rpiB are also found always

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downstream of a putative dihydroxyacetone kinase in bacterialgenomes devoid of eryA and eryB (Fig. S8). We propose that theyare involved in the metabolism of L-erythrulose (i.e., D-3-tetru-lose), which would be phosphorylated by the “dihydroxyacetonekinase” to D-3-tetrulose-4-P and isomerized to D-erythrose-4-Pby the TpiA2 and RpiB homologs.The revised erythritol pathway is remarkable not only because

it involves three enzymes that have never been previously iden-tified but also because it provides the first example, to ourknowledge, of a substrate that feeds carbohydrate metabolismexclusively via D-erythrose-4-P. We propose that the latter isconverted to intermediates of glycolysis and gluconeogenesis byreactions involving the pentose phosphate pathway enzymes(Fig. 1B). The balance of this set of reactions is the net formationof two molecules of glyceraldehyde-3-P and one molecule offructose-6-P from three molecules of D-erythrose-4-P.A flow of metabolites coming from erythritol and reaching first

the erythrose-4-P pool may be advantageous to Brucella. Indeed,this tetrose-P feeds, via chorismate, the synthesis of aromatic aminoacids and of the siderophore 2,3-dihydroxybenzoic acid (22), theproduction of which is, in fact, increased by erythritol under iron-limiting conditions (23). These characteristics of the revised path-way may explain why the brucellae reach exceedingly high numbersin genital tissues. The highly abortifacient effect of some currentlyused brucellosis vaccines, most notably Rev1 (Revertant 1), may berelated with their ability to multiply in genital organs and to con-sume erythritol (24). Accordingly, B. abortus S19, a vaccine that isnot able to catabolize erythritol, is associated with a low risk forabortion (25). The risk for abortion is a serious drawback in massvaccination, which is the only control strategy applicable in largeareas of the world (26). A full understanding of erythritol metab-olism may provide clues for developing safer vaccines. Further-more, as none of the five enzymes involved in erythritol metabolismis shared with the host, it opens perspectives for the development ofnew therapeutic agents aimed at inhibiting this pathway.

Materials and MethodsConstruction of Deletion Strains and Growth of B. abortus. Clean deletionmutants of B. abortus 2308 for fbp, glpx, tpiA2, and rpiB were made byallelic replacement and complemented as described in the SI Materials andMethods. Brucella was grown at 37 °C on rich medium 2YT or on a chemi-cally defined medium (modified Plommet medium), as detailed in SI Mate-rials and Methods. The growth was monitored by following the OD (600 nm)during 48–72 h in an automated plate reader (Bioscreen C, Lab Systems) withcontinuous shaking.

Protein Purification. Bifidus crudilactis phosphoketolase gene and BrucellaeryA, eryB, eryC, tpiA2, and rpiB were inserted into pET15b vectors, whichallow expression of N-terminal His-tagged proteins in E. coli Bl21pLysS.Proteins were purified by chromatography on His-trap columns to at least80% homogeneity. A membrane pellet derived from E. coli expressing EryBwas used as source of this enzyme, as it could not be purified. Details areprovided in SI Materials and Methods.

Synthesis of L-Erythritol-4-P, L-3-Tetrulose-4-P, and D-Erythrose-4-P. L-Erythritol-4-phosphate was prepared enzymatically (11), using EryA, and purified. L-3-tetrulose-4-P was prepared by oxidation of L-erythritol-4-P, using EryB andp-benzoquinone as an electron acceptor. D-Erythrose-4-P was produced en-zymatically with fructose-6-P phosphoketolase from B. crudilactis. See SIMaterials and Methods for details.

Enzymatic Assays. The erythritol kinase activity of EryA was assayed at 30 °Cspectrophotometrically by following ADP formation, coupled with the oxi-dation of NADH. The A340nm was measured either on a spectrophotometeror on a plate reader. The L-erythritol-4-P dehydrogenase activity of EryB was

Fig. 2. Conversion of L-3-tetrulose-4-P to D-erythrose-4-P: requirement ofEryC, TpiA2, and RpiB (A), and determination of the sequence of theirreactions (B and C). (A) L-3-tetrulose-4-P was incubated with EryC, TpiA2, andRpiB, alone or in combinations, and the formation of D-erythrose-4-P wasmonitored spectrophotometrically in the presence of NAD and erythrose-4-Pdehydrogenase (E4PDH). (B) L-3-tetrulose-4-P was preincubated with EryC,alone or in combination with TpiA2 or RpiB or both. The preincubation wasstopped with EDTA, and then TpiA2 or RpiB were added to the incubationmixtures from which they had been omitted, and the formation ofD-erythrose-4-P was monitored with E4PDH. The five values shown are signifi-cantly different from each other (P < 0.05 by Student t test), with the exceptionof the comparison of “EryC” with “EryC+RpiB,” where there is no difference.(C) D-erythrose-4-P was incubated with EryC, TpiA2, or RpiB. These enzymeswere removed from the incubation medium before spectrophotometric mea-surement of the remaining D-erythrose-4-P in the presence of E4PDH. After-ward, the enzyme used during the preincubation was added back, as indicated,to ensure that the metabolite had not been depleted because of an unrelated

reaction (e.g., dephosphorylation by a hypothetical contaminating phos-phatase). In all panels, error bars represent the SD for three distinctexperiments. These error bars are too small to be visible in some cases.

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assayed at 30 °C by following A600nm in a mixture containing 2,6-dichloro-phenolindophenol (DCPIP) as an artificial electron acceptor.

The activity of EryC, TpiA2, and RpiB weremeasured indirectly bymeasuringthe formation of D-erythrose-4-P from L-3-tetrulose-4-P or the depletion of D-

erythrose-4-P on incubation with the indicated enzymes. Briefly, L-3-tetrulose-4-P (0.1 mM) was incubated (30 min at 30 °C) in a mixture (100 μL) containing25 mM Tris at pH 8.0 and 1 μg EryC, TpiA2, or RpiB (alone or in combination).EDTA was added to a 2-mM final concentration, and TpiA2 or RpiB were

Fig. 3. Analysis by [3H]borohydride labeling/periodate oxidation of the products made from L-3-tetrulose-4-P (A–C) and D-erythrose-4-P (D–F) by EryC, TpiA2,and RpiB. Erythrose-4-P and L-3-tetrulose-4-P were incubated alone, with EryC, TpiA2, and Rpib or in combination, as indicated. [3H]borohydride was added tothe reaction mixture and the incubation pursued for 2 h. The samples were acidified with perchloric acid and neutralized. The tetritol-phosphates werepurified by anion exchange chromatography and submitted to periodate oxidation. The products were then analyzed by anion-exchange chromatography toseparate [3H]-formaldehyde (A and D), [3H]-formate (B and E), and [3H]-phosphoglycolaldehyde (C and F). Tritiated formaldehyde, formate, and phospho-glycolaldehyde are produced in this manner from D-erythrose-4-P, D-erythrulose-4-P, and D- or L-3-tetrulose-4-P, respectively, because of the fixation of tritiumon the carbons boxed in the right panels. Mean of two experiments, with the two extremities of the error bars corresponding to the individual values.

Fig. 4. (A) Origin of the different carbons in phenylalanine and tyrosine and their expected (B) and observed (C) mass isotopomer distribution formed inwild-type (WT) and FBPase-deficient (mut) cells grown on 20% [U-13C] erythritol. The analyzed [M-159] fragment ions (SI Materials and Methods) comprisecarbons C2–C9 of phenylalanine (Phe,m/z 234) or tyrosine (Tyr,m/z 364). These carbons are derived from one molecule of erythrose-4-P and from C2–C3 of twomolecules of phosphoenolpyruvate. The expected distributions of isotopomers were calculated as described in SI Materials and Methods for the “accepted”pathway and the “revised” pathway, either assuming (revised*) or not (revised) 25% exchange reactions between labeled and unlabeled D-erythrose-4-P.

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added to the incubation mixtures in which they were omitted and D-erythrose-4-P was quantified enzymatically. D-erythrose-4-P (0.1 mM) was incubated(30 min at 30 °C) in a mixture (800 μL) containing 25mM Tris at pH 8.0 and 5 μgEryC, TpiA2, or RpiB (alone or in combination). Enzymes were removed bymixing with 50 μL Ni-NTA resin for 30 min at room temperature, followed bycentrifugation, and D-erythrose-4-P was quantified enzymatically (SI Materialsand Methods).

Characterization of Tetrose-Phosphates by [3H]borohydride Reduction andPeriodate Oxidation. L-3-tetrulose-4-P or D-erythrose-4-P (0.25 mM) was in-cubated at 30 °C in a mixture (100 μL) containing 25 mM Hepes at pH 7.4,1 mM MgCl2, and 2 μg of the indicated enzyme or enzymes (see Results);after 30 min, 1 mCi tritiated sodium borohydride (final concentration,2.5 mM) was added and reduction was allowed to proceed on ice for 2 h.Perchloric acid was then added to a final concentration of 10% (wt/vol) todestroy residual borohydride and denatured enzymes. After 2 h at roomtemperature, samples were neutralized and the tetritol-phosphates werepurified and oxidized with periodate, and their breakdown product wasanalyzed, as described in SI Materials and Methods.

Isotopic Labeling Experiments and GC/MS Analysis. B. abortus 2308 was grownin chemically defined medium with erythritol as sole carbon source com-posed of 80% [U-12C] and 20% [U-13C]. During exponential growth, cellswere harvested, washed, and hydrolyzed with 6-M HCl at 100 °C for 24 h.Samples were then lyophilized, amino acid derivatization was carried out,and the tert-butyl-dimethylsilyl (tBDMS) derivatives were then analyzed byGC-MS (further details in SI Materials and Methods).

ACKNOWLEDGMENTS. The E.V.S. laboratory is supported by a Welbio grantof the Walloon Region and by a grant from the Fonds de la RechercheScientifique Médicale. J.-J.L.’s team is supported by an FNRS (Fonds Nationalde la Recherche Scientifique) grant (Fonds de la Recherche FondamentaleCollective Grant N° 2452110) and by the Interuniversity Attraction Poles Pro-gramme initiated by the Belgian Science Policy Office. T.B. has a PhD grant as“Aspirant FNRS.” I.M.’s laboratory is supported by grants from the Ministeriode Economía y Competitividad of Spain (AGL2011-30453-C04-00) and theFundación para la Investigación Médica Aplicada. A.Z.-R. has a postdoctoralgrant from FIMA. The research team of C.W. acknowledges financial supportfrom the German Research Foundation through Schwerpunktprogramm1316 “Host-Pathogen Interaction.”

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