Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of...

Crh, the paralogue of the phosphocarrier protein HPr,controls the methylglyoxal bypass of glycolysis inBacillus subtilismmi_7857 770..787

Jens J. Landmann, Ricarda A. Busse,Jan-Hendrik Latz, Kalpana D. Singh, Jörg Stülkeand Boris Görke*Department of General Microbiology, Institute ofMicrobiology and Genetics, Georg-August-University,Grisebachstrasse 8, 37077 Göttingen, Germany.

Summary

The histidine protein HPr has a key role in regulationof carbohydrate utilization in low-GC Gram-positivebacteria. Bacilli possess the paralogue Crh. Like HPr,Crh becomes phosphorylated by kinase HPrK/P inresponse to high fructose-1,6-bisphosphate concen-trations. However, Crh can only partially substitutefor the regulatory functions of HPr leaving its rolemysterious. Using protein co-purification, we identi-fied enzyme methylglyoxal synthase MgsA as interac-tion partner of Crh in Bacillus subtilis. MgsA convertsdihydroxyacetone-phosphate to methylglyoxal andthereby initiates a glycolytic bypass that prevents thedeleterious accumulation of phospho-sugars undercarbon overflow conditions. However, methylgyloxalis toxic and its production requires control. We showhere that exclusively the non-phosphorylated form ofCrh interacts with MgsA in vivo and inhibits MgsAactivity in vitro. Accordingly, Crh inhibits methylgly-oxal formation in vivo under nutritional famine condi-tions that favour a low HPr kinase activity. Thus, Crhsenses the metabolic state of the cell, as reflected byits phosphorylation state, and accordingly controlsflux through the harmful methylglyoxal pathway.Interestingly, HPr is unable to bind and regulateMgsA, making this a bona fide function of Crh. Fourresidues that differ in the interaction surfaces of HPrand Crh may account for this difference.

Introduction

Glycolysis is the universal pathway for degradation ofcarbohydrates in living organisms. It is required for thegeneration of energy and of building blocks for numerousbiosynthetic pathways. Glycolysis comprises a series ofreactions that convert sugars to triose-phosphates, whichare subsequently further metabolized to pyruvate in thebottom branch of the Embden–Meyerhof–Parnas pathway(EMP). Many organisms including bacteria, archaea aswell as eukaryotes possess an alternative route, the meth-ylglyoxal pathway, which bypasses the conventional routeof glycolysis (Cooper, 1984; Kalapos, 2008). This pathwayis initiated by enzyme methylglyoxal synthase MgsA,which converts dihydroxyacetone-phosphate (DHAP) tomethylglyoxal. Dependent on the organism, methylglyoxalis excreted (Baskaran et al., 1989) and/or converted tolactate and pyruvate by different pathways. Among these,the glutathione-dependent conversion of methylglyoxal toD-lactate by glyoxalases I and II might be the most wide-spread pathway (Mannervik, 2008).

There is no gain of energy associated with the meth-ylglyoxal pathway. Therefore, its role was often neglected,although it was once considered to be an intrinsic part ofglycolysis (Kalapos, 2008). Even more puzzling, methylg-lyoxal is highly cytotoxic, counteracting the idea of anutrient. It reacts with proteins, lipids and DNA, therebyimpairing protein functions and increasing DNA mutationrates (Thornalley, 2008). In bacteria such as Bacillus sub-tilis and Escherichia coli, methylglyoxal inhibits growthwhen present at mM concentrations (Tötemeyer et al.,1998; Nguyen et al., 2009). Studies in bacteria, mainly inE. coli, shed some light on the mysterious role of theenzymatic production of methylglyoxal. It was shown thatmethylglyoxal synthase activity is important for growth,when there is a sudden increase of carbohydrate uptakecausing an imbalance between flux through the upperbranch of glycolysis and the capacity of the lower branchof the EMP (Russell, 1993; Ferguson et al., 1998; Töte-meyer et al., 1998; Booth et al., 2003; Kim et al., 2004;Weber et al., 2005). Consequently, the methylglyoxalpathway is thought to function as an overflow mechanismthat prevents the accumulation of phosphorylated

Accepted 19 September, 2011. *For correspondence. [email protected]; Tel. (+49) 551 393796; Fax (+49) 551 393808.

Molecular Microbiology (2011) 82(3), 770–787 � doi:10.1111/j.1365-2958.2011.07857.xFirst published online 12 October 2011

© 2011 Blackwell Publishing Ltd

glycolytic intermediates, which are toxic for the bacteria(Böck and Neidhardt, 1966; Irani and Maitra, 1977;Kadner et al., 1992). Thus, bacteria produce a poison toget rid of phospho-sugar stress, but whether this is aregulated or a passive process has not been known.

Uptake and metabolism of carbohydrates are usuallytightly controlled. In many bacteria, the carbohydrate:phosphotransferase system (PTS) has a key role in thisregulation (Deutscher, 2008; Görke and Stülke, 2008).Primarily, the PTS represents a multi-enzyme sugar trans-port system, which phosphorylates its substrates duringuptake (Postma et al., 1993). The phosphoryl-groupsderive from phosphoenol-pyruvate and are transferred viahistidine protein (HPr) to the transporters. During this trans-fer, HPr becomes phosphorylated at His15. In B. subtilisand other Firmicutes, HPr has also numerous regulatoryfunctions (Deutscher et al., 2006; Fujita, 2009). In particu-lar, HPr is essential for carbon catabolite repression (CCR),i.e. it is required for the repression of genes for the utiliza-tion of secondary carbon sources, when a preferred sugarsuch as glucose is present (Deutscher, 2008; Görke andStülke, 2008). To be active in CCR, HPr must be phospho-rylated at a different site, the Ser46. HPr(Ser~P) serves ascofactor for the global regulatory protein CcpA, whichcontrols transcription of numerous catabolic genes. Thecomplex of both proteins binds to operator sites on theDNA and elicits CCR. The strength of repression is deter-mined by the amount of HPr(Ser~P) in the cell, which is inturn controlled by the quality of the carbon source. Pre-ferred carbon sources such as glucose or fructose triggerthe preferential phosphorylation of HPr at Ser46 and causea strong CCR. In contrast, HPr is predominantly non-phosphorylated and CCR is weak, when cells grow on lessfavourable substrates (Singh et al., 2008). Phosphoryla-tion as well as de-phosphorylation of Ser46 of HPr iscatalysed by the bi-functional enzyme HPr kinase/phosphorylase (HPrK/P) (Mijakovic et al., 2002). Theopposing activities of HPrK/P are regulated by the meta-bolic state of the cell. Phosphorylation of HPr requires ATPand fructose-1,6-bisphosphate (FBP), which allostericallyactivates the kinase function of HPrK/P. FBP levels arehighest, when cells utilize preferred carbon sources. Incontrast, phosphorylase activity of HPrK/P prevails, whenFBP levels are low and Pi concentrations are high (Jaultet al., 2000; Lavergne et al., 2002; Ramström et al., 2003;Singh et al., 2008).

Bacillus subtilis and other bacilli contain protein Crh,which exhibits 41–47% sequence identity with HPr (Deut-scher et al., 2006). Crh lacks the His15, but the Ser46 ispreserved and Crh is (de)phosphorylated at this site byHPrK/P like its homologue HPr (Galinier et al., 1997;Lavergne et al., 2002). Crh participates in CCR (hencethe name: catabolite repression HPr), but to a minorextent. Absence of Crh has no impact, because HPr is

sufficient for CCR. On the contrary, Crh can only partiallysubstitute for HPr(Ser~P) in CCR of many promoters(Galinier et al., 1997; Singh et al., 2008). However, in onecase a specific function of Crh in CCR has beensuggested. Expression of the citM gene encoding anMg2+-citrate transporter is repressed by Crh, but not byHPr, when bacteria grow in a medium containing succi-nate and citrate as carbon sources (Warner and Lolkema,2003). The weak contribution of Crh~P to global CCR, ascompared with HPr(Ser~P), can be attributed to its 10-foldlower binding affinity for CcpA (Seidel et al., 2005) and itsdrastically lower synthesis levels (Görke et al., 2004).Finally, the complex of CcpA with HPr(Ser~P), but not withCrh(Ser~P), is stabilized by binding of FBP or glucose-6-phosphate (Seidel et al., 2005). In the light of these dif-ferences, it was an open question whether Crh is merelyredundant for HPr in CCR or whether it has a distinctfunction.

In this study, we re-investigated the putative role of Crhin B. subtilis. Using a ligand-fishing approach we identi-fied methylglyoxal synthase MgsA as interaction partnerof Crh. The data show that MgsA preferably binds non-phosphorylated Crh and accordingly formation of theMgsA/Crh complex is triggered by the quality of thecarbon source. Consistently, we find that MgsA activity isinhibited in vitro by non-phosphorylated Crh, whereasCrh(Ser~P) is without effect. In agreement, Crh inhibitsmethylglyoxal production in vivo when cells grow on anunfavourable carbon source, whereas it has little impactunder carbon overflow conditions, which trigger phospho-rylation of Crh. Finally, we show that HPr is unable to bindand to inhibit MgsA. Mutational analysis suggests thatfour residues that differ between the interaction surfacesof Crh and HPr are responsible for this difference. Hence,our study reports a bona fide function for Crh. Crh couplesactivity of methylgyloxal synthase and thereby fluxthrough a glycolytic by-pass to the nutritional state of thecell and might therefore be central for combatingphospho-sugar stress. We propose to rename Crh to‘carbon-flux regulating HPr’.

Results

Identification of a potential interaction partner of Crh byligand fishing

To identify novel interaction partners of Crh, we performedligand fishing experiments using Crh as bait. To this end,plasmid pGP734 was used, which directs synthesis ofrecombinant Crh carrying a C-terminal Strep-tag epitopefrom a constitutive promoter in B. subtilis. Crh-strep couldbe easily affinity-purified from B. subtilis cells carrying thisplasmid (Fig. S1A). The Crh-strep protein was also activein CCR as indicated by a complementation analysis

Regulation of methylglyoxal synthase by Crh 771

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 770–787

(Fig. S1B). Therefore, the Strep-tag had no deleteriouseffect on the interactions of Crh with HPrK/P and CcpA.We carried out ligand fishing experiments, which wereperformed either as SPINE (Strep-protein interactionexperiment) or as conventional co-purification. The hall-mark of SPINE is the reversible in vivo cross-linking ofinteracting proteins by addition of formaldehyde prior toharvesting of cells. To this end, cells were grown inminimal glucose medium and proteins were eventuallycross-linked. The lysates were loaded on streptactincolumns, washed and eluted in three fractions (EI–EIII)from the columns. To evaluate suitability of the method,we first checked whether glyceraldehyde-3-phosphatedehydrogenase GapA, a further interaction partner of Crh(Pompeo et al., 2007), can be detected in the elutionfractions. Western blotting analysis detected GapA spe-cifically in the first elution fractions (EI) of the Crh-streppurifications, irrespective whether proteins were cross-linked or not (Fig. S2, lanes 3–4). In contrast, GapA wasundetectable in the EI fractions prepared from cells car-rying the empty bait plasmid pGP382 (negative control;Fig. S2, lanes 1–2). In conclusion, the experimentalset-up appeared to be suited for the detection of interac-tion partners of Crh.

To identify unknown interaction partners of Crh, theelution fractions obtained from the co-purification experi-ment were analysed by SDS-PAGE and subsequentsilver-staining to allow for sensitive detection of co-elutingproteins (Fig. 1). Most co-eluting proteins were detectablein both fractions, derived from the cells containing theCrh-strep bait protein as well as from the negative controlstrain and were thus non-specific. However, one prominentprotein band (marked with an asterisk in Fig. 1) was exclu-sively present in the EI and EII fractions of the Crh-streppurification and was therefore a candidate for an interac-tion partner of Crh (Fig. 1, compare lanes 4–5 with lane 3and lanes 7–8 with 6). Mass spectrometric analysis iden-tified in this band the metabolic enzyme methylglyoxalsynthase MgsA (for details see Supporting information).

Crh and the methylglyoxal synthase MgsA forma complex

In order to confirm interaction of Crh with MgsA, a reverseSPINE experiment was carried out using plasmid-encoded Strep-tagged MgsA as bait. To allow for thesensitive detection of Crh, the sequence encoding the3¥FLAG epitope was fused in frame to the 3′ end of crh atits natural chromosomal locus. The resulting strain GP69carrying either plasmid pGP1180 encoding Strep-MgsA orthe corresponding empty vector pGP380 as a negativecontrol was grown in minimal glucose medium and sub-sequently SPINE was carried out. Aliquots of the obtainedelution fractions were either heated for 25 min at 95°C,

which resolves the bonds formed during cross-linking,or heated for 5 min at 65°C, which preserves theformaldehyde-induced protein cross-links. Subsequently,the samples were analysed by Western blotting usingFLAG-antiserum for the detection of Crh and Strep-antiserum for the detection of MgsA (Fig. 2). Indeed, Crh-3¥FLAG co-purified with Strep-MgsA, since both proteinswere detectable at the expected positions on the gel in theEI and EII fractions of the Strep-MgsA purification, whilethey were missing in the corresponding fractions obtainedfrom the control strain (Fig. 2, left panels). In this case thesamples had been heated at 95°C to resolve cross-linking. Interestingly, an additional band at the position of~34 kDa was detected by both antisera, when the 95°Cheating step was omitted and cross-linking preserved(Fig. 2, right panels). The molecular weight of this bandapproximately fits with the sum of the molecular weightsof Crh-3¥FLAG (12.9 kDa) and Strep-MgsA (16.4 kDa).Alternatively, it could correspond to a complex of a Crh-3¥FLAG dimer with a Strep-MgsA monomer. Crh has pre-viously been shown to form dimers via domain swappingin vitro (Juy et al., 2003).

Fig. 1. Ligand fishing (co-purification) experiment for the detectionof interaction partners of Crh. B. subtilis strain GP860 (crh::aphA3)carrying the empty bait vector pGP382 (lanes marked with ‘e’) orthe crh-strep bait plasmid pGP734 (lanes marked with ‘crh’) wasgrown in CSE medium supplemented with 0.5% glucose. When thecultures reached an OD600 = 0.8, they were subjected toco-purification. Proteins were eluted in three steps (fractionsEI–EIII) from the streptactin columns. Sixty microlitres of the lastwashing steps and of the elution fractions were separated on a12.5% SDS-polyacrylamide gel and proteins were detected bysilver-staining. The positions of Crh-Strep and of MgsA that wassubsequently identified by mass spectrometry, are indicated byarrows and their molecular weights are given in brackets. Bothproteins were exclusively detectable in the elution fractions EI andEII obtained from the cells carrying the crh-strep bait plasmid, whilethey were undetectable in the corresponding fractions of controlcells carrying the empty bait plasmid.

772 J. J. Landmann et al. �


To obtain further evidence that the 34 kDa protein bandrepresents a complex of Crh-3¥FLAG and Strep-MgsA,we investigated whether Crh-3¥FLAG can also bedetected in this band in crude extracts after cross-linkingand whether appearance of this band depends on thepresence of MgsA. To this end, we used strain GP91,which carries the crh-3¥FLAG allele on the chromosomeand a DmgsA deletion to avoid interference with thenaturally encoded MgsA protein. Subsequently, plasmidpGP1180 carrying the strep-mgsA allele was introducedinto strain GP91. The resulting transformant as well as theempty strain lacking plasmid pGP1180 were cultivatedand protein cross-linking was carried out as described forthe SPINE protocol. Crude extracts were prepared andsamples were either heated or not in order to resolve orpreserve cross-linking. Finally, the samples were analy-sed by Western blotting using FLAG-antiserum for thedetection of Crh-3¥FLAG (Fig. S3). Indeed, the FLAG-antiserum detected the 34 kDa band in the strain thatexpressed strep-mgsA from a plasmid provided thatcross-linking was preserved (Fig. S3, lanes 3, 4). In con-trast, this band was undetectable in the strain lackingstrep-mgsA irrespective whether cross-linking wasretained or not (Fig. S3, lanes 1 and 2). These data show

that formation of the 34 kDa complex containing Crh-3¥FLAG depends on the presence of MgsA in the cell.Taken together, it is conclusive that the 34 kDa proteinband represents a complex of Crh and MgsA, indicating adirect interaction between both proteins.

Interaction of Crh and MgsA in a heterologous host

To confirm the interaction of Crh and MgsA by an addi-tional method, we used two different bacterial two-hybrid(BTH) systems operating both in the heterologous hostE. coli. In the first BTH system, the potential interactionpartners are fused either to the N- or to the C-termini ofthe T18 and T25 fragments of the Bordetella pertussisadenylate cyclase CyaA respectively (Karimova et al.,1998). When physically separated, the T18 and T25CyaA domains are inactive. However, fusion of thesedomains to interacting proteins restores adenylatecyclase activity, which allows activation of cAMP/CRP-controlled genes such as lacZ in the mutant E. coli strainBTH101, which lacks its endogenous CyaA protein. Toassess interaction, MgsA was fused to the N-terminus ofthe T25 fragment and tested together with constructs thatconsisted of the protein to be tested fused either to the

Fig. 2. MgsA and Crh form a complex in B. subtilis cells. A reverse SPINE experiment using Strep-MgsA as bait was carried out. To this end,strain GP69 was used, which carried a crh-3¥FLAG fusion gene in the chromosome and contained either plasmid pGP1180 encodingStrep-MgsA or the corresponding empty expression vector pGP380 (negative control). The transformants were grown in CSE glucose mediumand subjected to SPINE. Aliquots of the obtained elution fractions EI, EII and EIII were heated either for 25 min at 95°C (left panels), whichreverses cross-linking, or for 5 min at 65°C, which retains the cross-links (right panels). Of these samples 15 ml were separated by SDS-PAGEand analysed by Western blotting using aFLAG-antiserum to detect Crh-3¥FLAG (top panels) or aStrep-antiserum to detect Strep-MgsA(bottom panels). As a control, 10 mg of total protein of strain GP69/pGP1180 was loaded in the last lane respectively. The positions of themolecular weight marker and of the recombinant Crh and MgsA proteins are indicated on the left.



C- or to the N-terminus of the T18 fragment. The corre-sponding transformants of strain BTH101 were grown toexponential phase and the b-galactosidase activitieswere determined (Fig. 3). The transformants that coex-pressed the crh and mgsA fusion genes produced ~500units b-galactosidase, regardless whether Crh was fusedto the N- or C-terminus of the T18 domain. This corre-sponded to about 50% of the activity detected with thepositive control that detects homodimerization of theleucine zipper of the yeast transcriptional regulatorGCN4 (Fig. 3, columns 3, 4, 8). When MgsA–T25 wastested in combination with the GCN4 leucine zipperfused to T18, only 50 units b-galactosidase activity weredetectable (negative control; Fig. 3, column 7). In conclu-sion, the results indicated interaction of Crh and MgsA.Interestingly, high b-galactosidase activities were alsodetectable, when cells co-synthesized the MgsA–T25fusion and fusions of MgsA to the N- or C-terminus ofT18, indicating homomultimerization of MgsA (Fig. 3,columns 1 and 2). This is in agreement with the proper-ties of MgsA of E. coli, which was shown to form a homo-hexamer (Saadat and Harrison, 1999). Interaction of Crhand MgsA was also detectable with the second BTHsystem (see Fig. S4 and legend) that is based on a dif-ferent principle (Dove and Hochschild, 2004). Collec-tively, the SPINE and the BTH assays yielded congruentresults: They all show that Crh directly binds to MgsA.

MgsA binds Crh, but not its homologue HPr

All previously identified interaction partners of Crh alsointeracted with its homologue HPr, leading to the assump-tion that Crh has mainly a redundant role in the cell(Deutscher et al., 2006; Pompeo et al., 2007). Therefore,we were keen to learn whether MgsA could also interactwith HPr and addressed this question by making use of theBTH systems. We constructed fusions of HPr to the variousprotein domains as carried out before for Crh and testedthe resulting constructs for interaction with the respectiveMgsA fusion proteins. With the BTH system of Karimovaet al., only background levels of b-galactosidase activitieswere detected when interaction of MgsA–T25 with HPrfused either to the N- or to the C-terminus of the T18fragment was tested (Fig. 3, columns 5, 6). Similarly, theBTH system of Dove and Hochschild also detected onlylow b-galactosidase activities that did not exceed the levelof the negative control (Fig. S4, columns 3, 4). Thus,interaction of MgsA and HPr was undetectable by bothBTH systems.

To provide additional biochemical evidence for the inabil-ity of HPr to interact with MgsA, we once more carried outa SPINE experiment using Strep-MgsA as bait. In order toobtain more physiological protein concentrations, thestrep-mgsA gene was ectopically integrated into the chro-mosomal lacA gene and expressed from the PdegQ36 pro-moter (strain GP78; Fig. 4A). As a control, the isogenicstrain GP79 carrying the empty expression vector inte-grated in lacA was used. Both strains carried the crh-3¥FLAG allele at its natural locus (Fig. 4A). The eluatesobtained from SPINE were analysed by Western blottingusing antisera directed against the FLAG-tag, HPr and theStrep-tag respectively. Both Crh-3¥FLAG and Strep-MgsAwere readily detected in the elution fraction EI obtainedfrom strain GP78, while these signals were lacking in thenegative control strain GP79 (Fig. 4B, lanes 1–4, top andbottom panels). However, when the same samples weresubjected to Western analysis using the HPr-specific anti-serum, no signals were obtained, although this antiserumreadily detected HPr in the total protein extracts of thesestrains (Fig. 4B, medium panel). Taken together, Crh, butnot its homologue HPr, is able to bind MgsA.

MgsA preferably binds non-phosphorylated Crh

The next interesting question was whether phosphoryla-tion of Crh affects its interaction with MgsA. In E. coli anHPrK/P orthologue is missing and therefore Crh cannotbe phosphorylated at Ser46 in this host. Therefore, theBTH experiments in E. coli already indicated that non-phosphorylated Crh is capable to bind MgsA. To get firstinsight, whether phosphorylation of Crh has any impact oninteraction with MgsA, we made use of the CyaA-basedBTH system and tested whether coexpression of the

Fig. 3. Bacterial two-hybrid (BTH) assay demonstrating that MgsAinteracts with Crh, but not with its homologue HPr. The proteins tobe tested were fused to the N- or C-termini of the T18 and T25domains of the Bordetella pertussis adenylate cyclase CyaA,respectively, and co-synthesized in the E. coli strain BTH101, whichlacks endogenous CyaA. Interaction of the candidate proteinsrestores CyaA activity leading to cAMP synthesis, which allowsexpression of lacZ. The following plasmid combinations encodingthe fusions as indicated were tested: pGP1175/pGP1172 (column1), pGP1175/pGP1173 (column 2), pGP1175/pGP1164 (column 3),pGP1175/pGP1165 (column 4), pGP1175/pGP1160 (column 5),pGP1175/pGP1161 (column 6), pGP1175/pUT18C-zip (column 7,negative control), pKT25-zip/pUT18C-zip (column 8, positivecontrol). The co-transformants were grown in LB containing 0.5 mMIPTG to an OD600 = 0.6 and the b-galactosidase activities weredetermined.



B. subtilis hprK gene affects interaction of the Crh–T18and MgsA–T25 fusion proteins (see Fig. S5 and itslegend). These initial data suggested that presence ofHPrK/P kinase activity lowers interaction of Crh with MgsAin E. coli, supposedly through phosphorylation of Crh.

In the cognate host B. subtilis kinase activity of HPrK/Pis controlled by the FBP level, which reflects the quality ofthe utilized carbon source. So far, we performed theligand fishing experiments in minimal glucose medium,which triggers a high FBP level and thereby the efficientphosphorylation of HPr and Crh. Hence, predominantlyphosphorylated Crh was present in the cells. To corrobo-rate that phosphorylation of Crh impairs binding to MgsA,we performed SPINE but used a B. subtilis DhprK mutantthat carried the crh-3¥FLAG allele on the chromosomeand encoded the bait protein Strep-MgsA in the lacAlocus. The resulting strain GP83 and the isogenic wild-type (hprK+) strain GP78 were grown in minimal glucosemedium and subjected to SPINE. The EI and EII fractionswere analysed by Western blotting for the detection ofStrep-MgsA and Crh-FLAG (Fig. 5A). As already shown

before (Fig. 4), Crh-3¥FLAG co-purified with Strep-MgsAas both proteins were present in the eluates obtained fromthe wild-type strain GP78, while they were undetectable inthe eluates of the negative control strain GP79, whichcarried the empty strep expression vector integrated inlacA (Fig. 5A, lanes 3–6). Intriguingly, a much higheramount of Crh-3¥FLAG (11-fold increased) co-purifiedwith Strep-MgsA, when the DhprK mutant was used, inwhich Crh cannot be phosphorylated (Fig. 5A, comparelanes 1 and 3). Similar Crh-3¥FLAG amounts were detect-able in the total protein extracts of the wild-type and thehprK mutant, indicating that this difference is not causedby different Crh synthesis levels (Fig. 5A, lanes 7 and 8).Taken together, the data indicate that phosphorylation ofCrh inhibits its interaction with MgsA.

Interaction of Crh and MgsA is triggered by the qualityof the carbon source

The kinase activity of HPrK/P and thus the degree ofphosphorylation of HPr and Crh at their Ser46 sites

Fig. 4. Ligand fishing experiment (SPINE) detects interaction of MgsA with Crh, but not with its homologue HPr.A. Location of the recombinant crh-3¥FLAG and strep-mgsA genes on the chromosome of strain GP78 that was used for SPINE. The fusionof the 3¥FLAG encoding sequence to the 3′ end of crh gene was obtained by single cross-over with plasmid pGP1188. The strep-mgsA geneunder control of the constitutive PdegQ36 promoter was ectopically integrated into the lacA gene by double cross-over with plasmid pGP2207.Strain GP79, which was used as a control in (B) (lanes 3, 4, 6), is isogenic with strain GP78, but carries the empty expression vectorpGP1459 integrated in lacA.B. SPINE using chromosomally encoded Strep-MgsA as bait. Strains GP78 (lanes 1, 2) and GP79 (lanes 3, 4; negative control, see A) weregrown in CSE glucose medium and SPINE was carried out. Aliquots (25 ml) of the elution fractions EI and EII were separated by SDS-PAGEand subsequently subjected to Western blotting using antisera directed against the FLAG-tag (top panel), HPr (medium panel) and theStrep-tag (bottom panel). As controls, 10 mg of total protein of strains GP78 (lane 5) and GP79 (lane 6) were also loaded. Note that theaStrep-antiserum was not sensitive enough to detect chromosomally encoded Strep-MgsA in total-cell extracts, while it was readily detectablewhen encoded on a plasmid (Fig. 2, bottom panels, lane 7).



depends on the carbon source. Preferred carbon sourcessuch as glucose or mannitol trigger the preferential phos-phorylation of Crh and HPr by HPrK/P, whereas theseproteins are predominantly non-phosphorylated at Ser46when bacteria grow on secondary carbon sources suchas succinate, ribose or gluconate (Singh et al., 2008 andour unpublished results). If MgsA preferably interacts withnon-phosphorylated Crh, one might expect that theamount of Crh that is bound to MgsA, is determined by thequality of the utilized carbon source. This hypothesis wastested by SPINE. The wild-type strain GP78 carrying thecrh-3¥FLAG and strep-mgsA alleles on the chromosomewas grown in minimal medium supplemented with differ-

ent carbon sources. The cultures were subjected toSPINE and proteins in the EI fractions were subsequentlydetected by Western blotting (Fig. 5B). Indeed, theamount of Crh-3¥FLAG that co-purified with a givenamount of Strep-MgsA considerably varied with thecarbon source that was utilized by the bacteria: when cellsutilized the preferred carbon sources glucose or mannitol,only a small amount of Crh-3¥FLAG co-purified withStrep-MgsA, while up to 10-fold more Crh-3¥FLAG wasbound to Strep-MgsA when bacteria were grown on suc-cinate or ribose (Fig. 5B). When bacteria utilized glucon-ate, which triggers an intermediate level of HPrK/P kinaseactivity (Singh et al., 2008), fivefold more Crh-3¥FLAG

Fig. 5. Non-phosphorylated rather thanphosphorylated Crh interacts with MgsA.A. Absence of kinase HPrK/P stronglyenhances binding of Crh to MgsA in vivo.SPINE was carried out using B. subtilisstrains that encoded the bait (Strep-MgsA)and prey (Crh-3¥FLAG) proteins on thechromosome (see Fig. 4A). In addition to‘wild-type’ strain GP78 (lanes 3, 4 and 7), theisogenic DhprK mutant GP83 was employed(lanes 1, 2, 8). Strain GP79 carrying theempty strep-vector integrated in lacA servedas negative control (lanes 5, 6). The strainswere grown in CSE glucose medium andSPINE was performed as described for Fig. 4.The elution fractions were analysed byWestern blotting using antisera directedagainst the FLAG-tag (top panel) and theStrep-tag (bottom panel). As controls, 10 mgof total protein of strains GP78 (lane 7) andGP83 (lane 8) were analysed.B. Crh–MgsA complex formation is triggeredby carbon source quality. B. subtilis strainGP78 (see A) was grown in CSE mediumsupplemented with 0.5% of the carbon sourceindicated at the top and subsequently SPINEwas performed. Thirty-five microlitres of the EIelution fractions were separated bySDS-PAGE and analysed by Western blottingas in (A) (top and medium panel). The relativeamount of Crh-3¥FLAG co-purifying withStrep-MgsA is indicated. To determinewhether Crh synthesis or stability is affectedby the carbon source, strain GP69 carryingthe crh-3¥FLAG allele was grown in CSEmedium supplemented with the differentcarbon sources as indicated and crudeextracts were analysed by SDS-PAGE andWestern blotting using the aFLAG-antiserum(bottom panel, lanes 1–5). Wild-type strain168 served as control to demonstratespecificity of the antiserum (bottom panel,lane 6).



could be detected in comparison with glucose-grown cells(Fig. 5B, lanes 3 and 5). In principle, it is also possible thatdifferent Crh synthesis levels contribute to the carbonsource-dependent differences in Crh–MgsA complexformation. To address this possibility, the amounts of Crh-3¥FLAG were determined in crude extracts of cells grownon the various carbon sources. Slightly (i.e. 1.3–1.5¥)higher Crh-3¥FLAG amounts were detectable, when cellsutilized succinate, ribose or gluconate as compared withmannitol and glucose (Fig. 5B, bottom panel, comparelanes 1–3 with 4 and 5). In conclusion, the data show thatinteraction of Crh and MgsA is controlled by the qualityof the carbon source predominantly via the HPrK/P-mediated phosphorylation of Crh: the higher the degree ofCrh phosphorylation, the lower the amount of Crh boundto MgsA.

Non-phosphorylated Crh inhibits MgsA enzyme activityin vitro

The next important question was to elucidate the physi-ological function of the interaction of MgsA with non-phosphorylated Crh. To assess, whether this interactionaffects the enzymatic activity of MgsA, a colorimetric MgsAenzyme assay was used (Hopper and Cooper, 1972). Inthis assay, MgsA protein is incubated with its substrateDHAP and the formed methylglyoxal is detected by itsreaction with 2,4-dinitrophenylhydrazine to dinitrophenol-hydrazone that forms a coloured product in the presence ofNaOH, which can be measured spectrophotometrically.Using this assay, we determined a specific activity of0.12 mol g-1 min-1 for recombinant B. subtilis Strep-MgsA(Fig. 6B, column 1 and Fig. S6A), which is comparable tothe previously reported specific activity of MgsA fromE. coli (0.5 mol g-1 min-1), and considerably higher thanthe specific activities of MgsA from Proteus vulgaris(9.2 ¥ 10-3 mol g-1 min-1) and Pseudomonas saccharo-phila (5.8 ¥ 10-3 mol g-1 min-1) (Hopper and Cooper, 1972;Tsai and Gracy, 1976). Thus, the B. subtilis MgsA protein isa ‘bona fide’ methylglyoxal synthase. Next, we testedthe effects of phosphorylated and non-phosphorylatedrecombinant Crh-His6 protein on the MgsA enzyme activ-ity. Crh~P was obtained by in vitro phosphorylationusing purified Strep-HPrK/P (Fig. S6B) followed by itsre-purification by Ni2+-NTA affinity chromatography. BothCrh preparations were virtually free of the respective otherform, as demonstrated by non-denaturing PAA gel electro-phoresis that allows the separation of both forms of Crh(Fig. 6A, left panel, lanes 1 and 3). Intriguingly, increasingconcentrations of non-phosphorylated Crh incrementallyinhibited activity of MgsA (Fig. 6B, top panel). In contrast,phosphorylated Crh had no inhibitory effect but evenslightly stimulated MgsA activity when present at highconcentrations, although this difference appeared to be

within experimental error (Fig. 6B, bottom panel). Werepeated these experiments using HPr rather than Crh(Fig. 6A, right panel and Fig. 6C). HPr had no effecton MgsA activity, regardless whether it was phosphory-lated or not. Thus, the data consistently show that non-phosphorylated Crh binds to MgsA and inhibits itsenzymatic activity, whereas Crh~P, HPr or HPr(Ser)~P areunable to do so.

Carbon source quality triggers repression ofmethylglyoxal formation by Crh in vivo

Phosphorylation of Crh is catalysed by HPrK/P, whosekinase activity is triggered by carbon source availabilityand quality. Utilization of preferred sugars such asglucose triggers a high FBP level and thereby the phos-phorylation of Crh, whereas Crh is preferentially non-phosphorylated when cells grow on unfavourable carbonsources. Thus, Crh is expected to exert an effect on MgsAactivity in vivo predominantly in the absence of preferredsugars. To test this possibility, we compared methylgly-oxal production of the wild-type strain and the crh mutantin the presence or absence of preferred carbohydrates. Tothis end, we measured the methylglyoxal excreted by thebacteria into the medium. When grown on a mixture ofarabinose and glucose (feast conditions), the wild-typereleased up to 0.22 mM methylglyoxal into the medium,while much lower concentrations were produced whencells used succinate (Fig. 7, compare grey columns in Aand B), suggesting that B. subtilis produces methylglyoxalunder feast, but not under famine conditions. When grownon glucose + arabinose only slightly more methylglyoxalwas produced by the crh mutant in comparison with thewild-type (Fig. 7A, compare grey and black columns).However, during growth on succinate much higher meth-ylglyoxal concentrations were generated by the crhmutant in comparison with the wild-type (Fig. 7B, comparegrey and black columns) and these concentrations werecomparable with the methylglyoxal levels detected inglucose + arabinose-medium (Fig. 7, compare blackcolumns in A and B). These data indicate that methylgly-oxal production is inhibited, when B. subtilis grows on asecondary carbon source and that Crh is essential for thisrepression.

Molecular requirements for interaction with MgsA

According to our data Crh, but not HPr, binds and regu-lates MgsA activity. This finding was surprising since allother proteins known to interact with Crh, such as CcpA,HPrK/P and GapA, also interact with its homologue HPr.Therefore, we were keen to identify the molecular deter-minants underlying the specific recognition of Crh byMgsA. Crh and HPr exhibit very similar folds consisting of



three a-helices and four b-sheets (Favier et al., 2002).Generally, HPr-like proteins are known to interact withpartner proteins through a narrow region consisting ofhelices a1 and a2 and the loop preceding helix a1 (Deut-scher et al., 2006) and structural analyses showed thatthis region is also involved in interaction of Crh(Ser~P)and HPr(Ser~P) with CcpA (Schumacher et al., 2004;2006). The region encompassing helix a2 including theSer46 phosphorylation site is highly conserved in Crhas well as in cognate HPr proteins (Fig. 8A), making

it unlikely that it accounts for the differences in MgsAbinding. However, in the region encompassing helix a1several residues differ between the HPr and Crh proteins.These residues are present at positions 14 (Leu in Crh; Ileor Val in HPr), 15 (Gln in Crh; His in HPr), 20 (Ala in Crh;Thr in HPr) and 22 (Phe in Crh; Leu in HPr). To learn,whether these residues are important for interaction withMgsA, we exchanged them in Crh either individually orcollectively for the residues present in HPr at the respec-tive positions. To complement this analysis, mutant HPr

Fig. 6. Non-phosphorylated Crh inhibits methylglyoxal synthase MgsA in vitro.A. Preparation of phosphorylated and non-phosphorylated Crh and HPr proteins that were used in the MgsA enzyme assay. Purified Crh-His6

and His6-HPr proteins were phosphorylated in vitro using purified Strep-HPrK/P protein and subsequently re-purified by Ni2+-NTA affinitychromatography. The fractions obtained by elution with 50 mM (lanes 2, 5) and 80 mM imidazole (lanes 3, 6) and the non-phosphorylatedproteins (lanes 1, 4) were analysed by non-denaturing gel electrophoresis, which allows separation of the differently modified proteins.Proteins were detected by Coomassie blue staining. The preparations analysed in lanes 1, 3, 4 and 6 were used in the MgsA activity assays.B. Effect of Crh and Crh~P on MgsA activity. Purified Strep-MgsA (0.1 mg) was incubated with its substrate DHAP and increasing amounts ofCrh (top panel) or Crh~P (bottom panel) as indicated. Formation of methylglyoxal was determined by a spectrophotometric assay.C. Effect of HPr (top panel) and HPr~P (bottom panel) on MgsA activity. Same as (B), but HPr rather than Crh was added to the assay.



proteins carrying the residues from Crh at the respectivepositions were engineered. The interaction of the variousmutant proteins with MgsA was tested in the context of theCyaA-based BTH system. Reporter strain E. coli BTH101was co-transformed with the MgsA–T25 fusion constructand the plasmids encoding the Crh–T18 or HPr–T18 vari-ants respectively. The b-galactosidase activities of thesetransformants were monitored phenotypically on X-Galplates (data not shown) and quantitatively by measuringthe accumulating b-galactosidase activities (Fig. 8B). TheGln15His exchange in Crh had no effect on interactionwith MgsA and the Leu14Ile and Phe22Leu substitutionsreduced the b-galactosidase activities twofold (Fig. 8B,columns 1, 2, 3, 5). Notably, interaction with MgsA wascompletely abolished when Crh carried the Ala20Threxchange either individually or in combination with theother three substitutions (Fig. 8B, columns 1, 4, 6). Thus,Ala20 is essential and residues Leu14 and Phe22 maycontribute to the interaction of Crh with MgsA. Intriguingly,the HPr mutants behaved almost complementary to theircorresponding Crh mutants. When tested individually, theHis15Gln and also the Ile14Leu and Leu22Phe substitu-tions had no detectable effect on the inability of HPr tointeract with MgsA. In contrast, the T20A exchange con-ferred the ability on HPr to weakly interact with MgsA(Fig. 8B, columns 7–11). This interaction appeared to beeven further improved, when the T20A exchange was

combined with the other three exchanges tested in HPr.Intriguingly, interaction of MgsA with the HPr quadruplemutant appeared to be almost as strong as with Crh(Fig. 8B, columns 1, 12). Thus, the ability to interact withMgsA appears to be transmittable to HPr by substitution ofonly four residues in the interaction surface for their coun-terparts from Crh.

Discussion

The findings reported in this study allow two significantconclusions. First, our data indicate that in B. subtilis fluxthrough the methylglyoxal bypass of glycolysis is regu-lated by a sophisticated feed-forward loop. This controlinvolves inhibition of the key enzyme methylglyoxal syn-thase MgsA by interaction with Crh, a homologue of theHPr protein of the PTS (see model in Fig. 9). Phosphory-lation of Crh by kinase HPrK/P prevents this interactionand therefore releases MgsA from inhibition. The kinasefunction of HPrK/P requires activation by high FBPconcentrations. Consequently, high activity of the meth-ylglyoxal pathway is coupled via Crh to high intracellularFBP levels, which reflect a good carbon source supply oreven carbon overflow conditions. Second, we provide evi-dence for a ‘bona fide’ function of Crh, which cannot becarried out by its homologue HPr. Since its discovery 14years ago, the putative function of Crh has remained

Fig. 7. Impact of Crh on methylglyoxal production in vivo. The B. subtilis wild-type strain 168 (black squares and columns) and the Dcrhmutant QB7097 (grey diamonds and columns) were grown in minimal medium supplemented either with 0.5% glucose + 0.5% arabinose (A)or with 0.5% succinate (B). Samples were taken at periodic intervals and the methylglyoxal concentration in the medium was determined. Notethat the slight growth defect of the Dcrh mutant in (B) is within experimental error and therefore not significant.



Fig. 8. Molecular requirements for interaction of HPr-like proteins with methylglyoxal synthase MgsA. Transplantation of four amino acidsfrom Crh to HPr transforms HPr into an interaction partner for MgsA.A. Amino acid sequence alignment of Crh and their cognate HPr proteins from various bacilli. The amino acid positions and the location of theb-sheets and a-helices are indicated at the top. The two regions composing the interaction surface in HPr and Crh are marked with greenlines. Identical residues are depicted in red and residues conserved in at least half of the sequences are in blue. Four residues in theN-terminal half of the interaction surfaces deviate between Crh and HPr, but are conserved within each protein group (marked with arrows).These residues were substituted individually or collectively in Crh with their counterparts from HPr and vice versa. Interaction of the mutantswith MgsA was probed using the CyaA-based BTH assay.B. b-Galactosidase activities of the various transformants were monitored quantitatively by measuring enzyme activities. Shown are resultsobtained with reporter strain BTH101 carrying plasmid pGP1175 encoding the MgsA–T25 fusion protein and a compatible plasmid encodingthe indicated T18 fusion protein (see Table 2 for plasmid designations). Transformants carrying plasmids coding for fusions of the GCN4leucine zipper to the T25 and T18 domains served as positive control. In order to detect also weak interactions, cells were harvested at anOD600 = 3.0, which allows accumulation of b-galactosidase to higher levels.



controversial. Mostly, the role of Crh for CCR was consid-ered to be merely redundant or restricted to specificsystems such as the citM gene (Warner and Lolkema,2003). Our data suggest that regulation of MgsA activity isa primary function of Crh, while its role in CCR might be aminor one.

The methylglyoxal pathway is a paradox. It representsan energetically unfavourable bypass of glycolysis thatmoreover produces a poison. It is believed to serve asoverflow metabolism, when sugar uptake rates exceedthe capacity of the lower branch of the EMP, which wouldcause the accumulation of toxic sugar-phosphates. Thus,the cell must balance the production of two differentpoisons, which requires tight control. Many bacteria

produce methylglyoxal only when glucose is present oradded to a slow growing culture (Teixeira de Mattos et al.,1984; Baskaran et al., 1989; Russell, 1993; Weber et al.,2005). This seems to be also the case for B. subtilis(Fig. 7). In E. coli, flux through the methylglyoxal pathwaywas studied in glucose-limited chemostat cultures (Weberet al., 2005). Upshift of the dilution rate increased glucoseuptake rates to levels that exceeded the rate of biomassformation leading to an uncoupling of catabolism andanabolism. This resulted in accumulation of FBP, DHAPand concomitantly of methylglyoxal, while the glyceralde-hyde 3-phosphate concentration decreased. It was con-cluded that FBP or DHAP may have a key role inactivation of methylglyoxal production (Weber et al.,2005). Cells respond immediately to upshift by startingmethylglyoxal synthesis, indicating that this regulationtakes place at the level of enzymatic activity. MgsA fromE. coli is inhibited by phosphate and homotropically acti-vated by DHAP in vitro (Cooper, 1984). Inhibition ofB. subtilis MgsA by phosphate in mM ranges was alsoobserved in the current study (Fig. S7), indicating thatboth enzymes behave similarly in this respect. It wasspeculated that in vivo accumulation of DHAP may over-come inhibition of MgsA by phosphate, which could besupported by a transient depletion of cellular phosphateupon accumulation of sugar-phosphates (Cooper, 1984;Weber et al., 2005). However, the results obtained in thecurrent study suggest a more elaborate mode of regula-tion of MgsA activity control, at least in B. subtilis (Fig. 9).According to this model Crh transfers information aboutthe metabolic state of the cell to MgsA. This elegantmechanism may allow the cell to couple flux through themethylglyoxal bypass dynamically to the levels of FBPand other metabolites such as ATP, which modulateHPrK/P activity.

Interestingly, it has been reported that HPr(Ser)~P, andto minor degree also Crh~P, are capable to bind andto inhibit glyceraldehyde-3-phosphate dehydrogenaseGapA, which catalyses the first reaction in the bottombranch of the EMP (Pompeo et al., 2007). Binding ofGapA by Crh was also confirmed in the current study(Fig. S2). Hence, Crh and HPr might even have a moreglobal role for regulation of fluxes through glycolysis:under famine conditions Crh and HPr prevail in theirdephosphorylated forms. Therefore, MgsA activity isinhibited by Crh, while GapA activity is relieved fromrepression. This would allow for maximum flux through thelower branch of the EMP. At the same time, cells arerelieved from CCR and therefore primed for efficientuptake and metabolism of carbohydrates. Increasing FBPlevels, i.e. feast conditions, trigger phosphorylation of Crhand HPr. This will cause activation of MgsA, whereasGapA activity will be inhibited. Consequently, flux wouldbe redirected from the bottom branch of the EMP towards

Fig. 9. Model for regulation of the methylglyoxal pathway by Crh.The EMP and the embedded methylglyoxal pathway areschematically shown. Steps consuming or producing ATP and Pi

are indicated. H2 indicates involvement of NADH, NADPH or FADH.Secondary carbon sources such as succinate generate only lowFBP levels in the cell. Under these conditions the phosphorylasefunction of HPrK/P prevails leading to dephosphorylation of Crh.Dephosphorylated Crh binds MgsA and inhibits its enzymaticactivity, thereby blocking the methylglyoxal pathway. Under carbonoverflow conditions, i.e. when preferred substrates such as glucoseare present at high concentrations, the FBP levels increase andactivate the kinase function of HPrK/P. This triggers the formationof Crh~P, which relieves MgsA from inhibition leading to activationof the methylglyoxal bypass. Methylglyoxal either is excreted or isdegraded to pyruvate by several alternative pathways as has beenshown for E. coli (depicted in grey; Cooper, 1984; Weber et al.,2005; Ozyamak et al., 2010). These pathways may also exist inB. subtilis, since corresponding enzymatic activities have beendetected (Willetts and Turner, 1970), but the associated genes areunknown. In E. coli a fraction of the methylglyoxal is converted toL-lactate via lactaldehyde catalysed by methylglyoxal reductase andaldehyde dehydrogenase. Another fraction is detoxified throughglyoxalases I and II via formation of S-lactoylglutathione andthrough glyoxalase III, which generates D-lactate directly frommethylglyoxal. D- and L-lactate dehydrogenases convert lactate topyruvate completing the bypass.



the methyglyoxal pathway. In addition, HPr(Ser)~P andCrh~P will elicit CCR, limiting further uptake of carbo-hydrates. Thus, it is tempting to speculate that Crh andHPr dynamically redistribute fluxes through the methylgy-loxal pathway and the lower branch of the EMP inresponse to the physiological requirements. Undoubtedly,flux analyses addressing the roles of both proteins forregulation of glycolysis, including the methylglyoxalpathway, will be required to confirm this hypothesis.

How does Crh inhibit activity of MgsA? The role andfunction of MgsA in B. subtilis has so far not beenaddressed. However, MgsA from B. subtilis shares 45%sequence identity with the E. coli enzyme, which hasbeen characterized. The catalytically active form of MgsAis a homohexamer and many residues that are involved ininteraction between the monomers are conserved in allMgsA proteins (Saadat and Harrison, 1999). In agree-ment, the BTH analysis indicated that B. subtilis MgsAalso multimerizes (Fig. 3). Interestingly, a complex thatapproximately corresponded in size to one MgsAmonomer plus one Crh monomer, was detectable bySPINE when cross-linking was retained (Fig. 2). Hence,one possibility is that Crh inhibits multimerization of MgsAand thereby blocks its enzymatic activity. The identifica-tion of the Crh binding site in MgsA should yield insightinto this question. Our study also yielded first insight, whyCrh, but not HPr, is able to bind MgsA. HPr-like moleculesincluding Crh interact with their partner proteins througha-helices a1 and a2 and residues in their close vicinity(Deutscher et al., 2006). Region a2 in Crh is clearlyinvolved in interaction since phosphorylation of Ser46,which is located in a2, hinders interaction. However, themolecular determinants allowing Crh but not HPr tointeract appear to be located in helix a1. The mutationalanalysis identified Ala20 in Crh as critical determinantfor interaction with MgsA (Fig. 8). Substitution by a Thrresidue, which is present at this position in HPr, abolishedinteraction. Complementary, a Thr20Ala substitution con-ferred on HPr the ability to weakly interact with MgsA. Thisinteraction appeared to be further improved, when theresidues present at positions 14, 15 and 22 in HPr werealso substituted by their counterparts from Crh. Thus, anAla residue at position 20 is absolutely required for inter-action and residues Leu14, Gln15 and Phe22 may con-tribute to proper interaction. Interestingly, Thr20 of HPr isinvolved in complex formation with CcpA: it forms a hydro-gen bond to a Tyr residue of CcpA and the lack of thisbond was shown to be responsible for the weaker bindingof Crh~P to CcpA as compared with HPr(Ser)~P (Schu-macher et al., 2006). Thus, the residue present at position20 may determine whether an HPr-like molecule effi-ciently interacts with CcpA or with MgsA. Both functionsappear to be mutually exclusive. It is also interesting tonote that HPr proteins from Gram-negative bacteria carry

the same residues as Crh at positions 14, 20 and 22(Reichenbach et al., 2007). Hence, it is possible that inGram-negative bacteria, HPr binds and regulates MgsAactivity.

Once formed, methylglyoxal must be detoxified, buthow this is achieved in B. subtilis is still unknown. InE. coli, methylglyoxal can be degraded by several path-ways and corresponding enzymatic activities were alsodetected in B. subtilis (see Fig. 9; Willetts and Turner,1970), but the responsible genes are unknown. Onemajor route of methylglyoxal detoxification that is ubiqui-tously found occurs via the glutathione (GSH)-dependentglyoxalase system (Mannervik, 2008; Ozyamak et al.,2010). B. subtilis also encodes several candidate glyox-alases (http://subtiwiki.uni-goettingen.de/). However asa peculiarity, B. subtilis appears not to contain GSH.Instead, it synthesizes another low-molecular-weight thiol,bacillithiol. Interestingly, enzymes BshA and BshB1, whichare involved in bacillithiol synthesis, are encoded imme-diately downstream of MgsA in the same operon (Gaballaet al., 2010), suggesting a functional connection. It istempting to speculate that in B. subtilis the glyoxalasesystem is bacillithiol- rather than glutathione-dependent.

Our study highlights the importance of protein–proteininteractions for the regulation of central metabolic func-tions of the cell. As exemplified by Crh, these interactionsare often dynamic and a complete picture of the networkcontrolling carbohydrate metabolism can only beobtained, when the temporal character of the interactionsis taken into account. One major factor controlling thetransient nature of protein–protein interactions in the cellis protein phosphorylation as demonstrated here for theB. subtilis Crh protein.

Experimental procedures

Bacterial strains and growth conditions

Escherichia coli was routinely grown in LB at 37°C underagitation at 200 r.p.m. The following antibiotics were usedfor selection of E. coli strains, when necessary: ampicillin(100 mg ml-1), kanamycin (30 mg ml-1) and chloramphenicol(15 mg ml-1). B. subtilis strains were grown at 37°C in LB orCSE minimal medium (Martin-Verstraete et al., 1995) supple-mented with 50 mg l-1 tryptophan and with 0.5% of the indi-cated carbon source. If appropriate, the media containedspectinomycin (150 mg ml-1), tetracycline (10 mg ml-1), kana-mycin (10 mg ml-1), chloramphenicol (5 mg ml-1) or erythromy-cin (2 mg ml-1) plus lincomycin (25 mg ml-1).

Construction of plasmids and strains

Strains, plasmids and oligonucleotides used in this study arelisted in Tables 1, 2 and S1 respectively. Details on plasmidand strain constructions are provided under Supportinginformation. Standard procedures were used for isolation of



plasmids and for transformation of B. subtilis and E. coli withDNA. Transformants were selected on LB or SP plates con-taining the appropriate antibiotics.

Purification of His- and Strep-tagged proteins

His-tagged Crh-His6 and His6-HPr proteins were purified fromE. coli strain BL21(DE3) carrying plasmid pAG1 and fromE. coli NM522 carrying plasmid pAG2 respectively. The bac-teria were grown in 1 l of LB and synthesis of His-taggedproteins was induced by the addition of 1 mM IPTG when thecultures reached an OD600 = 0.8. After an additional hour ofgrowth, cells were harvested and crude lysates were pre-pared by passage through a French pressure cell or a oneshot model (Constant Systems). The lysates were cleared bylow and high speed (FiberLite rotor F50L-8¥39, 35000 r.p.m.,1 h, 4°C) centrifugations and supernatants were loaded onsuperflow columns containing 5 ml of Ni2+-NTA resin (Qiagen,Germany). After washing, proteins were eluted with an imi-dazole gradient containing up to 500 mM imidazole in 50 mMTris/HCl pH 7.5, 200 mM NaCl. For overproduction and sub-sequent purification of N-terminally Strep-tagged MgsA andHPrK/P proteins E. coli strain BL21(DE3) carrying plasmidpGP1301 and B. subtilis strain GP858 carrying plasmidpGP642 were used respectively. Cleared lysates were pre-pared as described for His-tagged proteins and subsequentlypassed over columns containing 1 ml of Strep-Tactin matrix(IBA, Germany). The columns were washed four times with2.5 ml of buffer W containing 100 mM Tris/HCl pH 8.0,150 mM NaCl, 1 mM EDTA and subsequently Strep-tag pro-teins were eluted in three fractions using 3 ¥ 1 ml of buffer Wcontaining 2.5 mM desthiobiotin. For subsequent in vitroexperiments, the fractions containing the respective His- orStrep-tagged recombinant protein were dialysed two timesagainst 10 mM Tris/HCl pH 7.5, 100 mM KCl, 1 mM DTT

containing 25% glycerol in the second step. Proteins werestored at -20°C until their use.

Protein co-purification and SPINE (Strep-proteininteraction experiment)

Ligand fishing experiments were performed using recombi-nant B. subtilis strains carrying the gene encoding the Strep-tagged bait protein under control of the constitutive degQ36promoter either on a plasmid or ectopically integrated into thelacA locus on the chromosome (Fig. 4A). Strains carrying therespective empty bait constructs (only Strep-tag encodeddownstream of PdegQ36) served as controls. The bacteria weregrown in 1 l of CSE medium containing 0.5% glucose if nototherwise indicated. For co-purification, cells were harvestedat an OD600 of 0.8 and subsequently the protocol of purifica-tion of Strep-tagged proteins was carried out. For SPINE(Herzberg et al., 2007), the culture was treated with 0.6%formaldehyde at an OD600 = 0.8 and incubated for additional20 min. Subsequently, the strep-tagged bait proteins werepurified as described. Aliquots of the elution fractionsobtained from the SPINE and the co-purification experimentswere separated by SDS-PAGE. To allow for reversal of theformaldehyde-induced cross-linking, the samples wereheated for 25 min at 95°C prior to SDS-PAGE. After SDS-PAGE, the gel was silver-stained in order to detect unknowninteraction partners or subjected to Western blotting for thedetection of defined proteins.

Western blot analysis

Proteins were separated by electrophoresis on polyacryla-mide gels and subsequently transferred to a polyvinylidenedifluoride membrane (Bio-Rad) by electroblotting (60 min at

Table 1. Strains used in this study.

Name Genotype Reference or construction

B. subtilis strains168 trpC2 Laboratory stockGP67 trpC2 mgsA::tet LFH→168, this workGP69 trpC2 crh-3¥FLAG spec pGP1188→168, this workGP78 trpC2 crh-3¥FLAG spec lacA::[PdegQ36::strep mgsA, aphA3] pGP2207→GP69, this workGP79 trpC2 crh-3¥FLAG spec lacA::[PdegQ36::strep aphA3] pGP1459→GP69, this workGP82 trpC2 DhprK::cat LFH→168, this workGP83 trpC2 DhprK::cat crh-3¥FLAG spec lacA::[PdegQ36-strep mgsA, aphA3] GP82→GP78, this workGP91 trpC2 mgsA::tet crh-3¥FLAG spec GP69→GP67, this workGP858 trpC2 hprK::aphA3 Singh et al. (2008)GP860 trpC2 crh::aphA3 QB7096→168, this workQB7096 trpC2 crh::aphA3 Presecan-Siedel et al. (1999)QB7097 trpC2 crh::spec Singh et al. (2008)QB7102 trpC2 crh::aphA3 ptsH1 Galinier et al. (1998)

E. coli strainsBL21(DE3) F- ompT lon gal dcm hsdSB(rB

- mB-) l(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Laboratory stock

BTH101 F- cya-99 araD139 galE15 galK16 rpsL1 (StrR) hsdR2 mcrA1 mcrB1 Karimova et al. (1998)DH5a f80d lacZDM15, recA1, endA1, gyrA96, thi-1, hsdR17 (rK

-, mK+), supE44, relA1, deoR,

D(lacZYA-argF) U169Laboratory stock

FW102 CSH142 strepR/F′ Kan OL2-62 lac Dove and Hochschild (2004)NM522 supE thi D(lac-proAB) hsd5 (r–, m–) [F′, proAB lacIq lacZDM15] Laboratory stock



0.8 mA cm-2). Polyclonal rabbit antisera directed againstGapA (Meinken et al., 2003), HPr (Monedero et al., 2001),the Strep-tag (Hames et al., 2009) and the Flag-tag (Sigma-Aldrich) were used to detect these proteins using dilutions of1:25 000 (anti-GapA), 1:10 000 (anti-HPr and anti-FLAG) and1:1000 (anti-Strep) respectively. The antibodies were visual-ized with goat anti-rabbit IgG secondary antibodies conju-gated to alkaline phosphatase diluted 1:100 000 (Promega)and the CDP* detection system (Roche Diagnostics).

BTH assays

Details on the BTH systems used in this study are providedunder Supporting information.

Phosphorylation of Crh and HPr by HPrK/P in vitro

Information on preparation of phosphorylated Crh-His6 andHis6-HPr proteins is provided under Supporting information.

Table 2. Plasmids used in this study.

Name Relevant structure Reference

p25-N Plac::MCS, cyaA 1–224 (T25), neo, ori p15A Claessen et al. (2008)pAC lcI lcI under PlacUV5 control, cat, ori p15A Dove and Hochschild (2004)pAC lcI-b 831–1057 Encodes fusion of aa 831–1057 of RNAP-b to lcI under PlacUV5 control, cat, ori

p15ADove and Hochschild (2004)

pAG1 crh-his6 under T7 promoter control in pT7-5, bla, ori ColEI Galinier et al. (1997)pAG2 his6-ptsH under T5 promoter control in pQE30, bla, ori ColEI Galinier et al. (1997)pBR a rpoA (encodes RNAP-a) under PlacUV5 control, bla, ori ColEI Dove and Hochschild (2004)pBR a-b 831–1057 Encodes fusion of aa 831–1057 of RNAP-b to RNAP-a under PlacUV5 control, bla,

ori ColEIDove and Hochschild (2004)

pBR a-s70 D581G Encodes fusion of aa 528–613 of s70 (with D581G substitution) to RNAP-a underPlacUV5 control, bla, ori ColEI

Dove and Hochschild (2004)

pFDX4291 Operatorless Ptac-RBSsacB-MCS, cat, ori pSC101 Kalamorz et al. (2007)pGP172 T7 promoter-streptag-MCS, bla, ori ColEI Merzbacher et al. (2004)pGP380 PdegQ36-streptag-MCS, ery, bla, ori pHT1030 (B.s.), ori pUC18 (E.c.) Herzberg et al. (2007)pGP382 PdegQ36-MCS-streptag, ery, bla, ori pHT1030 (B.s.), ori pUC18 (E.c.) Herzberg et al. (2007)pGP642 strep-hprK under PdegQ36 control in pGP380 This workpGP734 crh-strep with RBS of gapA under PdegQ36 control in pGP382 This workpGP744 Encodes fusion of Crh to lcI under PlacUV5 control, cat, ori p15A This workpGP745 Encodes fusion of MgsA to lcI under PlacUV5 control, cat, ori p15A This workpGP750 Encodes fusion of Crh to RNAP-a under PlacUV5 control, bla, ori ColEI This workpGP751 Encodes fusion of MgsA to RNAP-a under PlacUV5 control, bla, ori ColEI This workpGP1151 Encodes fusion of HPr to RNAP-a under PlacUV5 control, bla, ori ColEI This workpGP1152 Encodes fusion of HPr to lcI under PlacUV5 control, cat, ori p15A This workpGP1160 Encodes HPr–T18 fusion in pUT18 This workpGP1161 Encodes T18–HPr fusion in pUT18C This workpGP1164 Encodes Crh–T18 fusion in pUT18 This workpGP1165 Encodes T18–Crh fusion in pUT18C This workpGP1172 Encodes MgsA–T18 fusion in pUT18 This workpGP1173 Encodes T18–MgsA fusion in pUT18C This workpGP1175 Encodes MgsA–T25 fusion in p25-N This workpGP1180 strep-mgsA under PdegQ36 control in pGP380 This workpGP1188 Encodes fusion of 3¥FLAG to C-terminal of Crh (aa 4–85) in pGP1331 This workpGP1301 strep-mgsA under T7-promoter control in pGP172 This workpGP1331 spec, MCS, 3¥FLAG sequence, ori pUC19 Lehnik-Habrink et al. (2010)pGP1459 PdegQ36-streptag-MCS, aphA3 encompassed by lacA-5′ and lacA-3′, bla, ori pUC19 laboratory collectionpGP2207 strep-mgsA under PdegQ36 control in pGP1459 This workpGP2208 Encodes Crh(L14I)–T18 fusion in pUT18 This workpGP2209 Encodes HPr(I14L)–T18 fusion in pUT18 This workpGP2210 Encodes Crh(Q15H)–T18 fusion in pUT18 This workpGP2211 Encodes HPr(H15Q)–T18 fusion in pUT18 This workpGP2212 Encodes Crh(A20T)–T18 fusion in pUT18 This workpGP2213 Encodes HPr(T20A)–T18 fusion in pUT18 This workpGP2214 Encodes Crh(F22L)–T18 fusion in pUT18 This workpGP2215 Encodes HPr(L22F)–T18 fusion in pUT18 This workpGP2216 Encodes Crh(L14I, Q15H, A20T, F22L)–T18 fusion in pUT18 This workpGP2217 Encodes HPr(I14L, H15Q, T20A, L22F)–T18 fusion in pUT18 This workpGP2220 hprK under Ptac control in pFDX4291 This workpKT25 Plac::cyaA 1–224 (T25), MCS, neo, ori p15A Karimova et al. (1998)pKT25-zip Leucine zipper of GCN4 fused to T25 in pKT25 Karimova et al. (1998)pUT18 Plac::MCS, cyaA 225–399 (T18), bla, ori ColEI Karimova et al. (1998)pUT18C Plac::cyaA 225–399 (T18), MCS, bla, ori ColEI Karimova et al. (1998)pUT18C-zip Leucine zipper of GCN4 fused to T18 in pUT18C Karimova et al. (1998)

B.s., B. subtilis, E.c., E. coli; MCS, multiple cloning site; aa, amino acids; RNAP, RNA polymerase; RBS, ribosomal binding site.



Methylglyoxal synthase activity assay

Activity of B. subtilis Strep-MgsA, which was purified fromE. coli BL21 (DE3), was determined spectrophotometricallyby a direct assay described previously (Hopper and Cooper,1972). Briefly, the reaction mixture contained in a volume of0.5 ml of 40 mM imidazole buffer pH 7.5, 3 mM dihydroxyac-etone phosphate, 0.1 mg (1.12 mM) purified Strep-MgsA andvarying amounts (0–6 mg) of Crh-His6 or His6-HPr. After incu-bation at 30°C for 10 min, 100 ml of the reaction was removedand added to a solution containing 0.9 ml of H2O and 0.33 mlof 2,4-dinitrophenylhydrazine reagent (i.e. 0.1% 2,4-dinitrophenylhydrazine in 2 M HCl). After incubation at 30°Cfor 15 min 1.67 ml of 10% (w/v) NaOH was added and theOD555 was determined after an additional incubation at 30°Cfor 15 min. The readings were converted into mmol of meth-ylglyoxal using a molar extinction coefficient of 4.48 ¥ 104.

Methylglyoxal assay

To determine accumulation of methylglyoxal in the medium,cells were grown in minimal medium (Antelmann et al.,2000), which was supplemented with 0.5% of the respectivecarbon sources. Cells were harvested in periodic intervalsand the methylglyoxal concentration was determined by thereaction of methylglyoxal with 2,4-dinitrophenylhydrazine asdescribed (Huynh et al., 2000). The cells were removed bycentrifugation and various volumes (120 ml, 240 ml, 320 ml) ofthe supernatant were incubated with 120 ml of 2,4-dinitrophenylhydrazine (10 mg ml-1 in 2 M HCl) for 15 min at30°C. Thereafter, 560 ml of 10% (w/v) NaOH was added andthe mixture was further incubated for 10 min at roomtemperature. Subsequently, the OD550 was determined. Anabsorbance of 1.64 corresponds to 0.1 mmol of methylglyoxalpresent in the reaction mixture.

Acknowledgements

We thank Sabine Lentes for excellent technical assistanceand Maria Braun and Julia Nawrodt for help with constructionof plasmids. We are grateful to Hinnerk Eilers for the gift ofplasmid pGP1459 and to Katrin Gunka for helpfuldiscussions. Thanks are given to Karin Schnetz for criticalreading of the manuscript. This work was supported by theFederal Ministry of Education (Research SYSMO network) toJ.S. and by grant GO1355/7-1 of the Deutsche Forschungs-gemeinschaft to B.G. J.J.L. was supported by a stipend of theFonds of the Chemische Industrie.

References

Antelmann, H., Scharf, C., and Hecker, M. (2000) Phosphatestarvation-inducible proteins of Bacillus subtilis: proteomicsand transcriptional analysis. J Bacteriol 182: 4478–4490.

Baskaran, S., Rajan, D.P., and Balasubramanian, K.A. (1989)Formation of methylglyoxal by bacteria isolated fromhuman faeces. J Med Microbiol 28: 211–215.

Böck, A., and Neidhardt, F.C. (1966) Properties of a mutant ofEscherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase. J Bacteriol 92: 470–476.

Booth, I.R., Ferguson, G.P., Miller, S., Li, C., Gunasekera, B.,and Kinghorn, S. (2003) Bacterial production of methylgly-oxal: a survival strategy or death by misadventure?Biochem Soc Trans 31: 1406–1408.

Claessen, D., Emmins, R., Hamoen, L.W., Daniel, R.A., Err-ington, J., and Edwards, D.H. (2008) Control of the cellelongation-division cycle by shuttling of PBP1 protein inBacillus subtilis. Mol Microbiol 68: 1029–1046.

Cooper, R.A. (1984) Metabolism of methylglyoxal inmicroorganisms. Annu Rev Microbiol 38: 49–68.

Deutscher, J. (2008) The mechanisms of carbon cataboliterepression in bacteria. Curr Opin Microbiol 11: 87–93.

Deutscher, J., Francke, C., and Postma, P.W. (2006) Howphosphotransferase system-related protein phosphoryla-tion regulates carbohydrate metabolism in bacteria. Micro-biol Mol Biol Rev 70: 939–1031.

Dove, S.L., and Hochschild, A. (2004) A bacterial two-hybridsystem based on transcription activation. Methods Mol Biol261: 231–246.

Favier, A., Brutscher, B., Blackledge, M., Galinier, A., Deut-scher, J., Penin, F., and Marion, D. (2002) Solution struc-ture and dynamics of Crh, the Bacillus subtilis cataboliterepression HPr. J Mol Biol 317: 131–144.

Ferguson, G.P., Tötemeyer, S., MacLean, M.J., and Booth,I.R. (1998) Methylglyoxal production in bacteria: suicide orsurvival? Arch Microbiol 170: 209–219.

Fujita, Y. (2009) Carbon catabolite control of the metabolicnetwork in Bacillus subtilis. Biosci Biotechnol Biochem 73:245–259.

Gaballa, A., Newton, G.L., Antelmann, H., Parsonage, D.,Upton, H., Rawat, M., et al. (2010) Biosynthesis and func-tions of bacillithiol, a major low-molecular-weight thiol inBacilli. Proc Natl Acad Sci USA 107: 6482–6486.

Galinier, A., Haiech, J., Kilhoffer, M.C., Jaquinod, M., Stülke,J., Deutscher, J., and Martin-Verstraete, I. (1997) TheBacillus subtilis crh gene encodes a HPr-like proteininvolved in carbon catabolite repression. Proc Natl AcadSci USA 94: 8439–8444.

Galinier, A., Kravanja, M., Engelmann, R., Hengstenberg, W.,Kilhoffer, M.C., Deutscher, J., and Haiech, J. (1998) Newprotein kinase and protein phosphatase families mediatesignal transduction in bacterial catabolite repression. ProcNatl Acad Sci USA 95: 1823–1828.

Görke, B., and Stülke, J. (2008) Carbon catabolite repressionin bacteria: many ways to make the most out of nutrients.Nat Rev Microbiol 6: 613–624.

Görke, B., Fraysse, L., and Galinier, A. (2004) Drastic differ-ences in Crh and HPr synthesis levels reflect their differentimpacts on catabolite repression in Bacillus subtilis.J Bacteriol 186: 2992–2995.

Hames, C., Halbedel, S., Hoppert, M., Frey, J., and Stülke, J.(2009) Glycerol metabolism is important for cytotoxicity ofMycoplasma pneumoniae. J Bacteriol 191: 747–753.

Herzberg, C., Weidinger, L.A., Dörrbecker, B., Hübner, S.,Stülke, J., and Commichau, F.M. (2007) SPINE: a methodfor the rapid detection and analysis of protein–protein inter-actions in vivo. Proteomics 7: 4032–4035.

Hopper, D.J., and Cooper, R.A. (1972) The purification andproperties of Escherichia coli methylglyoxal synthase.Biochem J 128: 321–329.

Huynh, P.L., Jankovic, I., Schnell, N.F., and Brückner, R.



(2000) Characterization of an HPr kinase mutant of Sta-phylococcus xylosus. J Bacteriol 182: 1895–1902.

Irani, M.H., and Maitra, P.K. (1977) Properties of Escherichiacoli mutants deficient in enzymes of glycolysis. J Bacteriol132: 398–410.

Jault, J.M., Fieulaine, S., Nessler, S., Gonzalo, P., Di Pietro,A., Deutscher, J., and Galinier, A. (2000) The HPr kinasefrom Bacillus subtilis is a homo-oligomeric enzyme whichexhibits strong positive cooperativity for nucleotide andfructose 1,6-bisphosphate binding. J Biol Chem 275:1773–1780.

Juy, M., Penin, F., Favier, A., Galinier, A., Montserret, R.,Haser, R., et al. (2003) Dimerization of Crh by reversible3D domain swapping induces structural adjustments toits monomeric homologue Hpr. J Mol Biol 332: 767–776.

Kadner, R.J., Murphy, G.P., and Stephens, C.M. (1992) Twomechanisms for growth inhibition by elevated transport ofsugar phosphates in Escherichia coli. J Gen Microbiol 138:2007–2014.

Kalamorz, F., Reichenbach, B., März, W., Rak, B., and Görke,B. (2007) Feedback control of glucosamine-6-phosphatesynthase GlmS expression depends on the small RNAGlmZ and involves the novel protein YhbJ in Escherichiacoli. Mol Microbiol 65: 1518–1533.

Kalapos, M.P. (2008) Methylglyoxal and glucose metabolism:a historical perspective and future avenues for research.Drug Metabol Drug Interact 23: 69–91.

Karimova, G., Pidoux, J., Ullmann, A., and Ladant, D. (1998)A bacterial two-hybrid system based on a reconstitutedsignal transduction pathway. Proc Natl Acad Sci USA 95:5752–5756.

Kim, I., Kim, E., Yoo, S., Shin, D., Min, B., Song, J., and Park,C. (2004) Ribose utilization with an excess of mutarotasecauses cell death due to accumulation of methylglyoxal.J Bacteriol 186: 7229–7235.

Lavergne, J.P., Jault, J.M., and Galinier, A. (2002) Insightsinto the functioning of Bacillus subtilis HPr kinase/phosphatase: affinity for its protein substrates and role ofcations and phosphate. Biochemistry 41: 6218–6225.

Lehnik-Habrink, M., Pförtner, H., Rempeters, L., Pietack, N.,Herzberg, C., and Stülke, J. (2010) The RNA degradosomein Bacillus subtilis: identification of CshA as the major RNAhelicase in the multiprotein complex. Mol Microbiol 77:958–971.

Mannervik, B. (2008) Molecular enzymology of the glyox-alase system. Drug Metabol Drug Interact 23: 13–27.

Martin-Verstraete, I., Stülke, J., Klier, A., and Rapoport, G.(1995) Two different mechanisms mediate cataboliterepression of the Bacillus subtilis levanase operon.J Bacteriol 177: 6919–6927.

Meinken, C., Blencke, H.M., Ludwig, H., and Stülke, J. (2003)Expression of the glycolytic gapA operon in Bacillus subti-lis: differential syntheses of proteins encoded by theoperon. Microbiology 149: 751–761.

Merzbacher, M., Detsch, C., Hillen, W., and Stülke, J. (2004)Mycoplasma pneumoniae HPr kinase/phosphorylase. EurJ Biochem 271: 367–374.

Mijakovic, I., Poncet, S., Galinier, A., Monedero, V., Fieulaine,S., Janin, J., et al. (2002) Pyrophosphate-producingprotein dephosphorylation by HPr kinase/phosphorylase:

a relic of early life? Proc Natl Acad Sci USA 99: 13442–13447.

Monedero, V., Poncet, S., Mijakovic, I., Fieulaine, S., Dos-sonnet, V., Martin-Verstraete, I., et al. (2001) Mutationslowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism. EMBO J 20:3928–3937.

Nguyen, T.T., Eiamphungporn, W., Mäder, U., Liebeke, M.,Lalk, M., Hecker, M., et al. (2009) Genome-wide responsesto carbonyl electrophiles in Bacillus subtilis: control of thethiol-dependent formaldehyde dehydrogenase AdhA andcysteine proteinase YraA by the MerR-family regulatorYraB (AdhR). Mol Microbiol 71: 876–894.

Ozyamak, E., Black, S.S., Walker, C.A., Maclean, M.J., Bar-tlett, W., Miller, S., and Booth, I.R. (2010) The critical role ofS-lactoylglutathione formation during methylglyoxal detoxi-fication in Escherichia coli. Mol Microbiol 78: 1577–1590.

Pompeo, F., Luciano, J., and Galinier, A. (2007) Interaction ofGapA with HPr and its homologue, Crh: novel levels ofregulation of a key step of glycolysis in Bacillus subtilis?J Bacteriol 189: 1154–1157.

Postma, P.W., Lengeler, J.W., and Jacobson, G.R. (1993)Phosphoenolpyruvate: carbohydrate phosphotransferasesystems of bacteria. Microbiol Rev 57: 543–594.

Presecan-Siedel, E., Galinier, A., Longin, R., Deutscher, J.,Danchin, A., Glaser, P., and Martin-Verstraete, I. (1999)Catabolite regulation of the pta gene as part of carbon flowpathways in Bacillus subtilis. J Bacteriol 181: 6889–6897.

Ramström, H., Sanglier, S., Leize-Wagner, E., Philippe, C.,Van Dorsselaer, A., and Haiech, J. (2003) Propertiesand regulation of the bifunctional enzyme HPr kinase/phosphatase in Bacillus subtilis. J Biol Chem 278: 1174–1185.

Reichenbach, B., Breustedt, D.A., Stülke, J., Rak, B., andGörke, B. (2007) Genetic dissection of specificity determi-nants in the interaction of HPr with enzymes II of thebacterial phosphoenolpyruvate: sugar phosphotransferasesystem in Escherichia coli. J Bacteriol 189: 4603–4613.

Russell, J.B. (1993) Glucose toxicity in Prevotella ruminicola:methylglyoxal accumulation and its effect on membranephysiology. Appl Environ Microbiol 59: 2844–2850.

Saadat, D., and Harrison, D.H. (1999) The crystal structure ofmethylglyoxal synthase from Escherichia coli. Structure 7:309–317.

Schumacher, M.A., Allen, G.S., Diel, M., Seidel, G., Hillen,W., and Brennan, R.G. (2004) Structural basis for allostericcontrol of the transcription regulator CcpA by the phos-phoprotein HPr-Ser46-P. Cell 118: 731–741.

Schumacher, M.A., Seidel, G., Hillen, W., and Brennan, R.G.(2006) Phosphoprotein Crh-Ser46-P displays alteredbinding to CcpA to effect carbon catabolite regulation.J Biol Chem 281: 6793–6800.

Seidel, G., Diel, M., Fuchsbauer, N., and Hillen, W. (2005)Quantitative interdependence of coeffectors, CcpA and crein carbon catabolite regulation of Bacillus subtilis. FEBS J272: 2566–2577.

Singh, K.D., Schmalisch, M.H., Stülke, J., and Görke, B.(2008) Carbon catabolite repression in Bacillus subtilis:quantitative analysis of repression exerted by differentcarbon sources. J Bacteriol 190: 7275–7284.

Teixeira de Mattos, M., Streekstra, H., and Tempest, D.



(1984) Metabolic uncoupling of substrate level phosphory-lation in anaerobic glucose-limited chemostat cultures ofKlebsiella aerogenes NCTC418. Arch Microbiol 139: 260–264.

Thornalley, P.J. (2008) Protein and nucleotide damage byglyoxal and methylglyoxal in physiological systems – role inageing and disease. Drug Metabol Drug Interact 23: 125–150.

Tötemeyer, S., Booth, N.A., Nichols, W.W., Dunbar, B., andBooth, I.R. (1998) From famine to feast: the role of meth-ylglyoxal production in Escherichia coli. Mol Microbiol 27:553–562.

Tsai, P.K., and Gracy, R.W. (1976) Isolation and character-ization of crystalline methylglyoxal synthetase fromProteus vulgaris. J Biol Chem 251: 364–367.

Warner, J.B., and Lolkema, J.S. (2003) A Crh-specific func-tion in carbon catabolite repression in Bacillus subtilis.FEMS Microbiol Lett 220: 277–280.

Weber, J., Kayser, A., and Rinas, U. (2005) Metabolic fluxanalysis of Escherichia coli in glucose-limited continuousculture. II. Dynamic response to famine and feast, activa-tion of the methylglyoxal pathway and oscillatorybehaviour. Microbiology 151: 707–716.

Willetts, A.J., and Turner, J.M. (1970) Threonine metabolismin a strain of Bacillus subtilis: enzymes acting onmethylglyoxal. Biochim Biophys Acta 222: 668–670.

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