Characterization of MtfA, a Novel Regulatory Output Signal Proteinof the Glucose-Phosphotransferase System in Escherichia coli K-12

Anna-Katharina Göhler, Ariane Staab, Elisabeth Gabor, Karina Homann, Elisabeth Klang, Anne Kosfeld, Janna-Eleni Muus,Jana Selina Wulftange, and Knut Jahreis

Department of Biology and Chemistry, University of Osnabrück, Osnabrück, Germany

The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In additionto its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physio-logical status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICBGlc is primarily regulatedvia the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICBGlc,Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can alsobe inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the bindingsite for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, wedemonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICBGlc, by using in vivotitration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino4-nitroanilide substrates and show Mlc’s ability to enhance this activity. Based on our findings, we propose a model of MtfA as aglucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as theyenter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signalthat responds to changes in environmental conditions.

Despite the fact that Escherichia coli K-12 has been an impor-tant model organism for many decades, the function of

�20% of all putative open reading frames in its genome remainsunknown. Moreover, in many cases, no obvious sequence similar-ities with previously characterized proteins exist. The mtfA gene(formerly named yeeI), a monocistronic gene located at 44.1 minin the E. coli chromosome, has clearly belonged to this group.Intensive sequence similarity searches revealed that orthologs ofMtfA (mnemonic for Mlc titration factor A) exist in more than100 proteobacteria of the alpha, beta, and gamma subdivisions. Ina previous report (2), we were able to demonstrate that E. coliMtfA is a cytoplasmic protein that binds to the carboxy-terminalpart of the Mlc repressor protein (mnemonic for “making largecolonies” [gene, dgsA]) with a very high affinity. Mlc is one of theglobal regulators of carbohydrate metabolism in E. coli and is es-pecially involved in the regulation of the ptsG gene expression,which encodes the glucose transporter EIICBGlc. The EIICBGlc

together with the cytoplasmic protein EIIAGlc, encoded by thecrr gene (as part of the ptsHI-crr operon), forms the glucose-specific phosphoenolpyruvate (PEP)-dependent carbohydrate-phosphotransferase system (glucose-PTS). Both proteins takepart in a phosphorylation cascade, which begins with an auto-phosphorylation reaction of the so-called enzyme I (EI; gene, ptsI)at the expense of PEP. The phosphate group is subsequently trans-ferred from EI to the phosphohistidine carrier protein HPr (gene,ptsH) and then to the sugar-specific enzymes II and finally to thecarbohydrate (for a review, see reference 23). In addition to itstransport and carbohydrate phosphorylation functions, theglucose-PTS in enteric bacteria has a central regulatory role incarbon catabolite repression and inducer exclusion (reviewed inreferences 7 and 15). Many recent studies have revealed that theregulation of ptsG expression is very complex and takes place atboth the transcriptional and the posttranscriptional levels (re-viewed in references 3 and 12). In addition, there also exists a

direct regulation of transport activity that prevents intracellularhexose phosphate stress (10, 32). In the absence of glucose, ptsGexpression is repressed by Mlc. In the presence of glucose, dephos-phorylated EIICBGlc, which is generated during glucose uptake,binds Mlc and sequesters the repressor away from its DNA-binding sites (13, 16, 28, 34; for reviews, see references 3 and 21).Hence, in contrast to other regulatory systems, there is no intra-cellular low-molecular-weight inducer for Mlc. The EIICBGlc/Mlcsystem is considered to be an important glucose sensor in entericbacteria (13, 34), since Mlc is also involved in the glucose-dependent regulation of the ptsHI-crr operon (20, 29), the malTgene for transcriptional activation of the maltose regulon (6), andthe manXYZ operon encoding the EII proteins of the mannose-PTS (18).

Although MtfA has been shown to interact with Mlc and towork as an antirepressor, its true function has not yet been eluci-dated. We were interested in determining what the cellular role ofMtfA was. Thus, in the first part of this study we further charac-terized the specific interaction between MtfA and Mlc. By gener-ating a collection of MtfA mutants, we identified the Mlc-bindingdomain within this highly conserved protein. Furthermore, wecharacterized the interaction between MtfA and the Mlc*H86Rsuper-repressor, which is no longer capable of interacting with

Received 19 October 2011 Accepted 7 December 2011

Published ahead of print 16 December 2011

Address correspondence to Knut Jahreis, jahreis@biologie.uni-osnabrueck.de.

A.-K. Göhler and A. Staab contributed equally to this article.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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EIICBGlc (27). This allowed us to elucidate differences between theinactivation modes of Mlc by membrane sequestration throughEIICBGlc on the one hand and cytoplasmic titration by MtfA onthe other.

Recently, the crystal structure of the MtfA homologue fromKlebsiella pneumoniae was characterized (doi:10.2210/pdb3khi/pdb). Interestingly, the structure revealed similarities to the zinc-dependent metalloprotease lethal factor (LF) from Bacillus an-thracis, as well as to other metalloproteases of the gluzincin clan.Hence, the second goal of this study was to characterize the pep-tidase activity of MtfA and address the influence of Mlc on MtfA’sproteolytic activity. We present a model that postulates MtfA to bea glucose-regulated peptidase, whose activity is regulated by bind-ing to Mlc available in the cytoplasm, which in turn has beenreleased from EIICBGlc during times when no glucose is taken up.Finally, we speculate about putative biologically relevant MtfAtargets, which may have a function in growth transition underchanging environmental conditions.

MATERIALS AND METHODSMedia and growth conditions. Cells were grown routinely either in Len-nox broth without glucose and calcium ions (LBo) or in 2� TY mediumas described previously (1). Antibiotics were used at the following con-centrations: tetracycline, 10 mg/liter; ampicillin, 50 mg/liter; and chlor-amphenicol, 25 mg/liter.

Bacterial strains and plasmids. All strains used were E. coli K-12 de-rivatives. Table 1 list the genotypes and sources of the relevant bacterialstrains. Information on the plasmids and oligonucleotides is given in thesupplemental material. P1 transduction was performed as described pre-viously (2).

Isolation of chromosomal and plasmid DNA, restriction analysis,PCR, and DNA sequencing. All manipulations of chromosomal or re-combinant DNA were carried out using standard procedures as describedpreviously (1). Plasmid DNA was prepared using QIAprep spin miniprepkit (Qiagen, Hilden, Germany). Restriction enzymes were purchasedfrom New England Biolabs (Schwalbach, Germany) or Fermentas (St.Leon-Rot, Germany) and used according to supplier recommendations.Oligonucleotides for PCR were purchased from Thermo Fisher Scientific(Ulm, Germany). DNA sequencing was commissioned to Scientific Re-search and Development (Bad Homburg, Germany). Alternatively, DNAwas sequenced with an ABI 3100 Avant (Applied Biosystems, Foster City,CA) at the Center for Medical Genetics in Osnabrück. PCRs were per-formed as described by (24) using TaKaRa DNA polymerase from Lonza(Cologne, Germany).

Construction of pTM30mlchis. To purify Mlc, we used PCR to am-plify the corresponding dgsA gene using the oligonucleotides MlcBamHI

and MlcHindIII (see the supplemental material) and genomic DNA of E.coli K-12. Thus, BamHI and a HindIII restriction sites were inserted intothe PCR product. Afterward, the PCR product was cloned in the pTM30expression vector. An amino-terminal His tag was added to the proteinusing His tag-encoding complementary oligonucleotides MlcHis� andMlcHis�. Then, 10 �g of each oligonucleotide was dissolved in 10 �l ofsterile water. After the addition of 2 �l of annealing buffer (GE Healthcare,Munich, Germany) the mixture was heated to 95°C and incubated atroom temperature in a 1-liter beaker with 95°C water until the watertemperature reached 40°C. We then opened the vector pTM30mlc withBamHI and ligated the previously annealed oligonucleotides with thisvector to generate the vector pTM30mlchis. In addition, for the MtfAprotease activity assay, we amplified the dgsA gene using the oligonucleo-tides Mlchis-tev and MlcHindIII (see the supplemental material) and li-gated it into the pTM30 vector to generate the plasmid pTM30mlchis-tev.This plasmid encoded an amino-terminal His-tagged dgsA gene aspTM30mlchis, and a TEV-protease cleavage site between the tag and pro-tein. We treated the purified protein with TEV-protease (AcTEV protease;Invitrogen, Darmstadt, Germany) according to the manufacturer’s in-structions. This resulted in an untagged Mlc protein with an additionalglycine in front of the native amino acid sequence.

Site-directed mutagenesis. We generated defined mutations in themtfA gene using the pTMByeeI-S plasmid (2) and the QuickChange IIsite-directed mutagenesis kit according to the standard protocol (Strat-agene, Amsterdam, Netherlands). In addition, we introduced mutationsinto the dgsA gene with the same kit and using the pTM30mlchis plasmidas a template. The oligonucleotides used are listed in the supplementalmaterial.

Construction of strains JSW1 and JSW2. To characterize the interac-tion between MtfA and Mlc, we inserted a single copy of the dgsA wild-type gene or the dgsA* derivative encoding the Mlc*H86R super-repressormutation (in combination with a cat gene for a chloramphenicol resis-tance selection marker) into the chromosome of JEK5 at the pheU site viasite-directed recombination. In doing so, we applied the RecA-independent recombination mechanism of the conjugative transposonCTnscr94 (11) (the exact procedure is described elsewhere). PCR was usedto confirm correct integration of the desired gene copy.

Western blot analyses and �-galactosidase assays. Bacterial cells har-boring pTMByeeI-S as described previously (2) grown in LBo mediumwere harvested at an optical density at 650 nm (OD650) of 1 by centrifu-gation and resuspended in 100 �l of sterile water and 100 �l of SDS-PAGEloading buffer (125 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate[SDS], 10% glycerol, 5% �-mercaptoethanol, 0.01% bromophenol blue).If not stated otherwise, the samples were heated at 95°C for 10 min. Por-tions (15 �l) of the total cellular proteins were separated by electropho-resis on 0.1% SDS-containing 15% polyacrylamide gels and transferred toa nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Forthe detection of MtfA protein derivatives, we used a penta-His antibody(Qiagen, Hilden, Germany) except in the case of the two hybrid studies,where we used a polyclonal anti-MtfA antibody. Detection of antibody-binding was performed using infrared-labeled second antibodies (LI-COR Biosciences, Bad Homburg, Germany). Visualization and quantifi-cation were performed with an Odyssey infrared imager (LI-CORBiosciences). All �-galactosidase assays were performed as previously de-scribed (2).

Bacterial two-hybrid studies. Bacterial two-hybrid studies were per-formed as described previously (8). The genes mtfA, mtfA(�1– 83), anddgsA were amplified by PCR. PCR products were cloned into the pGEM-Tvector (Promega, Mannheim, Germany) and subsequently transferredinto the vectors pMS604 or pDL804 (8) using a combination of the re-striction enzymes BstEII/XhoI or XhoI/BglII, respectively, or, in the caseof pMM4, PstI/XhoI. Oligonucleotides were used in PCRs as follows:pMM2, BTH7 and BTH8; pDM2, BTH1 and BTH3; pMM3, MlcPX�/�;pDM3, MlcXB�/�; and pMM4, Ymlcpms�/�. All oligonucleotide se-quences are given in the supplemental material.

TABLE 1 Escherichia coli strains used in this study

Strain Relevant genotype or phenotypeSource orreference

JM109 thi-1 �(lac-proAB) recA1 hsdR1 F= traD36proA� B� lacIqZ M15

33

LZ110 LJ110 �lacU169 zah-735::Tn10 tet 34LZ150 LZ110 �(ptsG::cat�) 34JEK5 LZ110 �(dgsA::Tn10 kan) This studyJSW1 JEK5 pheU::dgsA� cat� This studyJSW2 JEK5 pheU::dgsA*(Mlc*H86R) cat� This studyXL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1

lac F= proAB� lacIqZ�M15 Tn10 Tcr

Stratagene

SU202 lexA71::Tn5 su1A211 sulA::lacZ �lacU169 F=lacIq lacZ�M15::Tn9

8

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Protein purification. For purification of MtfA-His5, cells harboringpTMByeeI-S or derivatives were grown in LBo with ampicillin and 500�M IPTG (isopropyl-�-D-thiogalactopyranoside). When culturesreached an OD650 of 3, the cells were harvested by centrifugation. Cellswere subsequently resuspended in buffer A (300 mM NaCl, 2 mM�-mercaptoethanol, 100 mM Potassium phosphate (KPi), 10% glycerol[pH 8.0]) and disrupted by sonication using a Branson Sonifier W-250D(Danbury, CT). Cell debris was removed by low-speed centrifugation(14,000 rpm). We then performed affinity chromatography using theAKTA-FPLC and His-Trap FF columns (GE Healthcare). Elution wasperformed with the buffer described above with an additional 300 mMimidazole. Subsequently, we obtained the samples and dialyzed themagainst 20 mM Tris buffer (pH 8.0). Finally, we stored them at 4°C orsupplemented them with 50% glycerol and stored them at �20°C.

For the purification of Mlc, cells harboring pTM30mlchis were grownin 2� TY or LBo with ampicillin until reaching an OD650 of 0.5, inducedwith 500 �M IPTG, and harvested at an OD650 of 2. Harvesting anddisruption were performed as described previously, using a 50 mM so-dium phosphate (NaPi) (pH 8.0), 300 mM NaCl, and 20 mM imidazolebuffer. For Mlc purification, it was also suitable to use affinity chromatog-raphy as described for MtfA. Elution was performed with a buffer con-taining 50 mM NaPi (pH 8.0), 300 mM NaCl, 500 mM imidazole, and 200�M ZnCl2. Afterward, the sample was dialyzed against HEPES buffer (100mM HEPES [pH 9.0], 25 mM sodium glutamate, 1 mM dithiothreitol)and stored at 4°C.

EMSAs. For the electrophoretic mobility shift assays (EMSAs), thefluorescence-labeled ptsG operator fragment according to (19) encodedby the complementary oligonucleotides ptsG01 (5= labeled with DY782)and ptsG02 (5= labeled with DY682) was annealed as described for the MlcHis tag. Then, 10 �g of bovine serum albumin (BSA), 0.5 �g of herringsperm-DNA as heterologous DNA, 100 nM ZnCl2, and 500 nM MgCl2were added to 1 ng of the operator fragment (4 nM final concentration inthe assay). The stated amounts of Mlc and MtfA were incubated for 10min at room temperature and afterward added to the mixture, which wasthen brought to a final volume of 20 �l using HEPES buffer containing 1�g of BSA/�l. Samples were incubated for an additional 10 min at roomtemperature and loaded on a 5% acrylamide-bisacrylamide gel. We per-formed electrophoresis at a constant voltage of 150 V and detection usingthe Odyssey infrared imaging system (LI-COR Biosciences).

Protease activity assays. A protease assay kit from Calbiochem(Merck, Darmstadt, Germany) was used to determine endoprotease ac-tivity of purified MtfA according to the manufacturer’s instructions. Wedetermined the amino peptidase activities of MtfA by measuring the hy-drolysis of L-alanine 4-nitroanilide, L-arginine 4-nitroanilide, L-proline4-nitroanilide, L-valine 4-nitroanilide, or L-alanine-L-alanine-L-alanine-4-nitronalide (Sigma, Taufkirchen, Germany) and performed an end-point measurement of the chromophoric end product 4-nitroanilide us-ing spectral photometric determination of the absorbance at 390 nm(Shimadzu UV mini 1240 photometer; molar extinction coefficient,11.5 � 106 cm2 mol�1). The assay was carried out in semi-microcuvettes(Sarstedt, Nümbrecht, Germany). We added 100 �g of purified protein to500 �l of 1 mM L-amino 4-nitroanilide. The samples were then filled to afinal volume of 1 ml with Tris-buffer (pH 8.0) and incubated at 37°C for 4h. The absorbance was measured against a blank sample without protein(500 �l of L-alanine 4-nitroanilide with 500 �l of Tris buffer) at 390 nm. Inthe case of hippuryl-L-phenylalanine and hippuryl-L-arginine (Sigma),the absorbance was determined at 254 nm. We conducted a subsequentendpoint measurement, determined the extinction difference (�E), andcalculated the specific activity in U/mg of protein of each preparation. Allspecific activities of purified proteins were compared to a fresh sample ofthe wild-type protein. If indicated, we added the inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride; Applichem, Darmstadt, Ger-many], EDTA (Roth, Karlsruhe, Germany), or phenanthroline (Sigma) inthe denoted concentrations to the assay.

RESULTSMultiple alignments to identify highly conserved amino acids.MtfA is a highly conserved protein that is not only restricted to thegroup of enterobacteria but can also be found in the alpha-, beta-,and gammaproteobacteria subdivisions, as well as in cyanobacte-ria. In order to further characterize the interaction between MtfAand Mlc, we created a multiple alignment of various MtfA pro-teins to identify highly conserved amino acid residues that mightbe of functional importance (Fig. 1). In this alignment, MtfA ho-mologs from 10 different bacteria and covering a broad spectrumof identity were used (39 to 89% identical amino acid residues).All of these bacteria harbor an Mlc homolog protein. Twenty-fiveamino acid residues were identified as absolutely invariant, andanother 32 residues were highly conserved. One important con-served motif is composed of the sequence 149HExxH153, whichcharacterizes zinc-dependent metalloproteases. Interestingly,many invariant amino acid residues were clustered at the carboxy-terminal end of MtfA, indicating another important region of theprotein. Based on this multiple alignment, a collection of highlyconserved amino acid residues were separately substituted by site-directed mutagenesis (marked in gray).

Substitution of conserved amino acids and determination ofprotein stability. To further analyze the interaction betweenMtfA and the repressor protein Mlc, we used site-directed mu-tagenesis to define mutants of MtfA. In order to identify specificeffects of single amino acid residues, we created substitutions withchemically related residues. Western blot analysis was used to testprotein stability of all MtfA derivatives. In almost all cases, a signalwas detected at the expected size of 32 kDa. Only the two deriva-tives with the substitutions I148E (in contrast to I148Y) andA215D (in contrast to A215S) exhibited massive protein decay(data not shown). Thus, compared to the wild type, the majorityof MtfA derivatives provided an equally stable protein, whichcould then be further characterized.

Titration of Mlc by MtfA and its derivatives in vivo. The in-tensity of the interaction between the Mlc and MtfA wild type orits derivatives was tested in vivo using a quantitative measure ofthe �-galactosidase activity of a single-copy ptsGop-lacZ fusion(34). The test system is depicted in Fig. 2. We introduced plasmidsencoding MtfA or its derivatives into the indicated strain back-ground (e.g., dgsA� strain, with deletions of ptsG and lacZ),induced mtfA expression, and measured the strength of Mlctitration by determining �-galactosidase activities. The�-galactosidase activity of completely induced MtfA wild type wasalways used as a control and set to 100%, basal expression levelswere determined using the empty expression vector pTM30(shown by gray columns in Fig. 3). A total of 25 of the 59 MtfAderivatives tested exhibited no or only minor reduction in Mlctitration (at least 80% wild-type activity, white columns in Fig. 3).However, 34 different MtfA mutants exhibited a significantly re-duced Mlc titration (indicated by black columns in Fig. 3). Withinthe amino-terminal part of the MtfA, only the two substitutionsL40E and A70D led to decreased interaction with Mlc. In contrast,a replacement of these two residues by other neutral amino acids(L40A or L40T and A70S, respectively) had almost no effect, indi-cating that negative charges at these positions either interfere withthe protein-protein interaction or with the overall conformationof the protein. Within the carboxy-terminal part of MtfA, manydifferent amino acid substitutions led to substantial changes in the

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FIG 1 Multiple alignment of MtfAEco and ortholog proteins from other bacteria. The amino acid sequence of MtfAEco was aligned with the presented homologuesusing the CLUSTALW program. Abbreviations are as follows: Eco, Escherichia coli gi|90111364; Efe, Escherichia fergusonii gi|22034302; Kpn, Klebsiella pneu-moniae gi|152970975; Sty, Salmonella enterica serovar Typhimurium gi|16420534; Ype, Yersinia pestis gi|22126457; Cvi, Chromobacterium violaceum ATCCgi|34104211; Psy, Pseudomonas syringae pv. tomato DC3000 gi|28851187; Xca, Xanthomonas campestris pv. campestris strain ATCC 33913 gi|21115196; Rso,Ralstonia solanacearum GMI 1000 gi|17428198; Npu, Nostoc punctiforme PCC 73102 gi|23129869. Identical residues are marked by an asterisk (�), highlyconserved residues are marked by a colon (:), and conserved residues are marked by a period (.). Numbers of identical residues in binary comparisons betweenE. coli MtfA and the respective homologues of all other bacteria are given in the box. Substituted amino acids are shaded in gray.

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interactions with Mlc. In particular, the two regions from posi-tions 132 to 149 and positions 214 to 243 seemed to be essential forthe binding of Mlc. Due to PCR artifacts, we found eight deriva-tives with an additional mutation next to the substitution of in-terest. In cases where strong effects were present, we reintroducedthe conserved amino acid residue to study the impacts of eachsingle substitution. In the case of the D156N/D161E double mu-tant, the latter mutation suppressed the phenotype of the first. Inthe Y205S/A207D double mutant, the decreasing interaction withMlc appeared to be due to a cumulative effect, since each singlemutation exhibited higher �-galactosidase activity. In the case ofthe V216L/L217I double mutant the decrease in interaction seemsto be caused by the latter change, because the single V216L muta-tion had almost no effect. However, changing V216 to an aspartateresidue again reduced the activity to less than 50%, indicating thatboth residues may be important. For all other double mutants, thesecond substitution did not change the result of the single substi-tution of the conserved amino acid.

The carboxy terminus of MtfA binds Mlc in a bacterial two-hybrid assay. Given the results from the in vivo titration assays,the carboxy-terminal part of MtfA appears to be responsible forthe binding of Mlc. To verify this hypothesis, we used a bacterialtwo-hybrid assay (8). This assay used a chromosomal lacZ geneunder the control of a hybrid lexA operator-promoter region inthe E. coli test strain SU202. One-half of this artificial operator isrecognized by the helix-turn-helix DNA-binding domain of theLexA wild-type repressor, the other half by a LexA* mutant. BothDNA-binding domains are encoded by compatible plasmids andcan be coupled to different proteins of interest. Expression of

these hybrid genes can be induced by IPTG. Only in the case ofcross talk between the two hybrid proteins did the two differentDNA-binding domains of LexA and LexA* come together to re-press lacZ expression, as was seen in the two control plasmidspMS604 (encoding a LexA-Fos-hybrid protein) and pDL804 (en-coding a LexA*-Jun-hybrid protein). As shown in Fig. 4, whenfused to the LexA DNA-binding domain, both full-length MtfAand MtfA�1-83, which is missing the amino-terminal part of theprotein, are able to interact with Mlc fused to LexA*. These resultsclearly demonstrate that the carboxy-terminal part of MtfA is suf-ficient for Mlc binding. Furthermore, as an internal control, wefused Mlc to both different DNA-binding domains of the LexArepressor. Expression of these two hybrid proteins resulted in alow �-galactosidase activity after induction, confirming the pre-viously published dimerization (tetramerization) of Mlc (26).Cells with only one of the two LexA-Mlc hybrid proteins alwaysshowed a very high �-galactosidase activity, indicating a highspecificity of the different LexA DNA-binding domains for theircognate operator sites (data not shown).

Gel shift assays confirm Mlc titration by MtfA. Our in vivoresults indicated that the interaction between MtfA and Mlc pre-vents binding of the repressor to the ptsG operator. In order tofurther characterize this effect, we used EMSAs with purifiedMlc and MtfA proteins. Addition of Mlc to a ptsG operator gene(ptsGop) fragment led to the formation of a repressor-operatorcomplex and thus a mobility shift (Fig. 5a). In contrast, addition ofMtfA to the labeled operator fragment had no effect, indicatingthat MtfA has no DNA-binding properties. Incubation of Mlc andMtfA prior to incubation with the DNA reduced the amount ofthe repressor-operator complex. Increasing amounts of MtfA ledto an increasing fraction of free ptsGop DNA. A supercomplex ofDNA, Mlc, and MtfA was never observed.

MtfA binds to the super repressor Mlc*H86R in vivo and invitro. In order to distinguish between the two currently knowninactivating mechanisms for Mlc, we used an Mlc super-repressor.This Mlc* derivative carries an amino acid substitution of histi-dine 86 to arginine and is unable to bind the unphosphorylated Bdomain of the EIICBGlc (27). Thus, in contrast to the wild type, thesuper-repressor cannot be inactivated by EIICBGlc. The in vivointeraction between the super-repressor Mlc*H86R and MtfA wasinvestigated using a similar system as presented in Fig. 2. In thecase of the Mlc wild type in strain LZ110, overexpression of MtfAcaused a high �-galactosidase activity of the single copy ptsGop-lacZ fusion, as expected (Fig. 6). In contrast, the introduction of adgsA deletion into this strain background (resulting in JEK5) ledto a constitutive �-galactosidase activity. Interestingly, overex-pression of MtfA caused a significant increase of ptsG expressionin the absence of Mlc. Since MtfA does not directly bind to theptsG operator as shown in Fig. 5, this might indicate some as-yet-unknown interaction of MtfA with a different regulator of ptsGexpression. Subsequently, we reintroduced into JEK5 a single copyof either a wild-type dgsA� (giving strain JSW1) or the dgsA* alleleencoding the Mlc*H86R super-repressor (giving strain JSW2).Both repressors strongly reduced the ptsGop-lacZ expression. Re-markably, JSW2 harboring the super-repressor Mlc*H86R exhib-ited almost no basal �-galactosidase activity. Induction ofplasmid-encoded mtfA� in both strain backgrounds resulted inhigh expression of the ptsGop-lacZ fusion, meaning that both wild-type Mlc and Mlc*H86R can be bound and inactivated by MtfA.However, corresponding to the very low basal expression level in

FIG 2 Test system to quantify the MtfA interaction with Mlc in vivo. Thisfigure illustrates the test system for phenotyping MtfA mutants. The strainbackground LZ150F=::�(ptsGop-lacZ) provides a chromosomally encodeddgsA� gene (mlc�), chromosomal deletions of the ptsG and the lacZ genes, anda single copy ptsGop-lacZ fusion on an F= plasmid. MtfA or its derivatives wereintroduced via the IPTG-inducible expression plasmid pTMByeeI-S or deriv-atives thereof, respectively. Since Mlc cannot bind to the EIIBGlc domain in thisstrain, it can only be inactivated by MtfA. Thus, inactivation of Mlc by MtfA isreflected by high �-galactosidase activities.

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the presence of the super-repressor, �-galactosidase activity inJSW2 after induction of the mtfA� strain were always lower com-pared to the wild type.

To further characterize the MtfA interaction with Mlc*H86Rin vitro, we purified the super-repressor and used it in an EMSAexperiment with the ptsGop fragment. In accordance to the in vivotitration data and the results from the band-shift assays with thewild-type repressor, the addition of MtfA to the complex ofMlc*H86R with the ptsGop fragment led to a release of the operatorDNA (Fig. 5b). This seemed to indicate that the inactivationmechanisms of Mlc by unphosphorylated EIIBGlc and MtfA differsignificantly.

MtfA shows amino-peptidase activity. The MtfA crystal struc-ture from Klebsiella pneumoniae was recently characterized (doi:10.2210/pdb3khi/pdb). A total of 76% of the amino acids areshared by MtfA of K. pneumoniae and E. coli MtfA. Analysis of theoverall MtfA architecture revealed structural similarities to thezinc-dependent metalloprotease lethal factor (LF) of Bacillus an-thracis or to the Mop protein, which is involved in the virulence ofVibrio cholerae (Q. Xu et al., unpublished data). Therefore, wetried to characterize the putative protease activity of E. coli MtfA.Since the natural target and the substrate specificity of MtfA arecurrently unknown, we made use of several relatively unspecificand artificial protease activity assays, which monitor the cleavage

FIG 3 �-Galactosidase activities for the quantification of the interactions between MtfA wild type or derivatives thereof with Mlc in vivo. The results ofphenotyping MtfA and defined mutants with regard to Mlc titration are shown. The test system presented in Fig. 2 was used. Cells harboring the pTMByeeI-Svector or derivatives thereof were grown in LBo, mtfA expression was induced with IPTG, and cells were harvested for �-galactosidase activity assays. The activityof the wild-type control (pTMByeeI-S), which was usually �850 nmol per mg of protein per min, was set to 100% in each set of experiments. The emptyexpression vector pTM30 served as a negative control (represented by gray bars). Mutants, which exhibited �-galactosidase activities of �80% of the wild-typelevel, were drawn in white columns. In contrast, MtfA mutants with �80% activity, which reflected a severe reduction of Mlc binding, are presented by blackcolumns. MtfA mutants K/Q/K/Q and A/A/A denote substitutions H149K/E150Q/H153K/E212Q and H149A/E150A/H153A, respectively. Averages of at leastthree independent tests with standard deviations are given.

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of peptide substrates fused to 4-nitroanilide. Originally, a4-nitroanilide substrate was used to characterize the in vitro activ-ity of LF from B. anthracis (30). Various different L-amino4-nitroanilide derivatives were chosen to look for aminopeptidaseactivity as indicated. The greatest activity and specificity was ob-served for L-alanine fused to 4-nitroanilide, as shown in Fig. 7.Other artificial substrates (arginine, proline, or valine fused to4-nitroanilide, respectively) were cleaved off with significantlylower activities. Moreover, L-glutamic-acid-4-nitroanilide as anacidic amino acid and substrates larger in size, such as L-alanine-

L-alanine-L-alanine-4-nitronalide, were not cleaved at all (datanot shown). Subsequently, hippuryl-L-phenylalanine and hippuryl-L-arginine were used to test for carboxypeptidase activity. Again, onlyvery low activities were obtained for these artificial substrates. Fur-thermore, we tested FTC-casein, a substrate for many endopeptidasesregarding proteolytic conversion by MtfA. In contrast to the artifi-cial aminopeptidase substrates, we did not detect any proteaseactivity after incubation of FTC-casein with purified MtfA (datanot shown). These results suggest that MtfA is an amino- ratherthan a carboxy- or endo-peptidase. Since the highest efficiency of

FIG 4 Bacterial two-hybrid assays to test in vivo interaction of MtfA with Mlc. Cells were grown in LBo without (�) and with (�) IPTG, harvested, and analyzedfor their �-galactosidase activities. Control samples [SU202, test strain without any plasmid as a negative control; pMS (LexA-Fos-hybrid protein)/pDL(LexA�-Jun-hybrid protein) as a positive control] are shown in gray. The results for the analyzed test samples (pMM3 and pDM3, Mlc full-length constructs;pMM2, MtfA full-length construct; pMM4, MtfA�1– 83 construct) are illustrated by the white bars. All test combinations showed reduced �-galactosidaseactivities (given in nmol per min and mg of total cell protein) under inducing conditions, which indicates an interaction of the fusion proteins. Averages of at leastthree independent measurements and standard deviations are given.

FIG 5 EMSAs with purified Mlc and MtfA proteins. (a) A fluorescence-labeled DNA fragment containing the Mlc-binding sites in front of the ptsG gene (ptsGop)was incubated with different combinations of purified Mlc and MtfA and subsequently run on a native gel. A control sample without any protein (�) led to anunshifted signal of the DNA. After incubation of the operator fragment with purified Mlc (�), a retarded Mlc-DNA complex could be detected. The addition ofpurified MtfA (150 nM in the assay) caused no band shift. Incubation of Mlc (usually 25 nM in the assay) with rising amounts of MtfA (usually 25 to 150 nM inthe assay) as indicated prior to the incubation with ptsGop led to an increasing release of operator fragments. (b) Incubation of the ptsG operator fragment withpurified super-repressor Mlc�H86R led to the generation of an Mlc-DNA complex. Similar to the results obtained for the wild-type repressor, addition of MtfAto the band shift assay prior to the incubation with ptsGop led to released operator fragments. (c) Incubation of the ptsG operator fragment with purified wild-typeMlc and increasing amounts of MtfA E150D. (d) Incubation of the ptsG operator fragment with purified wild-type Mlc and increasing amounts of MtfA E150Q(further explanation is given in the text).

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MtfA activity was monitored by cleaving off an amino acidfrom the artificial substrate L-alanine-p-nitroanilide, this sub-strate was used to further characterize the peptidase activity ofMtfA. Subsequently, we tested several protease inhibitors. Ad-dition of the chelators EDTA or phenanthroline significantlyreduced the peptidase activity of MtfA, whereas the addition ofother protease inhibitors (e.g., AEBSF) had much less effect.This finding is consistent with the idea that MtfA is a zinc-dependent protease. Addition of higher amounts of EDTA orphenanthroline caused an irreversible denaturation of MtfA,meaning that zinc-ions are absolutely required to stabilize theprotein structure and keep it in solution. Furthermore, a lack ofcomplete inhibition by the weaker chelator EDTA was previ-ously also observed with other HExxH metalloproteases, suchas PepN (4).

Aminopeptidase activities of MtfA mutants. In the next step,we examined the collection of MtfA mutants for their amino-peptidase activities. All mutants were purified and tested for theirability to cleave off the alanine residue from the L-alanine-p-nitroanilide substrate. Freshly prepared, wild-type MtfA proteinwas always taken as a control and set to 100%. The results for allMtfA derivatives are given in Fig. 8. Mutants with substitutions atpositions H149, E150, H153 (HExxH motif), or E212 displayedclearly reduced protease activities probably due to the fact that thecoordination of the zinc ion, the overall conformation of the pro-tein, and thus the catalytic activity were affected. With respect tothe histidine residues 149 and 153 and their involvement in zinccoordination, a substitution for lysine led to a stronger effect thana replacement by glutamine. A triple mutant with replacements(by alanine) of all three functionally important residues of theHExxH motif was insoluble and could not further be analyzed(data not shown). Substantial changes in the proteolytic activities

were also observed for mutants with substitutions of the two glu-tamate residues at positions 150 and 212. On the basis of the crys-tal structure of MtfA from Klebsiella pneumoniae (MtfAKpn), onewould expect these residues to be involved in catalysis. The sub-stitution E150Q influenced the protease activity most signifi-cantly. In contrast, as shown in Fig. 3, the replacement of thisresidue with aspartic acid or glutamine, respectively, did not affectMlc binding in vivo. Moreover, as illustrated in Fig. 5c and d,purified MtfAE150D or MtfAE150Q proteins are still capable ofcausing a release of the ptsGop fragment in a band-shift assay withpurified Mlc in vitro. These results demonstrate that the two MtfAfunctions of Mlc binding and proteolysis are clearly distinct fromone another. Other substitutions that led to reduced proteolyticactivities but retained Mlc binding include Y94S and P225S. MtfAderivatives with the substitutions S142F, D156N, A207D, V216D,or Y243S, respectively, exhibited both reduced enzyme activityand reduced Mlc binding, which might indicate severe changes instructure and function. Furthermore, replacements of the resi-dues isoleucine 148 with glutamic acid and alanine 215 with as-partic acid resulted in protein instability. Thus, the reduced activ-ities of MtfAI148Y and MtfAA215S may also be caused byenhanced protein degradation.

Mlc influences protease activity. The results described aboveraised the question as to whether Mlc is a substrate for MtfA orwhether it has an influence on its protease activity. Therefore,purified MtfA and Mlc were incubated together in a ratio of 1 to

FIG 6 �-Galactosidase assays for the characterization of the in vivo interactionbetween MtfA wild type and either Mlc wild type or Mlc�H86R super-repressor. The test system was similar to the one described in Fig. 2. LZ110 wasused as dgsA� wild-type control (as indicated by gray bars). JEK5 carries a dgsAdeletion. JSW1 carries a dgsA deletion and a single wild-type copy of dgsA� ata different position in the chromosome. JSW2 carries also a dgsA deletion anda single copy dgsA� gene encoding the Mlc�H86R super-repressor. All strainsharbor the mtfA wild-type expression plasmid pTMByeeIs. Cells were grown inthe absence (�) or presence of IPTG (�) and tested for their �-galactosidaseactivities, which are given in nmol per min and mg of total cell protein. Allactivities are mean values of at least three independent experiments with stan-dard deviations.

FIG 7 Peptidase assays of purified MtfA with different substrates. The activitymeasurements were carried out as described in methods. For L-amino4-nitroanilide substrates, activity was monitored by measuring the change ofthe optical density at 390 nm (�OD390) triggered by the release of4-nitroanilide after cleavage. The specific activity (U/mg·min) was calculated.The highest cleavage rate was reached with L-alanine 4-nitroanilide as sub-strate and set as 100%. The typical activity level under the test conditions forthis substrate was �9 mU per mg of protein and min. Lower activities weredetected for L-arginine 4-nitroanilide as a basic amino acid or for other nonepolar amino acid substrates such as L-proline or L-valine fused to4-nitroanilide. Glutamic-acid 4-nitroanilide as an acidic amino acid oralanine-alanine-alanine 4-nitroanilide (data not shown) could not be cleavedby MtfA. Substrates for carboxypeptidase were also investigated. Neitherhippuryl-arginine nor hippuryl-L-phenylalanine was cleaved sufficiently byMtfA. The cleavage of L-alanine 4-nitroanilide was not strongly inhibited bythe protease inhibitor AEBSF. In contrast, the chelator reagents EDTA andphenanthroline reduced proteolytic activity severely since it is shown in the lastthree columns. All activities are given in percentages relative to the cleavage ofL-alanine 4-nitroanilide. The data are mean values of three independent ex-periments with standard deviations.

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2 (MtfA is known to form dimers, Mlc to form tetramers) andtested for aminopeptidase activity. As illustrated in Fig. 9, theaddition of amino-terminal His-tagged Mlc to the standardassay led to an increased proteolytic activity. The effect waseven more severe with untagged Mlc. The addition of equalamounts of BSA caused no significant effect. The mutant pro-tein MtfAN145D, which displayed a reduced interaction withMlc in vivo (Fig. 3) but almost wild-type proteolytic activity(Fig. 8), was significantly less stimulated by His-tagged Mlc.The presence of some stimulation may have been caused by thein vitro test conditions, which required high amounts of puri-fied protein. Regardless, Mlc can clearly be excluded as a bio-logically relevant substrate.

DISCUSSION

In Escherichia coli K-12, one of the best-studied model organisms,the function of many putative open reading frames remains un-known. These had included mtfA (formerly yeeI), despite the factthat identification of orthologs of the E. coli mtfA gene in morethan 100 proteobacteria of the alpha, beta, and gamma subdivi-sions have implied an important function of this protein. Usingsurface plasmon resonance assays, we were able to demonstrate inan earlier study that MtfA binds with high affinity to the glucoserepressor Mlc (2). Moreover, we could demonstrate that MtfAbinds specifically to the carboxy-terminal segment of Mlc, whichis usually involved in protein tetramerization. In this manner,MtfA among other factors, seems to play an important role in the

FIG 8 Protease activity of MtfA wild type and defined MtfA mutants. MtfA and defined mutants (as indicated) were purified and the aminopeptidase activitiestoward the cleavage of L-alanine 4-nitroanilde were monitored. Each activity was calculated and is diagrammed relative to the activity of the MtfA wild-typeprotein, which was analyzed in parallel. Wild-type activity, which was typically �9 mU per mg of protein and min was set to 100% and is presented by the graybars. The activities of mutant proteins, which showed �80% wild-type activity, are shown by the white columns. In contrast, the black columns represent MtfAderivatives, which showed �80% wild-type proteolytic activity. The MtfA derivative 149/150/153/212 carries the substitutions H149K/E150Q/H153K/E212Q.Standard deviations and average values of at least three independent experiments are indicated.

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regulation of the glucose-PTS. In turn, the glucose-PTS has animportant function in the regulation of carbohydrate metabolismin E. coli. Mlc, for example, not only regulates ptsG gene expres-sion but is also a signal mediator for glucose induction of theptsHI-crr operon (20, 29, 34), the malT gene for the transcrip-tional activator of the maltose regulon (6), the manXYZ operonfor the mannose-PTS (18), and enzymes involved in glycolysis (5),respectively. Interestingly, DNA-binding activity of Mlc in E. coliis not regulated by a small molecular inducer. Instead, Mlc is in-activated by binding to the dephosphorylated EIIBGlc domain ofthe EIICBGlc during glucose transport (13, 16, 28, 34). The local-ization of EIIBGlc at the membrane appears to be essential for thiskind of inactivation mechanism (27), since overproduction of theliberated soluble EIIBGlc domain does not lead to Mlc inactivation(13, 34), although unphosphorylated EIIBGlc alone binds to Mlc,as shown in surface plasmon resonance assays (16). In contrast,membrane-bound fusion proteins consisting of the EIIBGlc do-main in combination with either the lactose permease LacY (27)or the bacteriophage M13 membrane coat protein Gp8 (26) areperfectly able to sequester Mlc. Thus, binding of EIIBGlc to Mlcmight either not cause any conformational change of the repres-sor, or there might be no similar conformational change as causedby MtfA binding. Instead, by anchoring the Mlc tetramer to themembrane via EIICBGlc, the structural flexibility of Mlc mono-mers within the complex becomes restricted, which seems to beresponsible for the loss of its DNA-binding activity (17). Crystalstructure analysis of E. coli Mlc (25) revealed the existence of threefunctional domains: the amino-terminal part containing thehelix-turn-helix DNA-binding motif (D domain), an oligomer-ization domain from residues 195 to 380 (O domain), and aninner EIIBGlc binding domain (E domain). There appears to beonly weak interactions between the three domains and in partic-

ular the O domain is separated from the residual protein by ahighly flexible hinge. However, structural rearrangements of the Odomain remotely affect the conformation of the DNA-bindingdomain by an unknown mechanism (17). In contrast to EIIBGlc,inactivation of Mlc by MtfA occurs in the cytosol and is not mem-brane dependent (2). Tanaka et al. (27) isolated an Mlc super-repressor, which carries an H86R amino acid substitution. Ac-cording to the domain model, Mlc*H86R cannot be inactivated byEIICBGlc. In the present study we could demonstrate that MtfA isstill able to bind and inactivate Mlc*H86R both in vivo and in vitro.This clearly supports the notion that MtfA-Mlc interaction defacto occurs at the C domain of Mlc, which may, in contrast to theEIIBGlc-Mlc interaction, result in either severe changes in the con-formation of the D domain or the prevention of repressor oli-gomerization. Both mechanisms would affect the DNA-bindingactivity of Mlc. Both the presence of a ternary complex in vivo andthat of cooperative binding effects remain uncertain and thus areareas in need of further elucidation.

Using systematic site-directed mutagenesis of conservedamino acid residues in MtfA and subsequent in vivo activity assays,we identified an Mlc interaction domain within the carboxy-terminal part of MtfA. The results of in vivo phenotyping of theinteraction between MtfA and its derivatives with Mlc led to theconclusion that amino acids from residue 132 onward appear tobe necessary for this interaction. Further experiments using a bac-terial two-hybrid assay have also supported this idea. However,two amino acid substitutions (L40E and A70D) in the amino ter-minus also caused a significant reduction in Mlc titration, whereasconserved exchanges at these two positions had no effect. Thismight indicate that there are two classes of amino acid substitu-tion that affect Mlc binding: substitutions of the first class disturbthe overall conformation of MtfA, whereas substitutions of thesecond class directly affect the MtfA-Mlc interaction.

Recently, the crystal structure of MtfA from Klebsiella pneu-moniae (MtfAKpn) was characterized at the Joint Center for Struc-tural Genomics (doi:10.2210/pdb3khi/pdb). A projection of theMtfAEc sequence onto the MtfAKpn structure revealed that at leasteight of the identified amino acid residues in the carboxy terminusare exposed on the surface of MtfA. Furthermore, these residuesare clearly clustered at one side of the protein (Fig. 10). Thus, it ishighly likely that these residues are directly involved in Mlc bind-ing. In contrast, other residues such as L40 or A70 are almost orcompletely buried inside the protein.

The MtfAKpn structure revealed overall similarities to the zinc-dependent metalloprotease Lethal Factor (LF) from Bacillus an-thracis as well as other metalloproteases in the gluzincin clan. Fur-thermore, we previously confirmed the presence of proteolyticactivity of the MtfAKpn protein and the existence of the artificialsubstrate L-alanine 4-nitroanilide (Xu et al., unpublished). In thepresent study, we were able to demonstrate a similar activity forthe E. coli protein. With an assortment of MtfA derivatives, ourresults support the notion of MtfA as a metal-dependent protease.Single substitutions of amino acids involved in zinc coordinationor catalysis of the substrate show, as deduced from the structure,significantly reduced in vitro activities. An MtfA mutant with atriple substitution at positions 149, 150, and 153 (zinc coordina-tion residues) for alanine, could not be investigated in vitro, sug-gesting a structural instability of the residual protein without themetal ion. Further analysis to identify a metal-bound and metal-unbound state by electrospray ionization mass spectrometry

FIG 9 Protease activities of MtfA wild type and MtfAN145D in the absence orpresence of Mlc. The cleavage efficiency of L-alanine 4-nitroanilide by MtfA orMtfAN145D was assayed in the presence or absence of Mlc as indicated. Con-trol samples were carried out incubating MtfA either without any supplementsor with accessory BSA. The addition of purified Mlc with an amino-terminalHis-tagged or addition of an untagged Mlc version stimulated MtfA enzymeactivity up to 175%. In contrast, the MtfA derivative N145D with reduced Mlcbinding capacity was only stimulated to 123% of the wild-type activity. Activityvalues are averages of at least three independent experiments with standarddeviations.

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failed due to irreversible unfolding of MtfA at low pH conditions.However, once again this result points to a metal-dependent pep-tidase activity of MtfA.

These findings now raise questions about the biological rele-vance of the MtfA-Mlc interaction. Mlc is clearly not a target pro-tein. On the contrary, addition of Mlc stimulates MtfA amino-peptidase activity. Preliminary analysis of the MtfKpn structurerevealed the existence of a self-inhibitory complex (Xu et al., un-published data). It is tempting to speculate that Mlc bindingcauses a conformational change in MtfA, which may override self-inhibition. Transcriptome analysis by Lemuth (14) under glucoselimitation revealed a significant upregulation of mtfA expressionunder growth conditions during the transition from the exponen-tial to the stationary phase. According to the Mlc regulationmodel, running out of glucose would lead to a rephosphorylationof EIICBGlc and thus to a release of Mlc into the cytoplasm. Botheffects would boost the overall MtfA protease activity at thisgrowth transition point. This leads to a model according to whichthe glucose sensor Mlc/EIICBGlc not only controls gene expressionby Mlc, but that at least in E. coli it also regulates the proteolyticactivity of MtfA in a glucose-dependent manner. This reflects anovel regulatory output signal by the glucose-PTS.

Questions still remain regarding which proteins are naturaltargets for MtfA. For example, the structurally related endopepti-dase LF of B. subtilis cleaves off the amino-terminal peptides ofmitogen-activated protein kinase kinases (MAPKKs) during hostinfections (9, 31). It is tempting to speculate that MtfA also maycleave off short peptides from its natural target proteins ratherthan working just as an aminopeptidase. In a series of unspecificcross-linking experiments to identify further interaction partnersof MtfA, several putative target proteins were identified (unpub-lished results). Among others, the �-subunit of the ATP synthasewas identified four times by five screening approaches under dif-ferent conditions. Variations in the number of ATP synthase com-plexes might be necessary for growth adaptation under changingenvironmental conditions, making the �-subunit a promising tar-get candidate of MtfA.

In conclusion, by elucidating that MtfA is a glucose-regulated

peptidase, we have identified a novel regulatory output mecha-nism of the glucose-PTS, which may be involved in the adaptationof E. coli cells during the transition from the glucose phase to thenext consecutive growth phase. Since the physiologically relevanttarget is not yet known, further investigation is necessary.

ACKNOWLEDGMENTS

We gratefully acknowledge Kurt Schmid, Hans-Peter Schmitz, Katja Bet-tenbrock, Gabriele Deckers-Hebestreit, Karin Lemuth, Qingping Xu, andJürgen Heinisch for helpful discussions; Katrin Fänger for excellent tech-nical support; and Lucille Schmieding and Jennifer Manne for help withthe manuscript.

This study was financially supported by the German Research Foun-dation (DFG) through the “Sonderforschungsbereich” 431 (TeilprojektP14 to K.J.) and by the German Ministry of Education and Researchthrough the FORSYS-program (grant FKZ 0315285C to K.J.).

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