Biochimica et Biophysica Acta 1834 (2013) 1415–1424

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Review

Transcription regulators controlled by interaction with enzyme IIB components of thephosphoenolpyruvate:sugar phosphotransferase system☆

Philippe Joyet a,b, Houda Bouraoui a,b,c, Francine Moussan Désirée Aké a,b, Meriem Derkaoui a,b,Arthur Constant Zébré a,b, Thanh Nguyen Cao a,b, Magali Ventroux a,b, Sylvie Nessler d,Marie-Françoise Noirot-Gros a,b, Josef Deutscher a,b,e,⁎, Eliane Milohanic a,b

a Institut National de la Recherche Agronomique, UMR1319 Microbiologie de l'alimentation au service de la santé humaine (Micalis), F-78350 Jouy-en-Josas, Franceb AgroParisTech, UMR Micalis, F-78350 Jouy-en-Josas, Francec Université El Hadj Lakhdar, Département de Biologie, Batna, Algéried Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC) UMR 8619 CNRS — Université Paris-Sud 11, F-91405 Orsay cedex, Francee Centre National de la Recherche Scientifique, SNC9130 Micalis, F-78350 Jouy-en-Josas, France

☆ This article is part of a Special Issue entitled: Inhibit⁎ Corresponding author at: Avenue Lucien Brétign

Thiverval-Grignon, France. Tel.: +33 1 30 81 54 47; fax: +E-mail address: Josef.Deutscher@grignon.inra.fr (J. D

1570-9639/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbapap.2013.01.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 October 2012Received in revised form 27 December 2012Accepted 4 January 2013Available online 11 January 2013

Keywords:Phosphoenolpyruvate:sugarphosphotransferase systemPTS regulation domainTranscription regulationMembrane sequestrationPTS inhibition

Numerous bacteria possess transcription activators and antiterminators composed of regulatory domainsphosphorylated by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS).These domains, called PTS regulation domains (PRDs), usually contain two conserved histidines as potentialphosphorylation sites. While antiterminators possess two PRDs with four phosphorylation sites, transcriptionactivators contain two PRDs plus two regulatory domains resembling PTS components (EIIA and EIIB). Theactivity of these transcription regulators is controlled by up to five phosphorylations catalyzed by PTS pro-teins. Phosphorylation by the general PTS components EI and HPr is usually essential for the activity ofPRD-containing transcription regulators, whereas phosphorylation by the sugar-specific components EIIAor EIIB lowers their activity. For a specific regulator, for example the Bacillus subtilis mtl operon activatorMtlR, the functional phosphorylation sites can be different in other bacteria and consequently the detailedmode of regulation varies. Some of these transcription regulators are also controlled by an interaction witha sugar-specific EIIB PTS component. The EIIBs are frequently fused to the membrane-spanning EIIC andEIIB-mediated membrane sequestration is sometimes crucial for the control of a transcription regulator.This is also true for the Escherichia coli repressor Mlc, which does not contain a PRD but nevertheless interactswith the EIIB domain of the glucose-specific PTS. In addition, some PRD-containing transcription activatorsinteract with a distinct EIIB protein located in the cytoplasm. The phosphorylation state of the EIIB compo-nents, which changes in response to the presence or absence of the corresponding carbon source, affectstheir interaction with transcription regulators. This article is part of a Special Issue entitled: Inhibitors of Pro-tein Kinases (2012).

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

1.1. The PTS and its catalytic functions

The phosphoenolpyruvate (PEP):sugar phosphotransferase sys-tem (PTS) is a bacterial multi-protein system catalyzing the transportand concomitant phosphorylation of numerous sugars and sugarderivatives (hexoses, pentitols, hexitols, disaccharides, amino- and

ors of Protein Kinases (2012).ières, bâtiment CBAI, 7885033 1 30 81 54 57.

eutscher).

rights reserved.

N-acetyl-aminosugars, gluconic acid, ascorbate, etc.) [1,2]. The twogeneral PTS proteins enzyme I (EI) and HPr and the sugar-specificcomponents enzyme IIA (EIIA) and enzyme IIB (EIIB) form a phos-phorylation cascade [3] (see also Fig. 1, left part). EI autophosphoryl-ates with PEP and passes the phosphoryl group on to His-15 in HPr.P~His-HPr transfers the phosphoryl group to one of usually severalsugar-specific EIIAs present in a bacterium. P~EIIA phosphorylatesan EIIB molecule of the same sugar specificity and in the last stepP~EIIB transfers the phosphoryl group to a sugar molecule bound tothe cognate membrane-spanning enzyme IIC (EIIC). The phosphory-lated sugar is subsequently released into the cytoplasm. The transportprocess is still poorly understood, although the crystal structure of apresumed N,N′-diacetylchitobiose-specific membrane-spanning EIICcomponent has recently been determined [4]. In phosphorylatedPTS components the phosphoryl group is attached either to the

P~

1

2

CAT

RAT

AUG

A: No inducer, no repressing sugar

B: Inducer is present

EIIA

EI

HPr HPr

EIIA

EIIC EIIB

EIIC EIIB

EI

P

~P

~P

~P

PEP Pyruvate

Inducer

P-Inducer mRNA

C: Inducer and repressing sugar are present (CCR)

mRNA

t

RAT

t

~ P~

P~

P~

P~

P~

CAT

1

2

1

2

CAT

P~

1

2

CAT

CAT

1

2

1

2

CAT

RAT

mRNA

t

Fig. 1. Common mechanism of antitermination carried out by PRD-containing transcription activators. Presented are three different conditions of bacterial growth. A: Inducer andrepressor of the transcription unit controlled by the antiterminator are absent. Under these conditions the general and the sugar-specific PTS components shown in the left part ofthe figure are mainly present in phosphorylated form and the antiterminator is phosphorylated at PRD2 by P~His-HPr and at PRD1 by the cognate P~EIIB. Although phosphorylationat PRD2 stimulates the activity of the antiterminator, phosphorylation at PRD1 is dominant and renders it inactive. The terminator (blue) will therefore be preferably formed. Thesequence indicated in red is part of both, the terminator and the RAT. Because under the described conditions the red part is used for the formation of the terminator, the RAT stemloop will not be formed. B: When the inducing carbohydrate is present it competes with the antiterminator for phosphorylation by P~EIIB. Because phosphorylation of sugars is veryfast, EIIB and consequently also PRD1 of the antiterminator will be dephosphorylated. However, there remains enough P~His-HPr in the cell in order to allow efficient phosphor-ylation of the antiterminator at PRD2. Under these conditions the antiterminator will be active and favour formation of the RAT stem loop by binding to the corresponding RNAsequence, thus preventing formation of the terminator. C: When the inducing and a repressing carbohydrate, such as glucose, are present, the rapid uptake and metabolism of glu-cose will also cause poor phosphorylation of P~His-HPr. As a consequence the antiterminator will be dephosphorylated not only at PRD1 but also at PRD2 and therefore be inactive.

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N(ε)-3 position (in EI and EIIAs) or the N(δ)-1 position of a histidine(in HPr and EIIBs of the mannose type PTS). In all other EIIBs thephosphoryl group is bound to a cysteyl residue located in a regionexhibiting similarity to the active site of certain phospho-tyrosinephosphatases. The components of the mannose-type PTS arecompletely different from the proteins of the other PTS families,which are usually more or less conserved. One characteristic of themannose type PTS is that they contain two unrelated integral mem-brane proteins EIIC and EIID and both were found to be necessaryfor sugar transport. Although the EIIA and EIIB components of theascorbate/galactitol PTS superfamily exhibit a certain degree of simi-larity to EIIA and EIIB proteins of other families, their EIIC componentsalso seem to have evolved independently from all other PTS EIICs [5].The PTS components can exist either as distinct proteins, such as inthe Bacillus subtilis lichenan/cellobiose-specific PTS [6], or can befused to another component. For example, the EIIB and EIIC compo-nents are frequently fused to each other and sometimes also EIIAand EIIB; other more extended fusions, such as EIIC–EIIB–EIIA in theB. subtilis glucose transporter, exist as well.

1.2. The regulatory functions of the PTS in enterobacteria

In addition to its catalytic activities, the PTS carries out numerousregulatory functions [3,7,8]. In enterobacteria, EI was found to medi-ate the coordinate utilization of nitrogen and carbon sources. Themetabolic intermediate α-ketoglutarate accumulates under nitrogen

limiting growth conditions and blocks the activity of EI and conse-quently PTS-mediated as well as non-PTS-mediated carbohydratetransport [9]. The low EI activity during nitrogen starvation probablyleads to poor phosphorylation of the other PTS components, includingthe constitutively synthesized glucose-specific EIIA (EIIAGlc). Inenterobacteria, this protein is the central regulator in inducer exclu-sion and carbon catabolite repression. For inducer exclusion,unphosphorylated EIIAGlc interacts with non-PTS permeases andmetabolic enzymes, such as the lactose permease and glycerol kinase,and inhibits their activity [3]. Unphosphorylated EIIAGlc probablyprevails in the cells not only during nitrogen starvation but alsowhen glucose or other rapidly metabolizable PTS or non-PTS carbonsources are taken up. Under these conditions it is the PEP to pyruvateratio that determines the phosphorylation state of EIIAGlc [10]. Induc-er exclusion therefore primarily prevents the uptake and/or metabo-lism of less favourable carbon sources when a preferred carbohydrateis present. There is also evidence that phosphorylated EIIAGlc interactswith adenylate cyclase and thus enhances the formation of thesecond messenger cyclic AMP (cAMP) [11], which forms a complexwith the transcription regulator Crp (cAMP receptor protein), thatstimulates the expression of catabolic genes. This regulatory systemwas proposed to be involved in the phenomenon of diauxie, wherethe simultaneous presence of a preferred and a less favourable carbonsource leads to two growth phases. The preferred carbon source isutilized during the first growth phase, which is followed by a moreor less extended lag phase, before finally the less favourable carbon

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source is metabolized. Adding cAMP to cells growing on glucose andlactose prevented the lag phase or diminished its duration [12].

1.3. The regulatory functions of the PTS in firmicutes

Similar to EIIAGlc in enterobacteria, HPr is the central regulatorprotein of carbohydrate transport and metabolism in firmicutes [3].In addition to its EI-catalyzed phosphorylation at His-15 with PEPthis 10 kDa protein becomes also phosphorylated at Ser-46 in anATP-dependent reaction [13]. The latter modification requires abifunctional enzyme, which also catalyzes the dephosphorylation ofseryl-phosphorylated HPr (P-Ser-HPr) [14]. P-Ser-HPr dephosphory-lation follows an unusual phosphorolysis mechanism, because theenzyme uses inorganic phosphate as substrate and the reaction prod-ucts are HPr and pyrophosphate [15]. The enzyme was thereforecalled HPr kinase/phosphorylase (HprK/P). The two antagonisticactivities of HprK/P are controlled by the intracellular concentrationsof its substrates or reaction products Pi, PPi, ATP and ADP and certainglycolytic intermediates, such as fructose-1,6-bisphosphate (FBP)[16]. The metabolism of glucose or other efficiently metabolizedcarbon sources leads to an increase of the PPi, ATP and FBP concentra-tions, which stimulates the kinase and inhibits the phosphorylasefunction of HprK/P and therefore leads to the transformation ofmore than 60% of the B. subtilis HPr into P-Ser-HPr [17]. P-Ser-HPrplays a major role in carbon catabolite repression by acting asa co-repressor for the catabolite control protein A (CcpA) [18], aLacI type repressor [19]. The P-Ser-HPr/CcpA complex binds tospecific operator sites called catabolite response elements (cre) [20].In firmicutes, these imperfect palindromes are located within orprecede numerous catabolic genes or operons and binding of theP-Ser-HPr/CcpA complex inhibits their transcription. Transcriptomestudies revealed that B. subtilis ccpA inactivation increased theexpression of more than 50 genes and lowered the transcription of35. Similar results were obtained with an hprK mutant, suggestingthat most of these genes are controlled by the P-Ser-HPr/CcpAmechanism [21]. B. subtilis possesses a paralogue of HPr calledcatabolite repression HPr (Crh). Crh can also be phosphorylated atSer-46 [22], but it lacks the His-15 and therefore cannot carry outany catalytic functions in sugar transport and phosphorylation. Itsseryl-phosphorylated form is involved in catabolite repression ofseveral genes and operons [3] and has also been shown to play aspecific role in citM repression during growth on glutamate/succinate[23] whereas unphosphorylated Crh interacts with methylglyoxalsynthase and thus controls the methylglyoxal bypass of glycolysis [24].

In firmicutes, P-Ser-HPr seems to regulate also inducer exclusion. Theuptake of maltose by Lactobacillus casei or of maltose or ribose byLactococcus lactis was found to be immediately arrested when glucosewas added to the transport assay mixture [25–27]. However, mutantsunable to form P-Ser-HPr owing either to inactivation of the hprK geneor replacement of Ser-46 in HPr with an alanine [28] had lost glucose-triggered maltose or ribose exclusion and simultaneously transportedglucose and either one of the less favourable carbon sources. In addition,a L. casei strain producing a mutant HprK/P (Val-267-Phe) which even inthe absence of extracellular glucose converted most of its HPr intoP-Ser-HPr [17] was unable to utilize maltose [29].

HPr and P~His-HPr also carry out several regulatory functions. Forexample, B. subtilis HPr was reported to bind to the glyceraldehyde-3-Pdehydrogenase GapA and to stimulate its activity [30] and to YesS, thetranscription activator of the pectin/galacturonan utilization genes,which is also activated by the interaction with HPr [31]. In firmicutes,P~His-HPr phosphorylates the non-PTS protein glycerol kinase andphosphorylation stimulates its activity about 10-fold [32]. Glycerolutilization in firmicutes therefore also depends on functional EI andHPr. Several non-PTS transporters of firmicutes, such as the lactosetransporter LacS, contain a C-terminal EIIAGlc-like domain and theseproteins are phosphorylated by P~His-HPr. Phosphorylation of LacS

from Streptococcus thermophilus was found to specifically increase thelactose/galactose exchange reaction [33].

2. PRD-containing transcription regulators

2.1. Occurrence of PRD-containing transcription regulators

The most abundant targets of P~His-HPr-mediated phosphoryla-tion are transcription regulators containing two specific domains,each of which usually possesses two conserved histidines as potentialphosphorylation sites for PTS proteins. These domains were thereforecalled PTS regulation domains (PRD). PRDs were so far detectedin two groups of transcription regulators: antiterminators and tran-scription activators. Both usually control the expression of operonscontaining genes encoding PTS proteins and/or catabolic enzymes.PRD-containing transcription regulators are more frequently found infirmicutes than in proteobacteria. For example, B. subtilis strain 168contains 4 antiterminators and 4 transcription activators [34], whereasEscherichia coli K12 contains only one antiterminator [35] and notranscription activator. In proteobacteria, PRD-containing transcriptionregulators are mainly found in the γ-subdivision. None of the bacteriaof the δ- and ε-subdivision, for which the genome sequence hasbeen determined, possesses a PRD-containing regulator. Among theα-proteobacteria only Oligotropha carboxidovorans and among theβ-proteobacteria only Chromobacterium sp. C-61 were found to containone plasmid-encoded PRD-containing antiterminator. In pathogenicbacteria, some of the operons controlled by PRD-containing transcrip-tion activators play a role in their virulence. For example, the tran-scription activator FrzR of the extraintestinal pathogenic E. colistrain BEN2908 controls the expression of genes related to patho-genicity. Deletion of frzR therefore drastically lowered the viru-lence of this organism [36]. In Listeria monocytogenes inactivationof the antiterminator BvrA prevented the inhibitory effect of theβ-glucosides, salicin and cellobiose, on the expression of PrfA-controlled virulence genes [37].

2.2. General characteristics of PRD-containing antiterminators

Antiterminators are usually composed of an N-terminal RNA bind-ing domain (first about 60 amino acids) [38] and two PRDs [39]. Theyinteract with specific inverted repeats able to form a low energy stemloop. These sequences are located in the leader region of mRNAs andthey were called ribonucleotidic antiterminator targets (RAT). [40].The RAT motifs usually overlap with a downstream sequence able toform a ρ-independent terminator (Fig. 1A) [3]. The formation of theterminator was calculated to be energetically more favourable thanthe formation of the RAT stem loop. As a consequence transcriptionstops at the terminator. Among others this has been demonstratedfor the B. subtilis bgl operon. Transcription from the constitutive bglpromoter was found to stop almost completely at the terminatorthus leading to the synthesis of a 110 nucleotide mRNA fragmentunder conditions where the antiterminator was not activated(absence of the inducers salicin or arbutin, Fig. 1A; or CcpA-independent carbon catabolite repression, Fig. 1C) [41]. However,when the antiterminator is active it will bind to the RAT and stabilizethe stem loop, thus preventing the formation of the terminator andallowing transcription of the full length mRNA (Fig. 1B). The mecha-nisms controlling the activity of the antiterminators will be discussedbelow.

2.3. General characteristics of PRD-containing transcription activators

PRD-containing transcription activators are DNA binding proteinspossessing an N-terminal helix-turn-helix motif. According to theirmode of interaction with their DNA target two types of transcription ac-tivators can be distinguished [3]. Regulators containing a domain

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resembling the central domain of NifA/NtrC type transcription activators(also called AAA+ transcription activators) containWalker motifs A andB for nucleotide hydrolysis. For the response regulator NifA nucleotidehydrolysis was found to be necessary for the interaction with and activa-tion of the σ54/RNA polymerase complex [42]. In addition, activationof this complex requires the specific sequencemotif G[A,S]FTGA locatedat the beginning of the central domain [43]. Their DNA binding site canbe located up to 1 kb upstream from the promoter. PRD-containingNifA/NtrC-like transcription activators usually control the expressionof operons encoding various mannose-type PTS, which transport alsoother carbohydrates such as fructose, sorbose, gluconate and pentitols.However, there exist several exceptions to this rule. For example, theL. monocytogenes NifA/NtrC-like regulator Lmo1721 was reported tocontrol the expression of two operons each encoding components of acellobiose-specific PTS belonging to the lactose/cellobiose PTS family[44]. The genetic results suggest that the activity of Lmo1721 is con-trolled by the proteins encoded by one of the cellobiose operons. Thesecond class of PRD-containing transcription activators contains aDeoR-like DNA binding domain, which usually interacts with a palin-dromic sequence locatednotmore than100 bpupstream from the pro-moter. It is followed by a potential second DNA interaction site,whichresembles the N-terminal domain in the Streptococcus pyogenes viru-lence regulator Mga. These transcription activators generally controlthe expression of operons encoding PTS of the fructose/mannitol andthe lactose/β-glucoside families.

While antiterminators contain only two PRDs, transcription activatorspossess in addition two domains resembling PTS components. Theseregulators therefore can contain up to six potential PTS phosphorylationsites. Both, NifA/NtrC- and DeoR-like PRD-containing transcriptionactivators, possess a galactitol EIIB-like domain (EIIBGat) as the thirdregulatory domain. In NifA/NtrC-like transcription activators, whichhave a MW of about 90 kDa, this domain is preceded by PRD1 and anEIIAMan-like domain and followed by PRD2. The EIIBGat-like domaincan sometimes be absent, such as in the protein TepRe1_2536 ofTepidanaerobacter acetatoxydans; or PRD2 can be lacking, such as inthe regulator Cbei_2497 of Clostridium beijerinckii, or be replaced withan EIIAMtl-like domain, such as in the Thermoanaerobacter tengcongensisPspF regulator. In DeoR-like PRD-containing transcription activators,such as MtlR of B. subtilis, the EIIBGat-like domain is preceded by PRD1

PRD1HTH

+

Mga

P

Fig. 2. Hypothetical 3D structure of the MtlR dimer. In fact, yeast two hybrid experiments wifor MtlR [50]. The two subunits are represented as cartoons colored in cyan and magentaC-terminal extremities are labeled accordingly. The model used for each domain representthe sequence of the corresponding MtlR fragment as a query. The HTH-domain (residues 1E. coli (PDB ID: 1BIA, residues 1–67) [82]. The Mga-type DNA-binding domain of MtlR (residMga family transcriptional regulator EFD32_2596 from Enterococcus faecalis (PDB ID: 3SQN;185–405) are represented by the LicT transcriptional antiterminator from B. subtilis (PDB IDfrom the EIIB domain of the E. colimannitol-specific PTS transporter MtlA (PDB ID:1VKR, resnitrogen regulatory protein EIIANtr from E. coli (PDB ID: 1A6J, residues 1–157) [85].

and PRD2 and followed by an EIIAMtl-like domain (Fig. 2). TheC-terminal domain is sometimes lacking, for example in the E. coliBEN2908 FrzR [36].

Owing to the fact that PRDs are present in antiterminators andtranscription activators, numerous annotation errors in sequencedbacterial genomes occurred and transcription activators are frequent-ly referred to as BglG-like antiterminators. These mistakes can easilybe avoided by taking into account the MW, which is usually 30 kDafor antiterminators, but 65 for DeoR-like and 90 to 100 kDa forNifA/NtrC-like transcription activators.

3. Control of PRD-containing transcription regulators

3.1. Regulation of PRD-containing transcription regulatorsby phosphorylation

All PRD-containing transcription regulators studied so far werefound to be controlled via phosphorylation catalyzed by PTS compo-nents [3]. Phosphorylation by the sugar-specific components P~EIIAor P~EIIB leads to an inhibition of the transcription regulator activity.This phosphorylation occurs when no substrate for the correspondingPTS is present. The presence of the substrate leads to induction,because under these conditions the phosphoryl group of P~EIIA orP~EIIB is primarily used for the phosphorylation of the carbohydrateduring its transport and the PRD-containing transcription regulatorwill be dephosphorylated (Fig. 1B). In antiterminators, the inhibitoryphosphorylation usually occurs at one or both conserved histidines inthe first PRD (Fig. 1A) [45]. Only BglG of E. coli was reported to bephosphorylated by its cognate P~EIIB domain at His-207 in PRD2 [46].

The negative phosphorylation site of transcription activators ismore variable. In NifA/NtrC-like regulators phosphorylation usuallyoccurs in the C-terminal PRD2 domain and it is catalyzed by thecognate P~EIIB [47]. In DeoR-like regulators the inhibitory phosphor-ylation site can either be the conserved histidine in the C-terminalEIIAMtl-like domain, which becomes phosphorylated by the corre-sponding P~EIIB (in MtlR of Geobacillus stearothermophilus) [48]; orit can be the conserved cysteine in the EIIBGat-like domain, whichbecomes phosphorylated by the cognate P~EIIA (Cys-419 in MtlR ofB. subtilis) (Fig. 2) [49]. As outlined in the lower part of Fig. 2, MtlR

EIIBGat EIIAMtlPRD2

~H-342 P~C-419

th the entire protein and its two C-terminal domains had suggested a dimeric structure, respectively. The domain borders are highlighted by geometrical forms. The N- ands the best hit from a similarity search performed against the Protein Data Bank using–60) corresponds to the structure of the homologous domain of the BirA protein fromues 80–150) is represented by the structure of the first 75 amino acids of the putativeJ. Osipiuk et al., deposited in PDB in 2011). The PRD1-PRD2 domains of MtlR (residues: 1TLV, residues 54–274) [83]. The EIIB domain (residues 410–505 of MtlR) was takenidues 375–471) [84]. Finally, the EIIA domain (residues 510–685) is represented by the

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probably forms dimers. Indeed, an interaction of MtlR with MtlR wasdemonstrated by yeast two hybrid experiments [50]. Thispresentation also reveals that the negative signal due to phosphoryla-tion at Cys-419 in the EIIBGat-like domain as well as the positive signaldue to phosphorylation of His-342 in PRD2 have to be transmitted allthe way to the N-terminal DNA binding domain. Activation of LicT byP~His-HPr-mediated phosphorylation was indeed accompanied bydrastic structural changes in its PRD2, which probably also lead tostructural rearrangements in the RNA binding domain [35].

In order to be activemost PRD-containing transcription regulators notonly have to be dephosphorylated at the inhibitory regulation site by thecorresponding sugar-specific PTS components, but also need to be phos-phorylated by the general PTS components EI and HPr. In mutants defec-tive in EI or HPr these PRD-containing transcription activators aretherefore inactive. The P~His-HPr-catalyzedphosphorylation is preventedwhen a carbohydrate, such as glucose, is rapidly transported via the PTS.Under these conditions, little P~His-HPr is present in the cells [17], be-cause the phosphoryl group of HPr is rapidly transferred to the carbohy-drate. As a consequence, PRD-containing transcription regulators arebarely phosphorylated by PEP, EI and HPr and remain inactive. This regu-latory phenomenon therefore serves as a CcpA-independent carbon ca-tabolite repression mechanism. However, this mechanism depends onthe formation of P-Ser-HPr, which accumulates in large quantities in B.subtilis cells grown on glucose [17] or other rapidly metabolizable carbonsources [51]. P-Ser-HPr is a very poor substrate for phosphorylation byPEP and EI [52]. A mutant producing HPr in which Ser-46 is replacedwith a non-phosphorylatable alanine did not exhibit CcpA-independentcarbon catabolite repression [41,53]. In some cases, phosphomimetic re-placement of the histidine phosphorylated by P~His-HPr with an aspar-tate also caused a relief from CcpA-independent carbon cataboliterepression [49,54].

In antiterminators, the P~His-HPr-catalyzed phosphorylation occursat one or both conserved histidines in PRD2 [45]. P~His-HPr-mediatedphosphorylation of LicT, the antiterminator controlling the B. subtilisbglPH operon, induces drastic conformational changes [39] which aretransferred via PRD1 to the N-terminal catalytic domain and lead to in-creased affinity for the RAT sequence. NifA/NtrC-like regulators can bephosphorylated by P~His-HPr either at the conserved histidine in theEIIAMan-like domain, as was observed for LevR from B. subtilis [47] andL. casei [55], or at PRD1, as was reported for ManR, the regulator of themannose/glucose PTS in Listeria innocua [56]. P~His-HPr-mediatedphosphorylation in DeoR type PRD-containing transcription activatorsvaries even more. In LicR of B. subtilis, replacement of either one of thefour histidines in the two PRDs prevented LicR activity. However, it isnot knownwhether all four histidines are indeed phosphorylated. Phos-phorylation of MtlR from B. subtilis and G. stearothermophilus byP~His-HPr was prevented when either one of the two conserved histi-dines in PRD2 was replaced with an alanine [48,49]. However, only re-placement of the second histidine with a presumed phosphomimeticaspartate allowed expression from the mannitol promoter in anEI-deficient mutant [49]. This histidine is lacking in MtlR of L. casei andpreliminary data suggest that the L. casei transcription regulator is activewithout phosphorylation by EI and HPr (P. Joyet, unpublished results).

There are a few antiterminators, such as GlcT [57] or SacY [58] ofB. subtilis, which similar to the transcription activator MtlR from L. caseiwere found to be functional in EI- or HPr-deficient mutants; they werecalled PTS-independent antiterminators (pia). A series of B. subtilis licTpia

mutants could be isolated in which LicT was functional without beingphosphorylated by PEP, EI and HPr. Some of these mutants expressedthe bglPH operon constitutively, others remained inducible. However,all of them had lost the CcpA-independent carbon catabolite repressionmechanism operative for the bglPH operon in the wild-type strain [59].These results unequivocally confirmed that the absence of phosphoryla-tion of most PRD-containing transcription activators by P~His-HPr dur-ing the uptake of a rapidly metabolizable PTS carbon source leads tocarbon catabolite repression.

3.2. Regulation of a PRD-containing antiterminator by protein–proteininteraction

In addition to being controlled by phosphorylation catalyzed byPTS proteins, certain PRD-containing transcription regulators werealso found to interact with PTS components. These protein/proteininteractions can have stimulatory or inhibitory effects on the activityof the transcription regulator. The first well established example wasthe E. coli antiterminator BglG. In the absence of salicin it was found tointeract with the EIIB domain of the PTS permease BglF (EIIACBBgl),which renders the antiterminator inactive [60]. In the absence of sal-icin the EIIBBgl domain is mainly phosphorylated. When salicin, thesubstrate for BglF, is present the EIIBBgl domain is dephosphorylatedand BglG is released into the cytoplasm and becomes active. Induc-tion of the bgl operon is therefore controlled by two parallel mecha-nisms: inactivation of BglG is mediated by P~EIIBBgl-catalyzedphosphorylation and by P~EIIBBgl-mediated membrane sequestration.In addition, BglG was reported to require EI and HPr in order to beactive. There are conflicting reports concerning the mechanism onhow EI and HPr activate E. coli BglG. In one study evidence wasprovided for a protein/protein interaction mechanism [61], whereasin another study BglG was reported to be activated by phosphoryla-tion at histidine-208 mediated either by P~His-HPr or P~FruB [62].FruB is a fructose-specific PTS component containing an N-terminalEIIAFru domain and a C-terminal HPr-like domain called FPr, whichis phosphorylated by P~EI at its conserved histidine. P~FPr transfersits phosphoryl group not only to EIIAFru but also to other EIIAs andapparently also to histidine-208 in BglG. However, histidine-208 haspreviously been claimed to be the site for the P~EIIBBgl-mediatedinhibitory phosphorylation of BglG [46], which in a later study wasreported to occur at histidine-101 [63]. These discrepancies mightindicate that the mechanisms regulating BglG activity are not yetfully understood.

3.3. Regulation of PRD-containing transcription activators by protein–protein interaction

Two PRD-containing transcription activators were also reported tobe regulated by interaction with their cognate EIIB domain. One is theDeoR-type regulator MtlR of B. subtilis (Fig. 2), which controls theexpression of the mtlAFD operon, which encodes the mannitol-specific PTS components EIICB and EIIA and the enzyme mannitol-1-P dehydrogenase. The gene encoding MtlR is located about14.5 kb downstream from the mtl operon [64,65]. As already men-tioned, B. subtilis MtlR needs to be phosphorylated by PEP, EI andHPr in PRD2 in order to be active. In contrast, phosphorylation byP~EIIAMtl at the conserved cysteine in the EIIBGat-like domain led tothe inactivation of the transcription activator. MtlR was found to beinactive in an EI-deficient mutant, in which both phosphorylationsare prevented [49,54]. This result suggested that the loss of theactivating effect due to phosphorylation at PRD2 dominates over theloss of the inhibitory effect caused by phosphorylation of the EIIBGat

domain. In agreement with this model, deletion of the EIIAMtl-encoding mtlF gene led to constitutive MtlR activity. However, addi-tional deletion of the EIICBMtl-encoding mtlA gene or of only the3′-mtlA DNA fragment encoding the EIIBMtl domain caused MtlRinactivation [50]. This result suggested that the EIIBMtl domain ofMtlA is required to render MtlR active. Yeast two hybrid studiesindeed revealed an interaction between MtlR and the EIIBMtl domain.This interaction specifically occurs with the two fused C-terminalEIIBGat- and EIIAMtl-like domains [50].

Constitutive MtlR activity could be restored by complementingthe mutants deleted for MtlA or only the EIIBMtl domain of MtlAwith entire mtlA (EIICBMtl). However, MtlR remained inactive whenthese mutants were complemented with only the 3′-mtlA fragment(EIIBMtl domain). Moreover, synthesis of the hydrophilic EIIBMtl

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domain in a wild-type strain prevented induction of the mtl promot-er. Finally, its synthesis in mutants exhibiting constitutive MtlR activ-ity caused a complete loss of MtlR-dependent transcription [50]. Onepossible explanation for these unexpected results was that MtlRneeds to be sequestered to the membrane in order to be active.Overexpression of the 3′-mtlA fragment in wild-type cells leads tothe formation of cytoplasmic and membrane-associated EIIBMtl,which compete for interaction with MtlR. With the cytoplasmicform being overexpressed and therefore more abundant, most ofthe MtlR will also stay in the cytoplasm and only little will be seques-tered to themembrane, which we thought is essential for MtlR activa-tion. In order to test this hypothesis we fused the EIIBMtl domain tothe C-terminus of another integral membrane protein, the B. subtilistyrosine kinase modulator YwqC (also called TkmA) [66]. Comple-mentation of the mutant carrying a deletion of the EIIBMtl domain ofMtlA with a plasmid expressing the allele encoding theYwqC-EIIBMtl fusion protein indeed fully restored constitutive MtlRactivity. The EIICMtl domain plays no or only a minor role in MtlR ac-tivation, because complementation of the mutant lacking EIICBMtl

with the fusion protein caused only slightly lower MtlR activity [50].Similar to the results obtained for BglG, phosphorylation of the

EIIBMtl domain of the PTS permease MtlA seems to prevent the interac-tion with and activation of MtlR. The presumed phosphomimeticreplacement of the phosphorylatable cysteine of EIIBMtl in MtlA or theYwqC-EIIBMtl fusion protein with an aspartate significantly loweredMtlR activity compared to complementation with the wild-type alleles[50]. In addition, in yeast two hybrid experiments, which were carriedout as described in [50], the same phosphomimetic replacement inthe EIIBMtl domain prevented the interaction with the two C-terminaldomains of MtlR (Fig. 3A). In contrast, phosphomimetic replacementsof the phosphorylatable amino acids in the C-terminal EIIBGat- andEIIAMtl-like domains of MtlR had no effect on their interaction withthe EIIBMtl domain (Fig. 3B).

Experiments carried out with a mutant EIIBMtl domain, in which thephosphorylatable Cys-8 had been replacedwith a Ser further supportedthe concept that only the unphosphorylated EIIBMtl domain of MtlAinteracts with MtlR. EIIBMtl(Cys-Ser) has previously been shown tobecome phosphorylated by EI, HPr and EIIAMtl at the serine mutationsite [67,68]. When the soluble B. subtilis EIIBMtl(Cys-Ser) mutantdomain was produced in a B. subtilis mtlF mutant lacking EIIAMtl itinhibited MtlR activity similar to the wild-type protein [50]. Owing tothe absence of EIIAMtl neither wild-type EIIBMtl nor the EIIBMtl(Cys8Ser)mutant protein can become phosphorylated. In a wild-type strain, MtlRactivity is induced by the presence of mannitol (Fig. 4A). As alreadymentioned before, induction of MtlR activity was prevented when the

A

EIIBGat-EIIAMtl EIIBGat(

pGAD

MtlR

pGB

DU

EIIB

Mtl

EIIB

Mtl (

Cys

Asp

)

Fig. 3. Yeast two hybrid protein/protein interaction studies were carried out as previously deas prey (in vector pGAD) and wild-type EIIBMtl and Cys8Asp mutant EIIBMtl as bate (in vector(Cys419Asp)EIIBGat/(His599Asp)EIIAMtl were used as prey and wild-type EIIBMtl as bate. Co

cytoplasmic wild-type EIIBMtl domain was produced (Fig. 4B). It candonate its phosphoryl group tomannitol bound toMtlA and is thereforemainly present in unphosphorylated form. It thus competes withthe EIIBMtl domain of MtlA for binding of MtlR and because it isoverproduced the majority of MtlR will stay in the cytoplasm and littleMtlR will be sequestered to the membrane. In contrast, under the sameconditions the EIIBMtl(Cys8Ser) mutant protein did not inhibit MtlRfunction (Fig. 4C) [50]. The most likely explanation for this result isthat owing to its low phosphoryl group transfer potential theEIIBMtl(Cys8Ser) mutant protein accumulates in phosphorylated formin the cytoplasm and phosphorylated EIIBMtl has apparently lost thecapacity to interact with MtlR. The transcription activator is thereforenormally sequestered to the membrane via the EIIBMtl domain of MtlA(Fig. 4C).

This result strongly supports the concept that only theunphosphorylated EIIBMtl domain interacts with MtlR. A model ofMtlR-mediated control of B. subtilis mtl operon expression is presentedin Fig. 5. It predicts that three conditions need to be fulfilled in order torender MtlR active: a) MtlR needs to be phosphorylated at His-342 byP~His-HPr; b) it needs to be dephosphorylated at Cys-419; andc)MtlR needs to interact with unphosphorylated EIIBMtl, which seques-ters it to the membrane.

The second PRD-containing transcription activator reported tobe regulated by interaction with a PTS component is ManR fromL. monocytogenes. This NifA/NtrC type regulator controls the expres-sion of the man operon, which encodes the PTS components of themain glucose/mannose transporter of this pathogenic organism. How-ever, ManR activity is not controlled by the components of the PTSMan,but by one of the four proteins of a second glucose/mannose-specificPTS called PTSMpo [69]. This PTS is a low efficiency glucose/mannosetransport system [70], which seems to primarily serve as a regulatorof ManR by sensing the glucose/mannose concentration in the environ-ment. Genetic studies suggested that the EIIBMpo component phosphor-ylates His-871 in the PRD2 domain and thereby inactivates ManR [56].Accordingly, a mutant lacking the EIIAMpo protein exhibited strong con-stitutive expression from the man promoter. However, deletion ofEIIBMpo or of both EIIAMpo and EIIBMpo caused a complete loss ofManR activity [70]. This result suggested that EIIBMpo is necessary forManR function and possibly interacts with ManR. Preliminary resultsobtained by carrying out yeast two hybrid experiments suggest thatthere is indeed an interaction between EIIBMpo and ManR (A. C. Zébré,M. Ventroux, M.-F. Noirot-Gros, J. Deutscher, E. Milohanic, unpublishedresults). However, ManR regulation by EIIBMpo certainly differs fromregulation of MtlR by its cognate EIIB. The hydrophilic EIIBMpo is notfused to a membrane integral protein, but can be isolated from the

CysAsp)-EIIAMtl(HisAsp)

B

EIIBGat-EIIAMtl

EIIBGat-EIIAMtl(HisAsp)

EIIBGat(CysAsp)-EIIAMtl

pGAD

MtlR

pGB

DU

EIIB

Mtl

scribed [50]. A: MtlR and its two C-terminal EIIBGat- and EIIAMtl-like domains were usedpGBDU). B: MtlR, wild-type EIIBGat/(His599Asp)EIIAMtl, (Cys419Asp)EIIBGat/EIIAMtl andntrol experiments with the empty vectors pGAD and pGDBU are also presented.

Membrane

MtIR activeMtIR inactive

EIIB

EIIBEIIB EIIB

EIIB

EIIB

EIIBEIIB

EIIBEIIB

EIIB

EIIBEIIB

EIIBEIIB

MtIR active

out

inMtIR

MtIR Cys

CysCys Ser-P

Ser-P

Ser-P

Ser-P

Ser-P

Ser-PSer

Cys

Cys~PCys~P

CysCys

MtIR

MtIR

EIIB EIIB EIIB

EIICMtl EIICMtl EIICMtl

Fig. 4. Presumed mechanism responsible for the effects exerted by cytoplasmic wild-type and Cys8Ser mutant EIIBMtl on MtlR activity. A: When mannitol is present in the medium,the EIIBMtl domain of MtlA in a wild-type B. subtilis strain is dephosphorylated and sequesters MtlR to the membrane, which is one of the conditions for its activation. B: Whensoluble cytoplasmic EIIBMtl is produced in the wild-type strain, it can interact with MtlA and donate its phosphoryl group for mannitol phosphorylation. Cytoplasmic EIIBMtl is there-fore present mainly in dephosphorylated form and competes with the EIIBMtl domain of membrane-integrated MtlA for binding of MtlR. Since soluble EIIBMtl is expressed from astrong promoter located on a plasmid it is more abundant than MtlA and MtlR is barely sequestered to the membrane. C: Cys8Ser mutant EIIBMtl can be phosphorylated by EI, HPrand EIIAMtl [67,68]. In an mtlF deletion mutant, in which cytoplasmic Cys8Ser mutant EIIBMtl cannot be phosphorylated owing to the absence of EIIAMtl, its production inhibits MtlRsimilar as observed for wild-type EIIBMtl [50]. However, when produced in a B. subtilis wild-type strain cytoplasmic Cys8Ser mutant EIIBMtl has no inhibitory effect on MtlR. It islikely that owing to the low phosphoryl group transfer potential phosphorylated Cys8Ser mutant EIIBMtl accumulates in the cytoplasm. The phosphorylated protein exhibits prob-ably only low affinity for MtlR, which is therefore sequestered to the membrane via the unphosphorylated EIIBMtl domain of MtlA and thus becomes active.

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cytoplasmic fraction. Membrane sequestration therefore does not seemto play a role in ManR activation by EIIBMpo.

3.4. Regulation of a PRD-less repressor by interaction with a PTS EIIBdomain

Regulation of transcription regulators by interaction with an EIIBdomain or protein is not restricted to PRD-containing regulators.The E. coli Mlc protein, a repressor and member of the ROK(repressors, open reading frames, and kinases) family, controls theexpression of several genes encoding proteins related to carbonmetabolism, such as ptsG, ptsHI-crr (encode EI, HPr and EIIAGlc),malT (encodes a positive regulator of the maltose regulon) and themannose operon. Mlc was found to be sequestered to the membraneby interaction with the unphosphorylated EIIB domain of theglucose-specific PTS component PtsG (EIICBGlc) [71–73]. This

ActiveMemb

EIIBGat EIIAMtlMtlR

P~H-342+

C-419 rane

MtlP

PEP

EI

P~EI P~HPr

HPr EIIAMtl

P~EIIAMtl

Mtl-1-Pin out

Pyruvate HPr EIIAMtlEIICEIIB

EIICEIIB

Fig. 5. Model of B. subtilis MtlR activation mediated via phosphorylation by or interac-tion with PTS components. When B. subtilis cells grow in the presence of mannitol, thehexitol-specific PTS components are dephosphorylated because their phosphorylgroup is used for mannitol phosphorylation and MtlR is therefore no longer inactivatedby phosphorylation at Cys-419. In contrast, cells contain sufficient P~His-HPr for thephosphorylation of His-342 in PRD2, which is required for activation of MtlR. In addi-tion, the unphosphorylated EIIBMtl domain of MtlA interacts with the two C-terminaldomains of MtlR and sequesters the regulator to the membrane. When the EIIBMtl do-main of MtlA becomes phosphorylated it probably exhibits only very low affinity forMtlR. In conclusion, three conditions need to be fulfilled to activate MtlR: Phosphory-lation of His-342, dephosphorylation of Cys-419 and EIIBMtl-mediated sequestrationto the membrane.

interaction leads to structural changes lowering the affinity of Mlcfor its target sites [74], and Mlc-controlled genes are thereforeexpressed. Similar to the activation of MtlR, inactivation of Mlcseems to be achieved by membrane sequestration and not by the in-teraction with the EIIBGlc domain [71]. The EIIBGlc domain isdephosphorylated when glucose is present in the medium andtransported via PtsG. In the absence of glucose the EIIBGlc domain isphosphorylated and no longer interacts with Mlc, which is thereforereleased into the cytoplasm and exerts its repressor function.

3.5. The different mechanisms controlling transcription regulators viaEIIB interaction

The above described results show that the control of transcriptionregulators by interaction with an EIIB domain can follow quite differ-ent mechanisms. The different modes of regulation found for the fourpresently known transcription regulators controlled by EIIB interac-tion are summarized in Fig. 6. The E. coli antiterminator BglG isinactivated by membrane sequestration mediated by P~EIIBBgl; it isreleased into the cytoplasm and activated when the substrate salicinis present in the medium, which leads to P~EIIBBgl dephosphorylation.Regulation of the E. coli repressor Mlc follows a membrane sequestra-tion mechanism resembling that of BglG. However, Mlc is sequesteredto the membrane via unphosphorylated EIIBGlc, which is mainlyformed when glucose is present in the medium. In the absence ofglucose EIIBGlc is present primarily in phosphorylated form and Mlcis released into the cytoplasm. Owing to the repressor function ofMlc the effects of membrane sequestration on gene expression areantagonistic to those of BglG.

In contrast to BglG and Mlc, B. subtilis MtlR is activated bymembrane sequestration mediated by its cognate EIIBMtl. In theabsence of mannitol, intracellular P~EIIBMtl prevails, which probablydoes not interact with MtlR. MtlR is therefore released into thecytoplasm. In the presence of mannitol, P~EIIBMtl is dephosphorylatedand sequesters MtlR to the membrane. Interaction with the mem-brane and not with EIIBMtl seems to be essential for MtlR activation.Finally, ManR is regulated by interaction with EIIBMpo by a mecha-nism identical to that of MtlR activation. However, the hydrophilicEIIBMpo is not fused to an integral membrane protein, but is a

Fig. 6. Comparison of the different modes of control of transcription regulators by interaction with an EIIB PTS component. Presented are the details of the control mechanisms forthe antiterminator BglG and the repressor Mlc of E. coli, ManR of L. monocytogenes and MtlR of B. subtilis. For further details see the text in chapter 3.5.

1422 P. Joyet et al. / Biochimica et Biophysica Acta 1834 (2013) 1415–1424

cytoplasmic component. ManR is therefore most likely activated byinteraction with EIIBMpo and not by membrane sequestration.

For completeness it should be mentioned that membrane seques-tration of transcription regulators is not only mediated by EIIBcomponents of the PTS. The E. coli transcription activator MalT,which controls the expression of the maltose regulon, is inhibitedby binding to the ATP binding cassette protein MalK [75]. MalT inac-tivation occurs when no maltose is present in the medium andrequires not only MalK but the entire ABC transporter complexMalFGK2. In fact, inhibition of MalT is mediated by membrane seques-tration [76]. In the presence of maltose MalT is released into thecytoplasm and functions as transcription activator.

4. In search of inhibitors for PTS components

4.1. Peptide inhibitors for EI

Phosphotransferase systems functional in sugar transport were sofar detected in numerous bacteria, including important pathogens, aswell as in a few archaea [77], but seem to be absent from plants andanimals. The PTS therefore represents an ideal target for antimicrobialagents, especially when taking into account their catalytic function incarbon source supply and their pleiotropic regulatory role in impor-tant cellular processes. Inactivation or inhibition of PTS componentscan therefore diminish the virulence of a pathogen [78]. Indeed,attempts have been made to isolate peptide inhibitors interfering inthe EI–HPr interaction. The C-terminal domain of EI binds PEP,whereas the N-terminal domain contains the intermediary phosphor-ylated histidine and the interaction site for HPr. Peptides interactingwith EI were identified by using combinatorial cellulose-boundpeptide libraries [79] or phage display [80]. Most of the peptides iden-tified by these means contained a histidine preceded or followed byan arginine in the first or second position. Some of them thereforestrongly resembled the histidine phosphorylation site in HPr(His-Ala-Arg). All these peptides became phosphorylated by PEPand EI. Interestingly, a few peptides interacting with EI contained aCys residue instead of a histidine [80]. They also became phosphory-lated by PEP and EI. This was surprising, because EI is known to trans-fer its phosphoryl group only to histidines in its protein substrates.Only most EIIAs have been reported to phosphorylate cysteyl residuesin their cognate EIIB components. The identified peptide inhibitors forEI exhibited IC values as low as 30 μM, which is almost as low as theKM value of EI for its natural substrate HPr.

In another study the binding of peptides derived from EI ofStreptomyces coelicolor to HPr of the same organism was determinedby using nuclear magnetic resonance and isothermal titrationcalorimetry techniques. Several peptides exhibiting KD values for

their interaction with HPr between 5 and 15 μM were identified.They all showed antimicrobial activity against a S. coelicolor strain[81].

4.2. PRD-containing transcription regulators, powerful tools for detectingPTS inhibitors

PRD-containing transcription regulators and their target genes canprovide a powerful tool for the identification of peptide and non-peptide inhibitors of PTS components. Specifically those regulators,which do not require activation by phosphorylation catalyzed by PEP,EI andHPr, seem to bewell suited. As explained before, this type of tran-scription regulators is still phosphorylated by their cognate P~EIIA orP~EIIB proteins and this phosphorylation renders them inactive.Interrupting the phosphoryl transfer by a chemical compound at anystep of the phosphorylation cascade will lead to constitutive expressionfrom the corresponding promoter. The transcription-stimulating effectcan be visualized by fusing an easily detectable reporter gene, such asthe gene encoding the green fluorescence protein, to the promoter.Cells constitutively synthesizing the green fluorescence protein or anyother reporter enzyme can easily be identified. This approach willallow detection of inhibitors not only for EI and HPr, but also for someof the sugar-specific PTS components.

Acknowledgement

This research was supported by the Agence National de laRecherche, grant number ANR-09-BLANC-0273-01.

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