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J Mol Microbiol Biotechnol 2003;5:206–215DOI: 10.1159/000071072

Transcription Regulators PotentiallyControlled by HPr Kinase/Phosphorylasein Gram-Negative Bacteria

Grégory Boëla Ivan Mijakovica Alain Mazéa Sandrine Ponceta

Muhamed-Kheir Tahab Mireille Larribeb Emmanuelle Darbona

Arbia Khemiria Anne Galinierc Josef Deutschera

aMicrobiologie et Génétique Moléculaire, CNRS UMR2585, Thiverval-Grignon, bUnité des Neisseria,Institut Pasteur, Paris, et cLaboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie,CNRS UPR9043, Marseille, France

Josef DeutscherMicrobiologie et Génétique MoléculaireCNRS UMR2585FR–78850 Thiverval-Grignon (France)Tel. +33 1 30815447, Fax +33 1 30815457, E-Mail jdeu@grignon.inra.fr

ABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com

© 2003 S. Karger AG, Basel1464–1801/03/0054–0206$19.50/0

Accessible online at:www.karger.com/mmb

Key WordsHPr kinase/phosphorylase W Gram-negative bacteria W

Cell adhesion W Two component system W rpoN operon

AbstractPhosphorylation and dephosphorylation at Ser-46 inHPr, a phosphocarrier protein of the phosphoenolpyru-vate:carbohydrate phosphotransferase system (PTS) iscontrolled by the bifunctional HPr kinase/phosphorylase(HprK/P). In Gram-positive bacteria, P-Ser-HPr controls(1) sugar uptake via the PTS; (2) catabolite control proteinA (CcpA)-mediated carbon catabolite repression, and(3) inducer exclusion. Genome sequencing revealed thatHprK/P is absent from Gram-negative enteric bacteria,but present in many other proteobacteria. These organ-isms also possess (1) HPr, the substrate for HprK/P;(2) enzyme I, which phosphorylates HPr at His-15, and(3) one or several enzymes IIA, which receive the phos-phoryl group from PFHis-HPr. The genes encoding thePTS proteins are often organized in an operon with hprK.However, most of these organisms miss CcpA and afunctional PTS, as enzymes IIB and membrane-inte-grated enzymes IIC seem to be absent. HprK/P and the

rudimentary PTS phosphorylation cascade in Gram-neg-ative bacteria must therefore carry out functions differentfrom those in Gram-positive organisms. The gene organi-zation in many HprK/P-containing Gram-negative bacteriaas well as some preliminary experiments suggest thatHprK/P might control transcription regulators implicatedin cell adhesion and virulence. In ·-proteobacteria, hprK islocated downstream of genes encoding a two-componentsystem of the EnvZ/OmpR family. In several other proteo-bacteria, hprK is organized in an operon together withgenes from the rpoN region of Escherichia coli (rpoNencodes a Û54). We propose that HprK/P might control thephosphorylation state of HPr and EIIAs, which in turncould control the transcription regulators.

Copyright © 2003 S. Karger AG, Basel

Introduction

HPr kinase/phosphorylase (HprK/P) is a bifunctionalenzyme catalyzing the phosphorylation at Ser-46 in HPr[Deutscher et al., 1986], a phosphocarrier protein of thephosphoenolpyruvate (PEP):carbohydrate phosphotrans-ferase system (PTS) [Postma et al., 1993], as well as the

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dephosphorylation of P-Ser-HPr [Mijakovic et al., 2002].HprK/P was discovered in Streptococcus pyogenes[Deutscher and Saier, 1983], but the numerous genomesequences which recently became available suggest thatHprK/P occurs in all low G+C Gram-positive organisms.In S. pyogenes, HprK/P was thought to be implicated in aregulatory phenomenon called inducer expulsion [Reizeret al., 1983]. However, recent studies revealed that in lac-tobacilli and lactococci HprK/P is not necessary forinducer expulsion [Dossonnet et al., 2000; Monedero etal., 2001], although it sometimes seems to play an indirectrole in this regulatory process. Nevertheless, HprK/Pturned out to be an important regulatory protein control-ling at least three different cellular processes: (1) sugaruptake via the PTS; (2) catabolite control protein A(CcpA)-mediated carbon catabolite repression (CCR),and (3) inducer exclusion.

To carry out its different regulatory functions, thebifunctional HprK/P controls the intracellular level of P-Ser-HPr by using either ATP [Deutscher and Saier, 1983]or pyrophosphate (PPi) [Mijakovic et al., 2002] to phos-phorylate HPr at Ser-46, and inorganic phosphate (Pi) todephosphorylate P-Ser-HPr [Mijakovic et al., 2002]. TheATP-dependent kinase reaction is stimulated by glycolyt-ic intermediates, mainly fructose-1,6-bisphosphate. Phos-phorylation with both phosphoryl donors is inhibited byPi [Deutscher and Engelmann, 1984; Mijakovic et al.,2002], which was found to bind to the same site (P-loop)as ATP and PPi [Fieulaine et al., 2001]. Pi has also beenreported to stimulate the HprK/P-catalyzed dephospho-rylation of P-Ser-HPr [Deutscher et al., 1985], but wasrecently found to function as substrate and not as stimula-tor for this reaction [Mijakovic et al., 2002]. Duringdephosphorylation, Pi bound to the active site of HprK/Pcarries out a nucleophilic attack on the phosphoryl bondin P-Ser-HPr [Fieulaine et al., 2002] leading to the forma-tion of HPr and PPi (phosphorolysis instead of the usualhydrolysis) [Mijakovic et al., 2002]. P-Ser-HPr dephos-phorylation is therefore the reversal of the PPi-dependentkinase reaction.

If bacilli or lactococci grow on a rapidly metabolizablecarbon source, the intracellular concentrations of ATP,fructose-1,6-bisphosphate and PPi drastically increase,whereas the concentration of Pi is strongly diminished[Mason et al., 1981; Thompson and Torchia, 1984; Neveset al., 1998; Mijakovic et al., 2002]. The metabolism ofglucose, fructose or other well-metabolizable PTS carbonsources therefore favors the formation of P-Ser-HPr.However, P-Ser-HPr is a poor substrate for the PEP-dependent, enzyme-I-catalyzed phosphorylation at His-

15 (more than 100 times more slowly phosphorylatedthan HPr) [Deutscher et al., 1984; Reizer et al., 1992].Phosphorylation of HPr at His-15 is necessary for PTSactivity, as PFHis-HPr transfers its phosphoryl group viaEIIAs and EIIBs to the incoming sugar (fig. 1, upper part).Increasing amounts of P-Ser-HPr slow EIIA phosphoryla-tion (fig. 1, lower part) and eventually prevent PTS-mediated sugar uptake [Monedero et al., 2001]. Bacteriaprobably modulate the PFHis-HPr/P-Ser-HPr ratio insuch a way that they can adjust the sugar uptake rate tothe metabolic rate of the cell thereby preventing an exces-sive accumulation of glycolytic intermediates.

CCR, the second process controlled by HprK/P, regu-lates sugar uptake if more than one carbon source is avail-able. Sugars such as glucose or fructose, which are rapidlymetabolized by most bacteria, prevent the uptake of lessfavorable carbohydrates such as ribose, gluconate or ino-sitol by exerting a repressive effect on the expression ofthe genes encoding the enzymes necessary for the uptakeand metabolism of the less favorable carbon sources. CCRin low G+C Gram-positive bacteria depends on the for-mation of P-Ser-HPr [Deutscher et al., 1994] and there-fore clearly differs from cAMP/Crp-mediated CCR inGram-negative enteric bacteria [Postma et al., 1993]. Asalready mentioned, the efficient utilization of sugars suchas glucose or fructose by Gram-positive organisms favorsthe kinase activity of HprK/P and therefore the formationof P-Ser-HPr, which forms a complex with CcpA[Deutscher et al., 1995], a member of the LacI/GalRrepressor family [Henkin et al., 1991]. The interactionwith P-Ser-HPr allows CcpA to bind to the cataboliteresponse elements cre [Fujita et al., 1995], operator sitespreceding most catabolite-regulated genes and operons inGram-positive bacteria [Nicholson and Chambliss, 1985;Deutscher et al., 2001], thereby inhibiting, or in somecases activating, the expression of the downstream genes[Deutscher et al., 2001].

Similar to CCR, inducer exclusion also leads to theordered uptake of carbohydrates if more than one carbonsource is available. However, this process primarily regu-lates sugar metabolism at the carbohydrate transport stepand not at the gene expression level. For example, addingglucose to Lactobacillus casei or Lactococcus lactis cellshas been shown to immediately stop the uptake of riboseor maltose by the corresponding ABC transporters. P-Ser-HPr was found to be necessary for the inhibitory effect ofglucose on the non-PTS transporters. When the formationof P-Ser-HPr was prevented in the above organisms byeither inactivating hprK or replacing Ser-46 of HPr with anon-phosphorylatable alanine (ptsH1 mutation), glucose

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Fig. 1. Schematic presentation of the PTS phosphorylation cascade.Upper part: To transport and phosphorylate sugars via the PTS,enzyme I catalyzes the PEP-dependent phosphorylation of HPr atHis-15. PFHis-HPr can donate its phosphoryl group to one of thesugar-specific EIIAs. The corresponding EIIBs subsequently transferthe phosphoryl group to the sugar bound to the EIIC (or EIIC/EIIDfor the mannose class PTS). Lower part: The uptake of a rapidlymetabolizable carbohydrate activates the kinase function of HprK/Pleading to the formation of P-Ser-HPr. Phosphorylation at Ser-46

hinders phosphorylation of HPr by PFenzyme I at His-15 as well asthe interaction of HPr with EIIAs [Deutscher et al., 1984; Reizer etal., 1992]. P-Ser-HPr formation therefore drastically slows EIIAphosphorylation. Gram-negative hprK-containing bacteria probablydo not have a complete PTS as they are missing any of the knownEIIBs and EIICs. They only possess the proteins forming the first partof the PTS phosphorylation cascade indicated by the shaded rectan-gle.

lost its inhibitory effect on the uptake of the nonPTS sug-ars [Dossonnet et al., 2000; Monedero et al., 2001]. Dur-ing inducer exclusion in Gram-positive bacteria, P-Ser-HPr probably interacts directly with certain non-PTS per-meases and inhibits their transport activity [Ye et al.,1994].

HPr Kinase/Phosphorylase in Gram-NegativeBacteria

Although the absence of HprK/P from Gram-negativeenteric bacteria such as Escherichia coli and Salmonellatyphimurium originally led to the assumption that HprK/P would occur only in Gram-positive organisms, genomesequencing revealed that HprK/P is also present in manyGram-negative bacteria. Bacteria containing genes codingfor proteins with significant homology to HprK/P ofGram-positive organisms are listed in table 1. They in-clude ·-, ß-, Á- and ‰-proteobacteria, spirochetes, chloro-bia, fibrobacteres and fusobacteria. However, these bac-teria are devoid of CcpA and a functional PTS also seemsto be absent. HprK/P must therefore carry out functions

differing from those in Gram-positive organisms. Inter-estingly, hprK is frequently organized in an operon togeth-er with genes encoding PTS proteins (enzyme I, HPr,EIIAs) and transcription regulators, suggesting thatHprK/P together with the PTS proteins might control theactivity of the regulator proteins.

All Gram-negative bacteria listed in table 1 possess anHPr, in which the sequence around the phosphorylatableseryl residue strongly resembles the corresponding regionin HPr of Gram-positive bacteria (fig. 2). By contrast, theregion around the phosphorylatable histidyl residue ismuch less conserved. HPrs of Gram-negative bacteriawithout an hprK gene also possess a Ser-46, but the sur-rounding sequence is completely different from that inHPr of Gram-positive and hprK-containing Gram-nega-tive organisms. Nevertheless, a phylogenetic analysis withthe full-length proteins revealed that HPrs of Gram-posi-tive organisms are more closely related to HPrs of Gram-negative enteric bacteria (which do not contain hprK)than to HPrs of Gram-negative bacteria possessing hprK(data not shown). Similarly, a phylogenetic tree obtainedwith HprK/Ps showed that the proteins of ·-, ß- andÁ-proteobacteria form clusters clearly separated from

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Table 1. HprK/P in bacteria not belonging to Gram-positive organisms

Organism Classification Relevant gene up- ordownstream of hprKa

Truncated hprK,EI with NifA

Agrobacterium tumefaciens ·-proteobacteria us chvG, ds ptsEIIAMan +Brucella melitensis biovar Abortus ·-proteobacteria us chvG, ds ptsEIIAMan +Brucella melitensis biovar Suis ·-proteobacteria us chvG, ds ptsEIIAMan +Caulobacter crescentus ·-proteobacteria us chvG, ds ptsEIIAMan +Magnetospirillum magnetotacticum ·-proteobacteria ds yhbJ +Mesorhizobium loti ·-proteobacteria us chvG, ds ptsEIIAMan +Novosphingobium aromaticovorans ·-proteobacteria us chvG, ds yhbJ +Rhizobium leguminosarum ·-proteobacteria us chvG, ds ptsEIIAMan +Rhodobacter sphaeroides ·-proteobacteria us chvG, ds yhbJ +Rhodopseudomonas palustris ·-proteobacteria us chvG, ds ptsEIIAMan +Silicibacter pomeroyi ·-proteobacteria us chvG, ds yhbJ +Sinorhizobium meliloti ·-proteobacteria us chvG, ds ptsEIIAMan +

Bordetella bronchiseptica ß-proteobacteria us ptsEIIAFru

Bordetella parapertussis ß-proteobacteria us ptsEIIAFru

Bordetella pertussis ß-proteobacteria us ptsEIIAFru

Burkholderia cepacia ß-proteobacteria us ptsEIIAFru, ds yhbJBurkholderia fungorum ß-proteobacteria us ptsEIIAFru, ds yhbJBurkholderia mallei ß-proteobacteria us ptsEIIAFru, ds yhbJBurkholderia pseudomallei ß-proteobacteria us ptsEIIAFru, ds yhbJNeisseria gonorrhoeae ß-proteobacteria us ptsEIIAFru, ds yhbJNeisseria meningitidis ß-proteobacteria us ptsEIIAFru, ds yhbJNitrosomonas europaea ß-proteobacteria us ptsEIIAFru

Ralstonia metallidurans ß-proteobacteria us ptsEIIAFru

Ralstonia solanacearum ß-proteobacteria us ptsEIIAFru

Acidithiobacillus ferrooxidans Á-proteobacteria us ptsEIIAFru, ds yhbJCoxiella burnetii Á-proteobacteria ds ptsH +Xanthomonas axonopodis Á-proteobacteria us ptsEIIAFru, ds yhbJXanthomonas campestris Á-proteobacteria us ptsEIIAFru, ds yhbJXylella fastidiosa Á-proteobacteria ds yhbJ

Geobacter sulfurreducens ‰-proteobacteria ds yhbJ

Treponema pallidum spirochetes ds ptsHTreponema denticola spirochetes ds ptsHChlorobium tepidum chlorobia us yhbHFibrobacter succinogenes fibrobacteres us yhbHFusobacterium nucleatum fusobacteria ds ytqI B. subtilis

us = Upstream; ds = downstream.

Gram-positive HprK/Ps (fig. 3) [Hu and Saier, 2002].Interestingly, proteobacteria of the ·-subdivision [Hu andSaier, 2002] as well as Coxiella burnetii possess a trun-cated HprK/P missing the N-terminal domain (about 130amino acids), the role of which remains unknown. Whentruncated at about the same position, L. casei HprK/Pretained all its known catalytic and regulatory characteris-tics [Fieulaine et al., 2001], suggesting that the naturallytruncated HprK/Ps are also active. HprK/Ps from the

Gram-negative Neisseria meningitidis and Neisseria go-norrhoeae have been purified and they could phosphory-late their respective HPrs as well as HPr from the Gram-positive organism Bacillus subtilis [Poncet et al., unpubl.].Interestingly, the hprK gene in Fusobacterium nucleatumis duplicated giving rise to an enzyme composed of twoHprK/P entities. However, only the second HprK/Pseems to contain an intact Walker motif A for ATP andPPi binding.

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Fig. 2. Alignment of the sequence aroundthe phosphorylatable histidyl and seryl resi-dues (bold letters) of HPr from the indicatedorganisms. The gap between the two present-ed sequences is 18 amino acids, except forHPr from N. aromarticovorans, where it is20 amino acids. In hprK-containing Gram-negative bacteria, the region around thephosphorylatable serine strongly resemblesthe corresponding region in HPrs of Gram-positive organisms (first consensus se-quence). The sequence around the non-phos-phorylatable Ser-46 of HPr from Gram-neg-ative bacteria devoid of hprK is also wellconserved (second consensus sequence), butclearly differs from that in HPr from hprK-containing Gram-positive and Gram-nega-tive bacteria. The following bacteria havenot been mentioned before: Staphylococcuscarnosus and the Gram-negative bacteriaBorrelia bugdorferi, Haemophilus influen-zae, Pasteurella multicoda, Shewanella pu-trefasciens, Vibrio cholerae and Yersinia pes-tis.

low G+C Gram-positive bacteria B. subtilis Crh LKTGLQARPAALF---18 aa---KVNAKSIMGLMSL B. subtilis HPr ADSGIHARPATVL---18 aa---TVNLKSIMGVMSL E. faecalis AETGIHARPATLL---18 aa---SVNLKSIMGVMSL L. casei AETGIHARPATLL---18 aa---SVNLKSIMGVMSL L. lactis AETGIHARPATLL---18 aa---SVNLKSIMGVMSL S. pyogenes AETGIHARPATLL---18 aa---AVNLKSIMGVMSL S. carnosus DETGIHARPATML---18 aa---KVNLKSIMGVMSL Gram-negative bacteria with hprK A. ferooxidans NRLGLHARPSAKF---18 aa---RVNGKSIMGLMTL A. tumefaciens NKRGLHARASAKF---18 aa---TVGGTSIMGLMML B. pertussis NKLGLHARAAAKL---18 aa---RVNAKSIMGVMML B. melitensis Suis NKRGLHARASAKF---18 aa---TVGGTSIMGLMML B. fungorum NKLGLHARASAKL---18 aa---RINAKSIMGVMML C. crescentus NERGLHARASAKF---18 aa---SVDARSIMGLMML C. burnetii NKLGLHARAAIKL---18 aa---KVNAKSLMCLMVL G. sulfurreducens NKLGLHARASALL---18 aa---EVNGKSIMGIMML M. magnetotacticum NRRGLHARAAAKF---18 aa---EVSGLSIMGLMML M. loti NQRGLHARASAKF---18 aa---KVGGTSIMGLMML N. aromaticovorans NQRGLHARASAKF---20 aa---DANGASILGLMML N. meningitidis NKLGLHARASSKF---18 aa---RVNGKSIMGVMML N. europaea NKLGLHARASAKL---18 aa---RVNAKSIMGVMTL R. leguminosarum NKRGLHARASAKF---18 aa---TVGGTSIMGLMML R. sphaeroides NEKGLHARASAKF---18 aa---VVSGDSIMGLLML R. palustris NKRGLHARASAKF---18 aa---TVGGTSIMGLMML R. metallidurans NKLGLHARASAKL---18 aa---QVDAKSIMGVMML S. pomeroyi NEKGLHARASAKL---18 aa---SASGDSIMGLLML S. meliloti NKRGLHARASAKF---18 aa---TVGGTSIMGLMML X. campestris NRLGLHARATAKL---18 aa---EVNAKSIMGVMLL X. fastidiosa NKLGLHARATAKL---18 aa---EVNAKSIMGVMLL consensus G HAR V SIMG M L Gram-negative bacteria without hprK B. burgdorferi AVNGLHVRPASTF---18 aa---SVSGKSLFRLQTL E. coli APNGLHTRPAAQF---18 aa---SASAKSLFKLQTL H. influenzae ASNGLHTRPAAQF---18 aa---SASAKSLFKLQTL K. pneumoniae APNGLHTRPAAQF---18 aa---SASAKSLFKLQTL P. multicoda APNGLHTRPAAQF---18 aa---SASAKSLFKLQTL S. putrefasciens AKHGIHTRPAALL---18 aa---QASAKSLFKLQTL S. typhimurium APNGLHTRPAAQF---18 aa---SASAKSLFKLQTL V. cholerae AENGLHTRPAAQF---18 aa---SASAKSLFKLQTL Y. pestis APNGLHTRPAAQF---18 aa---SASAKSLFKLQTL consensus A G H RPA S KSLF LQTL

Colocalization of hprK and PTS Genes inGram-Negative Bacteria

In nearly all low G+C Gram-positive bacteria hprK isthe first gene of an operon. It is usually followed by theprolipoprotein diacylglyceryl transferase-encoding lgtgene. So far, no hprK operon of Gram-positive bacteriawas found to contain genes coding for PTS components.This is quite different in Gram-negative bacteria, wherehprK is almost always located close to at least one geneencoding a PTS protein. This can be the HPr-encoding

ptsH gene, as is the case in treponemae and C. burnetii(ptsH directly follows hprK; table 1). More often, a geneencoding an EIIA of the fructose class PTS precedes hprK(in ß- and some Á-proteobacteria) or an EIIA of the man-nose class PTS is located downstream of hprK (in ·- andsome Á-proteobacteria; table 1, fig. 4). In ·-proteobacte-ria, hprK can either be directly followed by an EIIAMan-encoding gene and ptsH (Agrobacterium tumefaciens), oras observed in Rhodobacter sphaeroides, an additionalgene (yhbJ-like) is inserted between hprK and theEIIAMan-encoding gene (fig. 4). In the ·-proteobacterium

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Fig. 3. Phylogenetic tree with HprK/Ps from various Gram-positiveand Gram-negative bacteria. HprK/Ps of the ·-, ß- and Á-subdivisionof proteobacteria and of low G+C Gram-positive bacteria areboxed.

T. pallidum

F. nucleatum

C. burnetii

R. palustris

B. suis

S. meliloti

A. tumefaciens

R. leguminosarum

M. magnetotacticum

C. crescentus

M. loti

N. aromaticovorans

R. sphaeroides

S. pomeroyi

A. ferrooxidans

B. pertussis

B. fungorum

R. metallidurans

N. meningitidis

N. europaea

X. fastidiosa

X. campestris

B. subtilis

L. casei

L. lactis

S. pneumoniae

G. sulfurreducens

C. tepidum

F. succinogenes

α

β

γ

G+

Magnetospirillum magnetotacticum the enzyme I-encod-ing ptsI gene (enzyme I phosphorylates HPr at His-15) islocated downstream of ptsH (fig. 4). An identical organi-zation (hprK followed by yhbJ, an EIIAMan-encoding gene,ptsH and ptsI) can be observed in most Á-proteobacteria(fig. 4). In addition, in several Á-proteobacteria (Acidithio-bacillus ferrooxidans, Xanthomonas axonopodis, Xantho-monas campestris) hprK is preceded by an EIIAFru-encod-ing gene, revealing a total of four PTS genes locatedaround hprK.

hprK of Gram-Negative Bacteria Is FrequentlyOrganized with Genes Encoding TranscriptionRegulators

In addition to the PTS protein-encoding genes, thehprK operon of Gram-negative bacteria often containsgenes encoding non-PTS proteins implicated in gene ex-pression regulation. Two major organizations can be dis-tinguished. In ·-proteobacteria, the truncated hprK isusually preceded by genes encoding a two-component sys-tem of the EnvZ/OmpR family (fig. 4) [Hu and Saier,2002]. This system is best studied in A. tumefaciens,where its components were called ChvI (response regula-tor) and ChvG (sensor kinase). It was found to be impli-cated in pathogenicity, as inactivation of either chvG orchvI prevented tumor formation on plants infectable bythis organism [Charles and Nester, 1993]. Chv stands forchromosomal virulence, as chvG and chvI are located onchromosome 1 and not on the Ti plasmid, which carriesmost of the virulence genes of this organism. Recently,ChvG has been shown to regulate expression of severalchromosome-encoded acid-inducible genes, but also theTi-plasmid-encoded virulence genes virB and virE [Li etal., 2002]. In the pathogenic animal endo-symbiont Bru-cella abortus hprK is also preceded by chvI-chvG-likegenes. They were called bvrR and bvrS (for Brucella viru-lence related genes), as their inactivation affected cellinvasion and virulence of this organism [Sola-Landa etal., 1998]. The ChvG-like sensor kinase of the plant sym-biont Sinorhizobium meliloti was called ExoS. It has beenshown to regulate the production of succinoglycan, themain exopolysaccharide necessary for successful invasionof nodules on its host plant alfalfa [Cheng and Walker,1998].

As already mentioned, in the four ·-proteobacteria, M.magnetotacticum, Novosphingobium aromaticovorans, R.sphaeroides and Silicibacter pomeroyi, a gene resemblingyhbJ from the E. coli rpoN operon is inserted betweenhprK and the EIIAMan-encoding gene (fig. 4). Similarly, inmost ß- and Á- proteobacteria and in Geobacter sulfurre-ducens (‰-proteobacterium), a gene resembling yhbJ islocated downstream of hprK (fig. 4). Interestingly, therpoN operon of E. coli or Klebsiella pneumoniae containsgenes coding for an EIIA of the fructose class PTS (PtsN)and for an HPr paralogue (Npr). In these organisms, theyhbJ gene is inserted between ptsN and npr (fig. 4). AnyhbJ-like gene (yvcJ) also precedes crh in B. subtilis. Thecrh gene encodes an HPr homologue phosphorylated byHprK/P, but not by enzyme I, as Crh contains a glutamineat position 15 in place of the phosphorylatable histidine

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Fig. 4. Colocalization of hprK and pts genes (shaded arrows) of several Gram-negative bacteria with genes encodingeither a two-component system (ChvI/ChvG-like) or proteins from the E. coli rpoN region. hprK* indicates trun-cated hprK genes. Sequences were retrieved from either NCBI (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html), The Sanger Institute (http://www.sanger.ac.uk/Projects/Microbes/), TIGR (http://www.tigr.org/tdb/mdb/mdbinprogress.html) or JGI (http://www.jgi.doe.gov/JGI_microbial/html/index.html).

[Galinier et al., 1997] (fig. 2). The hprK operon of neisse-riae is surrounded by only two genes exhibiting similarityto genes from the E. coli rpoN region (ptsN- and yhbJ-like). The hprK operon of most other ß- and Á-proteobac-teria and of G. sulfurreducens contains several genes fromthe E. coli rpoN region, including rpoN itself, which codesfor a sigma factor (Û54; fig. 4). The highest number ofrpoN-related genes is attained in A. ferrooxidans and thexanthomonads (10 genes including the ptsN- and npr-likegenes).

How Might HprK/P Control TranscriptionRegulators?

Most hprK-containing Gram-negative bacteria possessenzyme I, HPr and at least one EIIA. However, they areprobably not able to transport sugars via the PTS, as they

are missing homologues of the known EIIBs and EIICsand therefore contain only the first part of the PTS phos-phorylation cascade presented within the shaded rectan-gle in figure 1. In addition, none of these bacteria containsCcpA. As a consequence, HprK/P of these organisms isinvolved neither in CcpA-mediated CCR nor in the regu-lation of PTS-catalyzed sugar transport. The gene organi-zation in many hprK-containing Gram-negative bacteriastrongly suggests that HprK/P regulates either a two-com-ponent sensory transduction system implicated in celladhesion/virology or RpoN-like Û-factors. In Gram-nega-tive enteric bacteria, RpoN controls primarily the expres-sion of genes implicated in nitrogen metabolism [Reitzerand Schneider, 2001]. PtsN, which becomes in vitro phos-phorylated by PEP, enzyme I and HPr [Merrick et al.,1995], seems to regulate RpoN activity. Inactivation of K.pneumoniae ptsN led to elevated expression from severalÛ54-dependent promoters, suggesting that unphosphory-

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lated PtsN exerts a negative effect on RpoN activity [Mer-rick et al., 1995]. In agreement with the above concept,npr mutations, which probably slow in vivo phosphoryla-tion of PtsN, significantly decreased expression from thesame promoters. The phosphorylation state of the EIIAsin hprK-containing Gram-negative bacteria could be simi-larly important for the regulation of RpoN, its potentialcofactors (YhbH, YhbJ?) or the ChvI/ChvG-like two-component systems. The phosphorylation state of thePTS proteins is altered in response to the uptake andmetabolism of PTS and non-PTS sugars via a change ofthe PEP/pyruvate ratio in the cells [Hogema et al., 1998].As hprK-containing Gram-negative bacteria probablypossess only the three proteins forming the first part of thePTS phosphorylation chain (shaded rectangle in fig. 1),and as in ·- and Á-proteobacteria the corresponding PTSgenes are frequently organized in an operon with hprK,HprK/P might control the phosphorylation state of theEIIAs in hprK-containing Gram-negative organisms bythe mechanism outlined in figure 1. If the kinase activityof HprK/P is stimulated and/or its phosphorylase activitydiminished in response to a yet unknown signal, P-Ser-HPr will be formed. Ser-46 is part of the interface in theenzyme I/HPr [Garrett et al., 1999] and the HPr/EIIAcomplexes [Wang et al., 2000; Cornilescu et al., 2002].Phosphorylation at Ser-46 therefore hinders the interac-tion of HPr with its partners within the PTS phosphoryla-tion cascade. As a consequence, HprK/P-catalyzed phos-phorylation of HPr drastically slows the enzyme-I-me-diated phosphorylation at His-15 and therefore PFEIIAformation (about 500-fold) [Deutscher et al., 1984]. Simi-lar to the model proposed for RpoN regulation in K. pneu-moniae [Merrick et al., 1995], the PtsN-like proteinpresent in many hprK-containing Gram-negative bacteriacould stimulate the expression of Û54-controlled tran-scription units. Interestingly, inactivation of hprK in N.meningitidis was found to strongly reduce cell adhesion[Taha et al., unpubl.]. Neisseriae possess a Û54-encodinggene, which, however, was proposed to be inactive. Nev-ertheless, these bacteria contain a Û54 binding motif inthe promoter preceding pilE and this promoter was activewhen inserted into Pseudomonas aeruginosa [Laskos etal., 1998]. The pilE Û54 binding motif is not present in allN. gonorrhoeae and N. meningitidis strains [Taha et al.,1996; Taha, unpubl]. In addition to pilE, several otherpromoter regions in neisseriae were found to containmotifs resembling the binding site for Û54 [Taha et al.,1998]. The pilE gene codes for the protein pilin, whichpolymerizes to form the type IV pili, which are specific topathogenic neisseriae. The formation of these pili is nec-

essary for cell adhesion [Nassif et al., 1994]. The obviousquestion therefore arises whether HprK/P, HPr or EIIAFru

might play a role in the regulation of pilE expression, pos-sibly by interacting with YhbJ or the presumed inactiveRpoN, a hypothesis presently tested in our laboratory.

For the potential regulation of a two-component sys-tem by HprK/P and PTS proteins in ·-proteobacteria,which is suggested by the colocalization of the corre-sponding genes (fig. 4), Hu et al. [2002] proposed thatHprK/P, HPr and EIIAMan might interact with each otherto regulate the activity of the sensor kinase. However, asall ·-proteobacteria possess a ptsI gene (with a nifA-like5)-extension; table 1), which in M. magnetotacticum islocated in the hprK operon downstream of ptsH (fig. 4), itis also possible that the phosphorylation state of theEIIAMan, which again would be regulated via HprK/P-catalyzed P-Ser-HPr formation (fig. 1), might controlthe activity of the two-component system. Based onthe known regulatory/catalytic non-PTS functions ofEIIAMan, two principal modes of regulation can be envis-aged. An EIIAMan-like domain is present in LevR-like pro-teins [Deutscher et al., 2001] of Gram-positive and Gram-negative bacteria. LevR belongs to the NifA/NtrC familyof transcription activators for Û54-dependent promoters[Débarbouillé et al., 1991]. Phosphorylation of itsEIIAMan domain by enzyme I and HPr was found to stim-ulate the activity of LevR [Martin-Verstraete et al., 1998].Similarly, EIIAMan or PFEIIAMan could interact withthe sensor kinase and control its activity. However,PFEIIAMan can also function as phosphoryl donor fornon-PTS proteins. E. coli possesses a homodimeric multi-phosphoryl transfer protein DhaM (previously YcgC),which is composed of three domains resembling EIIAMan,HPr and the N-terminal part of enzyme I [Paulsen et al.,2000; Gutknecht et al., 2001]. DhaM becomes phosphor-ylated by PEP, enzyme I and HPr at the truncated enzymeI domain. The phosphoryl group is then transferred to theHPr and EIIAMan domains within DhaM. Interestingly,the phosphoryl group bound to the EIIA domain of DhaMis subsequently used by dihydroxyacetone kinase to phos-phorylate dihydroxyacetone [Gutknecht et al., 2001]. Inmany other bacteria, dihydroxyacetone kinase uses ATPinstead of PFEIIAMan as phosphoryl donor. It can there-fore be envisaged that the sensor kinase encoded by thegene preceding hprK in ·-proteobacteria might be able touse PFEIIAMan as phosphoryl donor. PFEIIAMan-me-diated phosphorylation of the sensor kinase at a histidylresidue would then be controlled by HprK/P via P-Ser-HPr formation and HprK/P would thus regulate amongothers virB and virE expression in A. tumefaciens [Li et

214 J Mol Microbiol Biotechnol 2003;5:206–215 Boël/Mijakovic/Mazé/Poncet/Taha/Larribe/Darbon/Khemiri/Galinier/Deutscher

al., 2002] and succinoglycan production in S. meliloti[Cheng and Walker, 1998]. We are presently testingwhether the sensor kinases ChvG of A. tumefaciens andExoS in S. meliloti are indeed controlled by one of thepredicted modes of EIIAMan-mediated regulation.

Acknowledgments

This research was supported by the CNRS, the INRA and theINA-PG.

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