ORIGINAL PAPER

Rosa Viana Æ Gaspar Perez-Martınez Æ Josef Deutscher

Vicente Monedero

The glycolytic genes pfk and pyk from Lactobacillus casei are inducedby sugars transported by the phosphoenolpyruvate:sugarphosphotransferase system and repressed by CcpA

Received: 31 January 2005 / Revised: 5 May 2005 / Accepted: 9 May 2005 / Published online: 17 June 2005� Springer-Verlag 2005

Abstract In Lactobacillus casei BL23, phosphofructo-kinase activity was higher in cells utilizing sugarstransported by the phosphoenolpyruvate:sugar phos-photransferase system (PTS). The phosphofructokinasegene (pfk) was cloned from L. casei and shown to beclustered with the gene encoding pyruvate kinase (pyk).pfk and pyk genes are cotranscribed and induced upongrowth on sugars transported by the PTS. Contrarilyto the model proposed for Lactococcus lactis, where theglobal catabolite regulator protein (CcpA) is involvedin PTS-induced transcription of pfk and pyk, a ccpAmutation resulted in a slight increase in pfk–pykexpression in L. casei. This weak regulation was evi-denced by CcpA binding to a region of the pfk–pykpromoter which contained two cre sequences signifi-cantly deviated from the consensus. The PTS inductionof pfk–pyk seems to be counteracted by the CcpA-mediated repression. Our results suggest that the needto accommodate the levels of pfk–pyk mRNA to theavailability of sugars is fulfilled in L. casei by a PTS/CcpA-mediated signal transduction different fromL. lactis.

Keywords Lactobacillus Æ Glycolysis Æ CcpA ÆPhosphotransferase system

Introduction

Lactobacillus casei is a lactic acid bacterium (LAB)which gains a major part of its energy through theglycolytic breakdown of carbohydrates leading to theproduction of lactic acid (Kandler 1983). In manymicroorganisms the control of the glycolytic flux de-pends on the activity of phosphofructokinase(Pfk), which catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. A secondcontrol point is the reaction catalyzed by pyruvate ki-nase (Pyk), which catalyzes the conversion of phos-phoenolpyruvate (PEP) and ADP into pyruvic acid andATP (Kandler 1983; Branny et al. 1993). Recentstudies on glycolysis flux control exerted by Pfk andPyk in Lactococcus lactis showed that the coordinatedexpression control of pfk and pyk genes provides thecells with a very potent mechanism for regulating gly-colytic flux (Koebmann et al. 2005). This is reflected atthe level of genetic structure, and pfk and pyk form anoperon in many LAB (Branny et al. 1993; Crispie et al.2002; Llanos et al. 1993). In L. lactis, transcription ofthe las operon, carrying pfk and pyk, is activated bythe presence of sugars transported via the PEP:sugarphosphotransferase system (PTS) by means of thecatabolite control protein A (CcpA) (Luesink et al.1999). This transcriptional regulator acts by binding toa consensus sequence called catabolite-responsive ele-ment (cre), usually found in the promoter region or the5¢ part of catabolite regulated genes (Fujita et al. 1995).The HPr protein of the PTS is an effector of CcpA.When cells grow on readily metabolizable sugars(i.e.:sugars transported by the PTS), the elevatedintracellular concentration of glycolytic intermediatessuch us fructose-1,6-bisphosphate activates the enzymeHPr kinase/phosphorylase (HprK/P), which catalyzesthe phosphorylation and dephosphorylation of HPr atseryl residue 46 (Dossonnet et al. 2000; Kravanja et al.1999). P-Ser-HPr interacts with CcpA allowing itsbinding to cre sequences in catabolite-regulated genes

R. Viana Æ G. Perez-Martınez Æ V. Monedero (&)Biotechnology Department,Instituto de Agroquımica y Tecnologıa de Alimentos,IATA-CSIC, P.O. Box 73,46100 Burjassot, Valencia, SpainE-mail: btcmon@iata.csic.esTel.: +34-96-3900022Fax: +34-96-3636301

J. DeutscherMicrobiologie et Genetique Moleculaire,CNRS-INRA-Institut National AgronomiqueParis-Grignon, UMR2585, 78850Thiverval-Grignon, France

Arch Microbiol (2005) 183: 385–393DOI 10.1007/s00203-005-0003-6

(Deutscher et al. 1995). HPr is also phosphorylated atthe His-15 residue by PEP in a reaction catalyzed bythe enzyme-I of the PTS. P�His-HPr plays an essentialrole in the PTS phosphorylation cascade leading tosugar transport and phosphorylation (Postma et al.1993).

Catabolite control protein A seems to be a globalmetabolic regulator (Deutscher et al. 2002; Titgemeyerand Hillen 2002) which, in Bacillus subtilis, regulatesabout ten percent of the total genes (Blencke et al. 2003;Moreno et al. 2001). CcpA activates glycolytic, carbon-excess excretion and nitrogen assimilation genes when arapidly metabolized sugar is being used by binding to cresequences located upstream of their promoters (catab-olite activation). At the same time, through binding tocres located in the promoter or in the structural gene,CcpA represses the expression of genes for the utiliza-tion of other carbon sources, tricarboxylic acid cycleenzymes and the respiratory chain (catabolite repres-sion). In L. casei, CcpA and P-Ser-HPr are implicated incatabolite repression of the lactose and sorbose-utiliza-tion operons and the N-acetylglucosaminidase gene(Gosalbes et al. 1997; Viana et al. 2000; Yebra et al.2000). In this work we present evidences indicating thatthe glycolytic pfk–pyk operon from L. casei is also reg-ulated by CcpA and responds to the presence of PTSsugars. However, the regulatory mechanism seems to becompletely different from that reported for L. lactis.

Materials and methods

Bacterial strains and growth media

The strains and plasmids used in this study are listed inTable 1. L. casei strains were grown at 37�C under staticconditions in MRS medium (Oxoid) or in MRS fer-mentation medium (Adsa-Micro, Barcelona, Spain)

containing 0.5% of the appropriate sugars. Escherichiacoli DH5a was used in all cloning experiments and wasgrown in Luria-Bertani (LB) medium at 37�C underagitation. E. coli TF1010 (Daldal 1983) was grown inM9 minimal plates supplemented with 0.2% carbohy-drates. When appropriate, erythromycin was used at5 lg/ml for L. casei and at 500 lg/ml for E. coli.Ampicilline (100 lg/ml) was used for E. coli.

Recombinant DNA techniques

Standard methods were used for plasmid DNA isola-tion, restriction analysis, ligation and transformationof E. coli (Sambrook et al. 1989). Restriction enzymesand T4 DNA ligase were purchased from Roche andNew England Biolabs. DNA from L. casei was iso-lated as described (Posno et al. 1991). PCR for theisolation of an L. casei pfk fragment was performed ina Techne PHC-3 thermocycler (Techne Ltd., Cam-bridge, England) with degenerate oligonucleotidesPFK1: 5¢-GGNATGAATGCNGCN(A/G)T-3¢ andPFK5: 5¢-CCNCG(C/T)TG(C/G)ATGTGNCCNA(A/G)AAC-3¢ (N is any nucleotide) which encode theconserved regions GMNAAI and VLGHIQR, respec-tively, detected in aligned Pfk sequences. The PCRreaction mixture (50 ll) contained: 1· Taq buffer(Finnzymes Oy, Riihitontuntie, Finland), 200 lMdNTPs, 20 pmol of each primer, 0.1 lg of L. caseiDNA and 1 U of Taq DNA polymerase (FinnzymesOy). It was subject to 30 cycles of 1 min at 94�C,0.5 min at 50�C and 1 min at 72�C. The PCR productswere inserted into SmaI-digested pUC19 using theSureClone kit (Pharmacia Biotech). The pfk gene wasisolated from a genomic library in pJDC9 (Chen andMorrison 1988; Gosalbes et al. 1997) after a PCRscreening of 1,000 E. coli clones as described (Griffinet al. 1993). Internal fragments of the pfk and pyk

Table 1 Strains and plasmidsused in this study Strain or

plasmidRelevant genotype or properties Origin or reference

L. caseiBL23 Plasmid free, wild type strain B. Chassy (U. Urbana, Illinois)BL71 ccpA::erm Monedero et al. (1997)BL126 ptsI Viana et al. (2000)E. coliDH5a General cloning host Sambrook et al. (1989)BL21 Protein overproduction strain (DE3, pLysS) Laboratory stockTF1010 DpfkB201 D(rhaD-pfkA)200 Daldal (1983)PlasmidspET3c-ccpA pET3c (AmR) containing ccpA of L. casei Mahr et al. (2002)pET15b-ptsH pET15b (AmR) containing ptsH of L. casei Esteban et al. (unpublished)pJDC9 Cloning vector, EmR Chen and Morrison (1988)pFK24 pJDC9 with 4 kb of L. casei DNA including

pfk gene and part of pyk, EmRThis work

pUC18 Cloning vector, AmR Gibco BRLpUCpfk pUC18 with 1.4 kb SalI fragment from pFK24 This workpRV300 pBluescript SK�. EmR from pAMb1 Leloup et al. (1997)pVBpfk pRV300 with a 640 bp pfk internal fragment This workpVBpyk pRV300 with a 495 bp pyk internal fragment This work

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genes with a length of 640 and 495 bp were amplifiedby PCR using the oligonucleotides PFK1/PFK5 andPYK6(5¢-TTGGACCTGCAAGTAACACC-3¢)/PYKI(5¢-GTGTCATCATAAAGGCCAGG-3¢), respectively,and cloned in the suicide plasmid pRV300 (Leloupet al. 1997) digested with SmaI, giving pVBpfk andpVBpyk. Plasmid DNA was sequenced with the DyeTerminator Cycle sequencing kit (Perkin Elmer) andan Abi Prism automated sequencer (Perkin Elmer)using custom primers. The sequences were analyzedwith the GCG package (Genetic Computer Group,Madison, WI, USA).

Measurement of enzymatic activities

Cells were grown in 10 ml of MRS fermentation mediumcontaining 0.5% of appropriate sugars to an optical den-sity at 550 nm of 0.8–1, washed with 20 mM Tris–HCl,pH 7.2 and resuspended in 1 ml 50 mM Tris–HCl,pH 7.2, containing 0.5 mM dithiothreitol, and 0.1 mMphenyl-methyl-sulphonyl fluoride. One gram of glassbeads (0.1-mm diameter) was added to the resuspendedcells and the cells were broken in a Fast Prep apparatus(Biospec, Bartlesville, OK, USA) by shaking three timesfor 30 s. Cell-free extracts were obtained after centrifu-gation at 12,000·g for 10 min at 4�C. Phosphofructoki-nase (EC 2.7.1.11) activity was measured at 25�Cby usinga coupled spectrophotometric assay carried out in 500 llsample buffer containing 20–50 lg of protein as describedby Takahashi et al. (1995). Protein concentrations weredetermined with the Bio-Rad dye-binding assay.

RNA isolation and Northern blotting

Lactobacillus casei strains were grown as described forthe preparation of cell-free extracts. The cells from a10 ml culture were collected by centrifugation, washedwith 50 mM EDTA and resuspended in 1 ml of Trizol(Gibco BRL). One gram of 0.1-mm glass beads wasadded and the cells were broken by shaking in aFastprep apparatus (Biospec) three times for 30 s.RNA was isolated as recommended by the manufac-turers of Trizol, separated on a formaldehyde-agarosegel and transferred to Hybond-N membranes (Amer-sham). The pfk and pyk RNA probes used in theNorthern blots were synthesized using either aninternal pfk or a pyk fragment cloned in pRV300(plasmids pVBpfk and pVBpfk). As these plasmids arepBlueScriptII SK-derivatives, antisense RNA could besynthesized in vitro from linearized plasmids with T3or T7 RNA polymerase using the reagents from theRoche digoxigenin-RNA labeling kit. Hybridization(55�C), washing and staining were performed as rec-ommended by the supplier using the CDP-starchemiluminescent substrate. The transcription initia-tion sites were mapped using the 5¢/3¢ RACE kit from

Roche according to the manufacturer indications andusing RNA isolated from wild type L. casei.

Mobility shift and DNaseI foot-printing assays

Four overlapping DNA fragments covering the pfk–pykpromoter and part of pfk (from �282 to +297 relative tothe translational initiation site) were synthesized by PCRby using the oligonucleotide pairs A, (5¢-ttgcaaactgcc-catgga-3¢/5¢-TCTCTATTCTTCTTGTCG- 3¢); B, (5¢-GGCTAATGTGGTGTTGCG-3¢/5¢-ATCCCCGCC-ACTGGTCAA-3¢); C, (5¢-TAATGAAACG CATTGGTA-3¢/5¢-CTTATCACCGACAGTTGA-3¢) and D,(5¢-GTTAGTTGCCGGCGACAT-3¢/ TGACGACC-AACGCCTCGA-3¢). Hundred nanogram of each frag-ment was incubated for 10 min at 37�C in 20 ll of100 mM Tris–HCl pH 7.4 buffer, containing 1 mMEDTA and 0.5 lg of linearized pWH802 plasmid(unspecific DNA) in the presence or absence of 0.6 lMCcpA. The mixtures were separated in 5% polyacryl-amide gels in 100 mM Tris–HCl pH 8.8, 1 mM EDTAbuffer at 70 V for 45 min and the gels were stained withethidium bromide. A PCR product corresponding to thepfk–pyk promoter region (from �148 to +36) wasamplified by using the oligonucleotide pair B. Each ofthese oligonucleotides was 5¢-labeled using T4 polynu-cleotide kinase and [c�32P]ATP and served as primer in aPCR together with the second unlabeled oligonucleotideandL. casei chromosomalDNAas template. The reactionmixture (50 ll) contained 50 mM Tris–HCl pH 7.5,5 mMMgCl2, 20.000 cpm of the labeled PCR fragments,1 lg of poly dI-dC and 0.3 lM of purified CcpA from L.casei with or without 1 lM L. casei P-Ser-HPr added.After incubation for 20 min at 37�C, the samples weretreated for 1 min with an appropriate concentration ofDNaseI (Roche). The reaction was stopped by adding140 ll of a solution containing 0.4 M sodium acetate,50 lg/ml calf thymusDNA and 2.5 mMEDTA. Sampleswere extracted with phenol/chloroform, ethanol-precipi-tated and loaded on a 6% polyacrylamide-6 M urea geltogether with a sequencing reaction performed with thesame labeled oligonucleotide. CcpA and HPr from L.casei were purified after overexpression in E. coli BL21(DE3)(pLysS) using plasmid pET3c-ccpA (Mahr et al.2002) and pET15b-ptsH (Esteban CD, Hillen W, Titge-meyer F, Perez-Martınez G, unpublished) as previouslydescribed (Mahr et al. 2002). P-Ser-HPr was obtained byATP-dependent phosphorylation using L. casei V267Fmutant HprK/P as described (Galinier et al. 1998) andpurified by Ni-NTA chromatography.

Nucleotide sequence accession number

The nucleotide sequence reported here has been depos-ited in the EMBL-Genbank under accession no.AY242843

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Results

Phosphofructokinase activity in L. casei

In the model LAB L. lactis the gene coding for the Pfkenzyme is positively regulated by growth on PTS sugarsvia the CcpA regulator (Luesink et al. 1998). We testedPfk activity in L. casei cells grown on glucose (a pre-ferred PTS sugar), lactose (a sugar transported by thePTS in L. casei) and ribose (a non-PTS sugar). Also, inorder to test the possible effect of the CcpA signaltransduction pathway on the expression of Pfk in thisbacterium, a wild type strain (BL23) as well as a ccpAmutant (BL71) were used. Pfk activity was slightlyhigher in cells grown on PTS sugars than in cells whichhad utilized the non-PTS sugar ribose (Fig. 1). Inter-estingly, the ccpA mutant showed approximately atwofold increase in Pfk activity in all sugars. These re-sults were not in agreement with the data reported for L.lactis, where a ccpA mutation led to decreased pfkexpression (Luesink et al. 1998). The L. lactis las operon,coding for Pfk, contains also the pyk gene. Pyk activitywas also measured in L. casei, but this activity was fairlyunstable in cell-free extracts and no conclusive resultscould be obtained (data not shown). Doubling time (t2)was measured for the wild type and the ccpA mutant onthe different sugars. In the wild type, t2 was 85, 122 and135 min for glucose, lactose and ribose, respectively,while in the ccpA strain it was 131, 142 and 151 min,respectively.

Isolation of the pfk and pyk genes from L. casei

We were interested to study whether the differences inPfk activity detected in L. casei were related to differ-ences in gene expression. In order to obtain an L. caseipfk probe, a pair of primers based on an alignment of L.

lactis and Lactobacillus bulgaricus Pfks was designed. Bycarrying out a PCR amplification using L. casei DNA astemplate, a single 640-bp band was obtained and se-quenced, showing an open reading frame (ORF) withsignificant homology to Pfks. This fragment was used toscreen an L. casei genomic library (Gosalbes et al. 1997),and a plasmid, pFK24, containing a 4-kb insert with thecomplete pfk homologue was obtained (Fig. 2a). Thecomplete pfk 3¢ flanking region was characterized byreverse PCR and determined thanks to the current L.casei BL23 genome sequencing project (Klaenhammeret al. 2002). The L. casei pfk homologue is preceded by aputative GAGG ribosome binding site. It encodes a 319amino acid protein with a MW of 34 kDa exhibiting 62and 63% identity to the Pfks from L. lactis and L. bul-garicus, respectively. Upstream of pfk, the end of anORF was found which exhibited 59 and 50% identity tothe a-subunit of the DNA polymerases III from Bacillushalodurans and L. lactis, respectively. Interestingly, in B.subtilis the corresponding dnaE gene is located about3.5 kb upstream of pfkA and a dnaE homologue is alsopresent immediately upstream of the pfk–pyk operon inStreptococcus thermophilus (Crispie et al. 2002). Fifty-eight nucleotides downstream of pfk a pyk homologuestarted which was preceded by a potential AGGAGribosome binding site. The putative L. casei Pyk is a 587-amino acids long 63 kDa protein which exhibits 79 and65% sequence identity to its L. bulgaricus and Bacillusstearothermophilus counterparts, respectively. Interest-ingly, the L. casei enzyme displayed a C-terminalextension consisting of 112-aa, which is also present inthe L. bulgaricus and B. subtilis proteins, but not in theL. lactis and S. thermophilus counterparts. This C-ter-minal domain has been shown to contain a motifhomologous to the PEP-phosphorylation site in enzyme-I of the PTS (539TSHAAV544, in L. casei Pyk), where thephosphorylated His is conserved (Nguyen and Saier1995). This raises the possibility of a likely regulation ofPyk by PEP- or P�His-HPr-dependent phosphoryla-tion.

Complementation of an E. coli Pfk-deficient strainwith L. casei pfk

To demonstrate the functionality of L. casei pfk and pykgenes we tried to construct their corresponding mutantsby Campbell-like integration of the suicide plasmidspVBpfk and pVBpyk. In spite of several attempts, onlyrandom integrations in the genome could be obtained, asconfirmed by Southern blots (data not shown). L. caseiis totally dependent on glycolysis and it is likely thatsuch integrants would severely affect growth.

To assign a role to pfk we tried a different approachof functional complementation. A 1,400 bp SalI frag-ment from pFK24 containing the complete pfk wascloned into pUC19, resulting in plasmid pUCpfk(Fig. 2a). The presence of pUCpfk in E. coli DH5acaused a 3.5-fold increase of Pfk activity in cell extracts:

BL23 BL71

nmol

/min

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prot

ein

0

500

1000

1500

2000

2500

3000

Glucose

Lactose

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Fig. 1 Phosphofructokinase activity in cell extracts from L. caseiBL23 (wild-type) and BL71 (ccpA) grown on different carbonsources. Results are from two experiments with cells coming fromdifferent cultures

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209±29 nmol/min mg prot for the pUC19-transformedstrain and 746±49 nmol/min mg prot for the pUCpfk-transformed strain. Furthermore, transformation of E.coli TF1010, a mutant lacking the two E. coli phosp-hofructokinases (Daldal 1983), with pUCpfk restoredthe growth of this strain in minimal medium supple-mented with mannitol or ribose. These results confirmedthat the presumed L. casei pfk encodes indeed a phos-phofructokinase.

pfk and pyk genes are cotranscribed

When L. casei total RNA was hybridized with a pfk or apyk probe in Northern blot experiments, a 2.9-kbmRNA was detected (Fig. 3a). The length of thismRNA was in good agreement with the size of a tran-script covering the pfk and pyk genes, indicating that

both genes are transcribed together, as is the case for theL. bulgaricus pfk and pyk genes (Branny et al. 1993). Thetranscription start point was mapped by 5¢-RACE andshown to coincide with a G present at position �48relative to the translation initiation site (Fig. 2b, c). Inaddition to the major 2.9 kb transcript, a weaker 1.9-kbband was obtained with the pyk probe and a 1-kb bandwas detected when using the pfk probe. This latter bandwas very faint and became visible in most lanes onlywhen the membrane was overexposed (Fig. 3a). The 5¢end of the pyk-specific 1.9 kb mRNA was determined by5¢-RACE and was identified as the A situated sevenbases downstream of the pfk stop codon. Likely �35 and�10 promoter sequences could be found in the pfkcoding region that would account for this pyk-specifictranscript (see Fig. 2a). However, no obvious terminatorcould be detected between pfk and pyk. As the size of theminor 1 kb messenger was in agreement with the size of

Fig. 2 a Schematicrepresentation of the sequencedL. casei region containing pfk–pyk and part of a dnaEhomologue. Stem-loopsrepresent transcriptionalterminators. The inserts presentin plasmids pFK24 andpUCpfk are indicated.b Sequence of the intergenicregions within the L. casei pfk–pyk operon (Acc. no.AY242843). Underlined basesshow the putative Shine-Dalgarno sequences and the�35 and �10 promotersequences, together with thetranscriptional initiation sitedetermined by 5¢ RACE.Inverted arrows represent thepresumed transcriptionalterminators. The 5¢ end of pyk-specific mRNA is underlined.c Sequencing reactions of theRACE products. The 5¢ ends ofpfk–pyk messengers areindicated by black triangles.The RNA stem-loop structurein the pfk–pyk intergenic regionand the putative cleavage site(marked by an arrow) areshown. The pfk stop codon isunderlined

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pfk, it was tempting to assume that both smaller bandswere the result of an endonucleolytic cleavage of a bi-cistronic mRNA. The pyk mRNA 5¢ end was located atthe base of a possible stem-loop structure (Fig. 2c). Al-though no consensus processing site for RNAses wasfound, the stem loop could represent a cleavage site foran endonuclease. The different amounts of pfk- and pyk-specific RNAs detected in the Northern blots suggestthat the two RNA fragments have very different halflives.

pfk–pyk expression is induced by PTS sugarsand repressed by CcpA

RNA was isolated from several L. casei strains grown onthe same sugars used for the determination of Pfkactivity and used for Northern blot analysis (Fig. 3). Inthe wild-type strain BL23, the level of pfk–pyk mRNAwas low in ribose-grown cells, clearly stronger in glu-cose- and the strongest in lactose-grown cells (Fig. 3a).The ccpA mutant exhibited an induction pattern similarto that of the wild type strain, except that the mRNAlevels in glucose and ribose-grown cells were clearlyhigher, indicating a likely repression of pfk–pyk byCcpA. Growth on the non-PTS sugar maltose, whichcontrarily to ribose enters glycolysis at an early step,

resulted in almost negligible pfk–pyk mRNA in the wildtype strain, while it was clearly detectable in the ccpAmutant (Fig. 3b). In both strains the signals in maltosewere much weaker than those obtained with glucose-grown cells. For the wild type and the ccpA mutant onmaltose, the t2 measured was 201 and 219 min, respec-tively. Non-PTS sugars such us ribose or maltose aresupposed to trigger no catabolite repression. However, itwas shown that some degree of CcpA-mediated repres-sion of pfk–pyk occurs on these sugars.

We measured expression of pfk–pyk in a ptsI strain(BL126). This strain is defective in the general PTSprotein enzyme-I and thus it is unable to transportglucose by this system (Viana et al. 2000). Nevertheless,it is still able to grow on glucose due to the presence ofan additional proton-driven glucose permease (Veyrat etal. 1994). In agreement with the results obtained withnon-PTS sugars, low pfk–pyk expression was detected inBL126 cells, suggesting dependence of pfk–pyk expres-sion on PTS sugar utilization (Fig. 3c).

The pfk–pyk messenger levels were not completely inagreement with the Pfk activity data. Nevertheless, theyfurther strengthened the main conclusions drawn fromthe Pfk activity studies and reinforced the facts that: (1)CcpA has a negative effect on pfk–pyk expression and (2)utilization of a PTS sugar leads to elevated pfk–pykexpression.

CcpA binds to the pfk–pyk promoter

Data from Northern blots suggested a direct regulationof pfk–pyk expression by CcpA. In order to determinewhether CcpA binds to the operon promoter region,four correlative 180-bp DNA fragments overlappingbetween 35 bp and 50 bp and spanning the region from�282 to +297 relative to the translational initiation sitewere used to perform band mobility shift assays. Onlythe fragment from �148 to +36 and thus containing thepromoter region was clearly retarded by CcpA (Fig. 4a).In order to define the binding region, DNaseI protectionassays were performed with this fragment. As shown inFig. 4b, CcpA protected a region expanding from �108to �60. This protection was strengthened by the pres-ence of P-Ser-HPr. A search for potential cre sites in thepfk–pyk promoter region containing less than threemismatches compared to the consensus cre failed.However, a careful inspection of the protected sequencestretch revealed the occurrence of two degenerate cre-like sites located within the �35 and �10 sequences thatcould be the target for CcpA binding (Fig. 4b).

Discussion

Analysis of the pfk and pyk genes from L. casei revealedthat they are colocalized on the genome and cotran-scribed as a single 2.9-kb mRNA. In contrast to B.subtilis (Doan and Aymerich 2003; Ludwig et al. 2001;

Fig. 3 Northern blot analysis of pfk–pyk transcription usingspecific probes. a RNA was extracted from L. casei wild type(BL23) and a ccpA (BL71) mutant grown in the presence of glucose(G), lactose (L) and ribose (R). Sizes of the transcripts are indicatedby arrows. b RNA was isolated from L. casei wild-type (BL23) anda ccpA (BL71) mutant, grown with glucose (G) or maltose (M).c RNA was extracted from the wild type (BL23), and a ptsI mutantstrain (BL126), affected in the EI component of the PTS grownunder the same conditions as in panel A, except for BL126, which isunable to grow on lactose (Viana et al. 2000). The staining of therRNA in each membrane is shown below the blots

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Ludwig et al. 2002), little is known about the regulationof glycolytic genes in LAB. CcpA-mediated activation ofthe lactate dehydrogenase (ldh) gene has been reportedfor Streptococcus bovis (Asanuma et al. 2004), S. ther-mophilus (van den Bogaard et al. 2000) and Enterococcusfaecalis (Leboeuf et al. 2000). Transcription of the L.lactis las operon, coding for Pfk, Pyk and Ldh is alsoactivated by CcpA through binding to a cre presentupstream of its promoter (Luesink et al. 1998). In L.lactis, las expression was reduced by fourfold whengrown on galactose (non-PTS sugar) compared to glu-cose (PTS sugar). An L. lactis ccpA mutant also showeda fourfold reduction in las expression and a two- tofourfold decrease in Pyk and Ldh activities on glucose.In accordance to this model of regulation, overexpres-sion of HPrSer46Asp mutant protein in L. lactis, which

mimics P-Ser-HPr, led to an increase in Pyk and Ldhactivities (Luesink et al. 1999). The levels of pfk–pykexpression in L. casei were also higher in PTS sugars(glucose, lactose), which favors the formation of P-Ser-HPr, than in non-PTS sugars (ribose, maltose, andglucose in the ptsI strain). However, two major differ-ences were found compared to L. lactis: induction byPTS sugars was independent of CcpA, as in a ccpAbackground PTS sugars had a stimulatory effect on pfk–pyk transcription, and CcpA had a weak negative effecton transcription. A comparison of Pfk–Pyk enzymaticactivities and pfk–pyk transcripts in L. casei showeddiscrepancies between enzyme and mRNA levels, sug-gesting that additional translational or posttranslationalregulatory mechanisms might exist. Similar results havebeen described when analyzing expression of glycolisisgenes in L. lactis by using DNA arrays and measure-ments of specific enzymatic activities (Even et al. 2001;Even et al. 2003). In these studies, pfk–pyk transcriptionwas also induced by glucose by a factor ranging from 1.8to 2.4 compared to galactose, while differences in specificactivities of the respective enzymes did not vary corre-spondingly. As pointed out by the authors, additionalfactors such as growth rate, protein turnover and morenotably, an observed difference in translational efficiencydepending on the sugar used for growth might accountfor this observation.

The repressive effect of CcpA was clearly observed onglucose, whereas pfk–pyk transcription remained un-changed on lactose. This would indicate that CcpAmight be counteracting the induction of glucose (strongrepressing sugar), while PTS induction could not becounteracted by the CcpA repression generated aftergrowth on lactose. Our results suggested that CcpA-mediated regulation occurred at the level of pfk–pykpromoter binding, as CcpA protected two degenerate cresites located in a position conceivable with a repressiveeffect. The low degree of conservation of the pfk–pyk cresequences might lead to weak CcpA binding, whichcould account for the low contribution of CcpA to pfk–pyk regulation (only two to threefold downregulation).In vitro CcpA binding to the pfk–pyk cres was slightlyenhanced by P-Ser-HPr.

The mechanism/s responsible for PTS induction ofpfk–pyk genes in L. casei is not known. However, it hasto be pointed out that growth rates on PTS sugars weregenerally higher compared to non-PTS sugars and thusthe efficiency of metabolism rather than the transportmechanism might control pfk–pyk expression.

We showed that pfk–pyk regulation in L. casei doesnot follow the mechanisms described for L. lactis.Depending on growth conditions, PTS- and CcpA-mediated induction/repression could permit a fine tuningof the expression of enzymes that control the glycolyticflux, which most probably leads to a more efficientexploitation of available carbohydrates. However, itseems that different microorganisms have developedslightly different regulatory pathways which differing in

Fig. 4 a Gel shift assays of DNA fragments from the pfk–pykpromoter in the presence of CcpA. Fragments are: A (�282 to�109); B (�148 to +36); C (+1 to +179) and D (+128 to +297).Positions are relative to the translational initiation site. b DNAfoot-printing analysis of CcpA binding to the pfk–pyk promoterregion (fragment B, �148 to +36). Protection of the coding andnon-coding strands in the presence of CcpA with or without its co-repressor P-Ser-HPr is shown. The bold vertical and horizontalbars represent CcpA-protected regions. Possible cre-like sites areindicated by a consensus sequence below the DNA sequence

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their action modes, though they are all based on PTS/CcpA-mediated signal transduction.

Acknowledgements We thank CD Esteban for pET plasmidsexpressing L. casei CcpA and HPr. Manuel Zuniga is acknowl-edged for valuable discussions. This work was supported by theBIO4-CT96-0380 European project and by funds of the SpanishMinisterio de Educacion y Ciencia (Project BIO2001-01616).Genome sequencing of L. casei BL23 was carried out at the Uni-versity of Caen, Laboratoire de Microbiologie de l’Environnementand at the INRA Thiverval-Grignon, Microbiologie et GenetiqueMoleculaire, with the financial aide from the Region Basse Nor-mandie and the INRA. RV was recipient of a fellowship from theMinisterio de Educacion y Ciencia from Spain.

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