Molecular Microbiology (2000) 36(3), 570±584

Enzyme I and HPr from Lactobacillus casei: their role insugar transport, carbon catabolite repression andinducer exclusion

Rosa Viana,1² Vicente Monedero,1,2²

ValeÂrie Dossonnet,2 Christian Vadeboncoeur,3

Gaspar PeÂrez-MartõÂnez1* and Josef Deutscher2

1Instituto de AgroquõÂmica y TecnologõÂa de Alimentos,

C.S.I.C., Ap. Correos 73, 46100 Burjassot, Valencia,

Spain.2Laboratoire de GeÂneÂtique des Microorganismes, INRA-

CNRS URA 1925, 78850 Thiverval-Grignon, France.3GREB, DeÂpartement de Biochimie et de Microbiologie,

Faculte des Sciences et de GeÂnie and Faculte de

MeÂdecine Dentaire, Universite Laval, Ste-Foy, QueÂbec,

Canada G1K 7P4.

Summary

We have cloned and sequenced the Lactobacillus

casei ptsH and ptsI genes, which encode enzyme I

and HPr, respectively, the general components of the

phosphoenolpyruvate±carbohydrate phosphotransfer-

ase system (PTS). Northern blot analysis revealed that

these two genes are organized in a single-transcrip-

tional unit whose expression is partially induced. The

PTS plays an important role in sugar transport in

L. casei, as was confirmed by constructing enzyme

I-deficient L. casei mutants, which were unable to

ferment a large number of carbohydrates (fructose,

mannose, mannitol, sorbose, sorbitol, amygdaline,

arbutine, salicine, cellobiose, lactose, tagatose, treha-

lose and turanose). Phosphorylation of HPr at Ser-46 is

assumed to be important for the regulation of sugar

metabolism in Gram-positive bacteria. L. casei ptsH

mutants were constructed in which phosphorylation

of HPr at Ser-46 was either prevented or diminished

(replacement of Ser-46 of HPr with Ala or Thr

respectively). In a third mutant, Ile-47 of HPr was

replaced with a threonine, which was assumed to

reduce the affinity of P±Ser±HPr for its target protein

CcpA. The ptsH mutants exhibited a less pronounced

lag phase during diauxic growth in a mixture of

glucose and lactose, two PTS sugars, and diauxie

was abolished when cells were cultured in a mixture

of glucose and the non-PTS sugars ribose or maltose.

The ptsH mutants synthesizing Ser-46±Ala or Ile-47±

Thr mutant HPr were partly or completely relieved

from carbon catabolite repression (CCR), suggesting

that the P±Ser±HPr/CcpA-mediated mechanism of CCR

is common to most low G1C Gram-positive bacteria.

In addition, in the three constructed ptsH mutants,

glucose had lost its inhibitory effect on maltose

transport, providing for the first time in vivo evidence

that P±Ser±HPr participates also in inducer exclusion.

Introduction

In Gram-positive bacteria, the phosphocarrier protein HPr,

one of the general components of the phosphoenol-

pyruvate (PEP)±sugar phosphotransferase system (PTS),

can be phosphorylated at two different sites. PEP-

dependent phosphorylation catalysed by enzyme I occurs

at the catalytic His-15. P±His±HPr transfers its phos-

phoryl group to the sugar-specific enzyme IIA 0s, which in

turn phosphorylate their corresponding enzyme IIB 0s. P-

enzyme IIB and the integral membrane protein enzyme

IIC catalyse the simultaneous uptake and phosphorylation

of carbohydrates (Postma et al., 1993). P±His±HPr can

transfer its phosphoryl group also to non-PTS proteins,

such as glycerol kinase (Charrier et al., 1997) or anti-

terminators and transcriptional activators possessing the

PTS regulation domain (PRD), which contains several

phosphorylation sites recognized by P±His±HPr (Tortosa

et al., 1997; StuÈ lke et al., 1998; Lindner et al., 1999). In all

cases, P±His±HPr-dependent phosphorylation leads to

the activation of the function of the non-PTS proteins and

this phosphorylation has been shown to serve as a

secondary carbon catabolite repression (CCR) mechan-

ism in Gram-positive bacteria (Deutscher et al., 1993;

KruÈger et al., 1996; Martin-Verstraete et al., 1998). In

Lactobacillus casei, the antiterminator LacT, which

regulates the expression of the lac operon, contains a

PRD and seems to be controlled by this mechanism.

Dephosphorylation of P±His±HPr and P±His±LacT in

cells growing in glucose-containing medium was assumed

to be partly responsible for the repressive effect exerted

by the presence of glucose on lac operon expression

(Gosalbes et al., 1997; Gosalbes et al., 1999).

The second, ATP-dependent phosphorylation of HPr in

Q 2000 Blackwell Science Ltd

Received 13 October, 1999; revised 18 January, 2000; accepted28 January, 2000. ²The first two authors contributed equally to thiswork. *For correspondence. E-mail gaspar.perez@iata.csic.es; Tel.(134) 963 900 022; Fax (134) 963 636 301.

Regulatory functions of enzyme I and HPr in L. casei 571

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 570±584

Gram-positive bacteria is catalysed by the bifunctional

HPr kinase±phosphatase (Galinier et al., 1998; Reizer

et al., 1998; Brochu and Vadeboncoeur, 1999; Kravanja

et al., 1999). In Bacillus subtilis, this phosphorylation,

which occurs at the regulatory Ser-46 (Deutscher et al.,

1986), is stimulated by fructose-1,6-bisphosphate and

inhibited by inorganic phosphate (Galinier et al., 1998).

P±Ser±HPr participates in the major mechanism of CCR±

carbon catabolite activation operative in bacilli and pre-

sumably other Gram-positive bacteria (Deutscher et al.,

1997). It functions as a corepressor for the catabolite

control protein CcpA, a member of the LacI±GalR family

of transcriptional repressors±activators (Henkin et al.,

1991). The complex formed between CcpA and P±Ser±

HPr has been shown to bind to catabolite response

elements (cre) (Fujita and Miwa, 1994; GoÈsseringer et al.,

1997; Kim et al., 1998; Galinier et al., 1999; Martin-

Verstraete et al., 1999), operator sites preceding or

overlapping the promoters or being located within the 5 0

region of catabolite repressed genes and operons (Hueck

et al., 1994).

Studies regarding the role of P±Ser±HPr in the CcpA-

mediated CCR mechanism in Gram-positive bacteria

have mainly been carried out with bacilli. A single Bacillus

subtilis ptsH1 mutation, which results in the replacement

of Ser-46 of HPr with an alanine, has so far been used to

study in vivo the involvement of Ser-46 phosphorylation of

HPr in CCR. This mutation was originally present in strain

SA003 (Deutscher et al., 1994) and has been transferred

in the meantime into many other strains. Similarly, hprK

mutants, which are defective in HPr kinase±phosphatase,

have been constructed only with B. subtilis (Galinier et al.,

1998; Reizer et al., 1998). To test whether the P±Ser±

HPr/CcpA-dependent CCR mechanism established for

bacilli is operative in a similar manner in other Gram-

positive bacteria, we have cloned and sequenced the L.

casei ptsHI operon and constructed various ptsH mutants,

including ptsH1. We studied the effect of these mutations

on diauxic growth and CCR. L. casei was an ideal

candidate for these studies, as it exhibits a strongly

expressed lag phase during diauxic growth. Cells grown in

a medium containing glucose and a less preferred carbon

source such as lactose, galactose, maltose or ribose,

stopped growing for up to 20 h when glucose had been

consumed, before they finally started to regrow on the

second carbon source (Veyrat et al., 1994).

Based on in vitro studies, P±Ser±HPr has been

suggested to also be implicated in inducer exclusion.

The Ser-46±Asp mutant HPr which structurally resembles

P±Ser±HPr (Wittekind et al., 1989), has been reported to

interact with the lactose- and glucose-specific non-PTS

permeases of Lactobacillus brevis (Ye and Saier,

1995a,b) and to inhibit the non-PTS sugar transporters

by uncoupling them from proton symport (Ye et al., 1994a,b).

The presence of the corresponding non-PTS substrate

was found to exert a co-operative effect on binding of Ser-

46±Asp mutant HPr to the non-PTS permeases. How-

ever, owing to the use of Ser-46±Asp mutant HPr in

place of the physiologically relevant P±Ser±HPr and

owing to the availability of only in vitro results, the

involvement of P±Ser±HPr in inducer exclusion in

Gram-positive bacteria remained questionable and was

still awaiting in vivo confirmation. This could not be

achieved with Bacillus subtilis strains, because until now

no transport system submitted to inducer exclusion has

been detected in this organism. As we had found that

maltose uptake by L. casei cells is strongly inhibited by

the presence of glucose, suggesting that the maltose

permease is regulated by an inducer exclusion mechan-

ism, we studied whether the above ptsH mutations would

influence the inhibitory effect of glucose on maltose

transport in L. casei.

Results

Purification and N-terminal sequence of HPr from L. casei

Purification of L. casei HPr was performed as described

under Experimental procedures by carrying out the

following steps: heat denaturation of a crude extract, gel

filtration of the supernatant containing the heat-resistant

HPr followed by reverse phase chromatography on a

high performance liquid chromatography (HPLC) column.

After reverse phase chromatography, HPr was found to

be nearly homogenous (more than 95% pure), as was

judged from SDS±PAGE on which 10 ml aliquots of HPr-

containing fractions had been separated. Aliquots (about

0.5 nmol) of the purified, lyophilized HPr were used to

determine the sequence of the first 21 amino acids by

automated Edman degradation. The following N-terminal

amino acid sequence was found for HPr from L. casei:

M±E±K±R±E±F±N±I±I±A±E±T±G±I±H±A±R±P±A±T±L.

The obtained sequence exhibited strong similarity to the

N-terminal sequence of HPr from other bacteria, notably

around the active centre His-15, the site of PEP-

dependent phosphorylation (data not shown), confirming

that the purified protein was indeed HPr of L. casei.

Cloning of PCR-amplified L. casei ptsHI fragments

To amplify L. casei DNA fragments containing ptsH and

part of ptsI, degenerate oligonucleotides were designed

based on the N-terminal sequence of HPr described

above and on strongly conserved regions in enzyme I,

which were detected by carrying out an alignment of

different enzyme I sequences (data not shown). Two

combinations of primers (PTS-H2±PTS-I3 and PTS-H2±

PTS-I4) gave PCR-amplified fragments of 1.6 kb and

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0.3 k respectively. Sequencing of the PCR products

revealed that the deduced amino acid sequences

exhibited strong similarity to the sequences of known

enzyme I and HPr. As expected, both DNA fragments

began with the 5 0 end of ptsH and extended to the region

in ptsI encoding the conserved sequence chosen as a

basis for the second primer (Fig. 1). The larger of the two

fragments obtained with primer PTS-I3 was cloned into

pUC18, providing plasmid pUCR±H1.

Disruption of ptsI and cloning and sequencing of DNA

fragments containing flanking regions of L. casei ptsH and

ptsI

A 865 bp EcoRI fragment, which contained an internal

part of the ptsI gene, (Fig. 1) was obtained from plasmid

pUCR±H1 and subcloned into the suicide vector pRV300

(Leloup et al., 1997), providing plasmid pVME800. This

plasmid was used to transform the L. casei wild-type

strain BL23 and integration of the plasmid at the correct

location (ptsI::pVME800) was verified by PCR and

Southern blot. The phenotype of two clones carrying the

correct integration was tested. In contrast to the wild-type

strain, the two mutants could no longer produce acid from

fructose, mannose, mannitol, sorbose, sorbitol, amygda-

line, arbutine, salicine, cellobiose, lactose, tagatose,

trehalose and turanose. However, they could still meta-

bolise ribose, galactose, glucose, N-acetylglucosamine,

aesculine, maltose and gluconate, suggesting that in L.

casei PTS-independent transport systems exist for this

second class of sugars.

Restriction analysis of the ptsHI region was carried out

by Southern hybridization using DNA isolated from one

integrant (BL124) with the aim of identifying restriction

enzymes allowing cloning of the ptsH and ptsI genes

together with their flanking regions. Digestion of BL124

Fig. 1. Schematic presentation of the sequenced chromosomal L. casei DNA fragment containing the ptsHI operon. Indicated are the three ORFsdetected in this fragment, the promoter and terminator of the ptsHI operon and several important restriction sites. A 400 bp DNA sequence,including the ptsHI promoter, the ribosome binding site (both underlined), the transcription start site mapped by primer extension (marked with anasterisk) and almost the complete ptsH gene, is shown underneath the schematic presentation of the total sequenced DNA fragment. The arrowindicates the position of the oligonucleotide used in the primer extension experiment. The phosphorylatable His-15 and Ser-46 residues are boxed.Amino acid substitutions present in HPr encoded by the three ptsH alleles are written below the wild-type sequence at positions Ser-46 and Ile-47.The initially isolated PCR fragments H2±I4 and H2±I3 (flanked by inverted arrows) and the 865 bp EcoRI fragment, which was called E800 andsubcloned into pRV300, are shown above the schematic presentation of the total sequenced DNA fragment. The plasmid containing the 865 bpEcoRI fragment was used to construct a mutant with a disrupted ptsI gene, which allowed us to clone the flanking regions of the ptsHI operon byisolating plasmids pVMH1 and pVMS1, the inserts of which are completely or partly presented in this figure.

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DNA with SacI and religation of the obtained DNA

fragments allowed to isolate plasmid pVMS1 carrying an

about 9 kb insert. Partial sequencing of this insert

revealed that it contained the 3 0 part of ptsI and its

downstream region (Fig. 1). The same experiment carried

out with HindIII allowed us to isolate plasmid pVMH1

carrying a 2.4 kb insert comprising the complete ptsH

gene together with part of its promoter region and the

5 0 part of ptsI (Fig. 1). The sequence containing the

complete ptsH promoter and 560 bp of the upstream

region was subsequently obtained by reverse PCR. In

total, a continuous stretch of 4150 bp has been

sequenced and has been submitted to the GenBank

database under accession number AF159589. It con-

tained the complete ptsH and ptsI genes and an open

reading frame (ORF) located downstream of ptsI. The

stop codon of ptsH was found to overlap with the initiation

codon of ptsI by 1 bp, suggesting that these two genes

are organized in an operon. Whereas the encoded L.

casei HPr and enzyme I exhibited sequence similarities

ranging from 65% to 85% when compared with their

homologues in B. subtilis, Lactococcus lactis, Lactoba-

cillus sakei, Streptococcus salivaruis or Enterococcus

faecalis, the protein encoded by the ORF located down-

stream of ptsI exhibited similarity to the sugar permeases

XylE (Davis and Henderson, 1987) and GalP (Pao et al.,

1998) from Escherichia coli. No ORF could be detected in

the 560 bp region upstream from the ptsHI promoter.

Transcriptional analysis of the L. casei ptsHI operon

To determine the size of the ptsHI transcripts and to test

the effect of a man (prevents the uptake of glucose via the

PTS) and a ccpA mutation on ptsHI expression, Northern

blots were performed with RNA isolated not only from the

L. casei wild-type BL23, but also from the mutant strains

BL30 (man) (Veyrat et al., 1994), BL71 (ccpA) (Monedero

et al., 1997) and BL72 (man ccpA) (Gosalbes et al.,

1997), which were grown in medium containing either

glucose, lactose or ribose. Hybridization experiments

were carried out with either ptsH (Fig. 2) or ptsI-specific

probes (data not shown). With both probes, a mRNA band

of about 2.1 kb could be detected, which is in good

agreement with the size expected for the combined ptsH

and ptsI genes, confirming that these two genes are

organized in an operon and that transcription stops at the

stem±loop structure located downstream of ptsI (Fig. 1).

Densitometric measurement of the hybridizing bands in

the RNA isolated from cells of the different mutants

grown in glucose-, lactose-, or ribose-containing medium

showed that expression of the ptsHI operon was

moderately induced by glucose in the wild type and

ccpA mutant, while this effect was less pronounced

in the strains carrying the man mutation (Fig. 2). The

transcriptional start point of the transcript of L. casei ptsHI

operon was mapped by primer extension using the primer

PTS-P.E. (Fig. 1) and was found to be at the G located

82 bp upstream of the ptsH start codon (Fig. 1).

Construction of ptsH mutants altered at Ser-46 or Ile-47

To construct chromosomal L. casei ptsH mutants by

double crossing over, we first tried to obtain a ptsI mutant

strain carrying a frameshift mutation. For this purpose,

plasmid pVMH1 was partially digested with EcoRI and

made blunt end (filled in with the Klenow fragment) before

it was religated and used to transform E. coli DH5a. From

one of the resulting transformants, a plasmid (pVMR10)

could be isolated bearing a frameshift mutation at the

EcoRI site located at nucleotide 327 of the ptsI gene, as

was confirmed by restriction analysis and DNA sequen-

cing (insertion of four additional bp). Plasmid pVMR10

was subsequently used to transform L. casei BL23 and an

erythromycin-resistant ptsI1 integrant resulting from a

Campbell-like recombination was isolated. From this

strain, a ptsI mutant (ptsI1, BL126) could be obtained by

a second recombination. BL126 was erythromycin-sensi-

tive and exhibited a fermentation pattern identical to that

found for the ptsI::pVME800 mutant BL124. Interestingly,

no ptsHI mRNA could be detected in BL126 by Northern

blot analysis (data not shown).

Fig. 2. Northern blot showing hybridization bands obtained with aptsH-specific probe. Experiments were carried out with RNA isolatedfrom L. casei BL23 (wild type), BL30 (man mutant), BL71 (ccpAmutant) and BL72 (man ccpA double mutant). Lanes are markedaccording to the carbon source used to grow the bacterial cells: G(glucose), L (lactose) or R (ribose). Numbers on the left and rightmargins indicate the position of the two closest RNA standards andthe estimated size of the observed band in kb respectively. Thehistogram below shows the average relative band intensities and theirerrors from three different experiments. The average relative bandintensities were calculated as the peak area of the bands observed inthe photographic film after hybridization divided by the peak area ofthe 23S rRNA.

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PCR-based site directed mutagenesis was carried out

with the L. casei ptsH gene present in plasmid pVMH1

(Table 1) to replace either Ser-46 with alanine, aspartic

acid or threonine, or Ile-47 with threonine. The four

resulting plasmids carrying the various ptsH alleles were

named pVMH2, pVMH3, pVMH4 and pVMH5, respec-

tively (Table 1), and were used to transform the L. casei

ptsI mutant BL126. Erythromycin-resistant recombinants

were obtained with all plasmids. As outlined in Fig. 3, the

Campbell-like first recombination can occur at three

different places (before the ptsH mutation, between the

ptsH and ptsI mutations and after the ptsI mutation)

resulting in three distinguishable DNA arrangements.

Whereas recombinants of type 1 and 2 were unable to

utilize lactose, recombinants of type 3 were able to grow

slowly on lactose. This was probably due to a readthrough

from a plasmid-located promoter allowing low ptsI

expression. For each ptsH allele, one `type 3' recombi-

nant was used for further experiments aimed to isolate

strains with a second recombination. Again, three

possibilities existed for the second recombination event

providing either the original ptsI mutant, a wild-type strain

or one of the desired ptsI1 ptsH strains (Fig. 3). In the last

case, the resulting strains were expected to contain a

functional PTS and to be able to grow on PTS substrates,

although slightly reduced growth on PTS substrates has

been reported for mutants in which Ser-46 or Ile-47 of HPr

were altered (Deutscher et al., 1994; Eisermann et al.,

1988; Reizer et al., 1989; Gauthier et al., 1997).

Erythromycin-sensitive clones able to ferment lactose

were therefore isolated after cells had been grown for 200

generations without selective pressure to allow the

second recombination to occur. As explained, these

clones could be either wild-type strains or ptsI1 ptsH

mutants (Fig. 3). Two types of erythromycin-sensitive

lactose-fermenting recombinants were obtained that

exhibited slightly different growth characteristics. Using

appropriate primers, the ptsH alleles of two clones of the

slower and faster growing recombinants were amplified by

PCR and sequenced. For each ptsH allele, the two faster

growing clones contained the wild-type ptsH, whereas the

slightly slower growing strains carried either the Ser-46±

Ala (ptsH1, BL121), the Ser-46±Thr (ptsH2, BL122) or the

Ile-47±Thr ptsH mutation (ptsH3, BL123). Only a strain

synthesizing Ser-46±Asp mutant HPr could not be

obtained with this method, although PCR amplification

followed by DNA sequencing was carried out with 15

erythromycin-sensitive clones constructed with plasmid

pVMH4. Because the Ser-46±Asp ptsH mutation is

known to drastically lower PTS-mediated sugar transport

Table 1. Lactobacillus casei: strains and plas-mids used in this study. Strain or plasmid Genotype or properties Origin

L. caseiBL23 wild type Bruce ChassyBL30 man Veyrat et al. (1994)BL71 ccpA Monedero et al. (1997)BL72 man ccpA Gosalbes et al. (1997)BL121 ptsH1 (S46AHPr) This workBL122 ptsH2 (S46THPr) This workBL123 ptsH3 (I47THPr) This workBL124 ptsI::pVME800 This workBL126 ptsI1 (frameshift introduced into the

first EcoRI site of ptsI)This work

PlasmidpUC18 Pharmacia-BiotechpRV300 pBluescript SK2 with the pAMb1 EmR gene Leloup et al. (1997)pUCR-HI pUC18 with 1.6 kb PCR fragment with

part of ptsH and ptsIThis work

pVME800 pRV300 with a 865-bp EcoRI internalptsI fragment

This work

pVMS1 pRV300 with 9 kb fragment downstreamfrom ptsI

This work

pVMH1 pRV300 with part of ptsI, complete ptsHand 105 bp upstream from ptsH

This work

pVMH2 pVMH1 derivative (codon 46 of ptsHis GCT for Ala)

This work

pVMH3 pVMH1 derivative (codon 46 of ptsHis ACT for Thr)

This work

pVMH4 pVMH1 derivative (codon 46 of ptsHis GAT for Asp)

This work

pVMH5 pVMH1 derivative (codon 47 of ptsHis ACC for Thr)

This work

pVMR10 pVMH1 derivative with a frameshift in thefirst EcoRI site of ptsI

This work

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Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 570±584

and phosphorylation (Reizer et al., 1989), clones able or

unable to ferment lactose were tested.

The ptsH mutations affect CCR and diauxic growth

In order to test the effect of the different amino acid

substitutions in HPr on diauxie, the growth behaviour of

the mutants on basal MRS broth supplemented with 0.1%

glucose and 0.2% lactose was compared with that of the

wild type and a ccpA mutant (Fig. 4). As previously

demonstrated (Veyrat et al., 1994; Gosalbes et al., 1997;

Gosalbes et al., 1999), the L. casei wild-type strain

exhibited strong diauxic growth in the presence of these

two sugars with a lag phase of about 15 h separating

the growth phases on glucose and lactose, whereas in the

ccpA mutant strain this lag phase was reduced to 5 h. The

diauxic growth observed with the ptsH2 mutant was very

similar to that of the wild-type strain. By contrast, the lag

phase was only about 6 h for the ptsH1 mutant and in

between the wild type and ptsH1 mutant for the ptsH3

strain (10 h).

A similar gradation was found when the relief from

glucose-mediated repression of N-acetyglucosaminidase

activity was investigated. Whereas high activity of this

enzyme could be measured in ribose-grown wild-type

cells, glucose was found to inhibit its activity about 10-fold

(Fig. 5). Similarly to the ccpA mutant, the repressive effect

of glucose on N-acetylglucosaminidase had completely

disappeared in the ptsH1 mutant. Inhibition of N-acetyl-

glucosaminidase activity by the presence of glucose in the

growth medium was also clearly diminished in the two

other ptsH mutants (about twofold inhibition in the ptsH3

mutant and 2.5-fold inhibition in the ptsH2 mutant),

confirming the importance of Ser-46 phosphorylation of

HPr and of the amino acids in the vicinity of Ser-46 for

CCR in L. casei. Therefore, these two tests indicated that

there was a remarkable and progressive loss of catabolite

repression in the different mutants: wild-type , ptsH2 ,

ptsH3 , ptsH1 � ccpA.

The ptsH mutations affect inducer exclusion in L. casei

To test a potential effect of the ptsH mutations on inducer

exclusion of non-PTS sugars, we decided to study ribose

Fig. 3. Schematic presentation of possible recombination events during the construction of ptsH mutants with the ptsI1 strain BL126 and the pVMHplasmids containing the various ptsH alleles. Integration of the pVMH plasmids carrying a mutation in ptsH (indicated by the filled circle) into thechromosome of BL126 carrying a frameshift mutation in ptsI (indicated by the filled triangle) by Campbell-like recombination could take place atthree different locations, resulting in the three different DNA arrangements presented under `1st recombination'. Integrants obtained by the first andsecond type of recombination exhibited a Lac2 phenotype, whereas type 3 integrants could slowly ferment lactose (probably due to a readthroughfrom a plasmid-located promoter). Type 3 integrants obtained with each of the three pVMH plasmids were grown for 200 generations withoutselective pressure to allow the second recombination leading to the excision of the pVMH plasmids. Again, three possibilities existed for the secondrecombination providing either a lac2 strain (3a), a wild-type strain (3b) or the desired ptsH mutant (3c) (see 2nd recombination). As only the Ser-46±Asp ptsH mutation was found to exert an inhibitory effect on the phosphocarrier activity of HPr, erythromycin-sensitive, lactose-fermentingcolonies were isolated from `type 3 0 integrants obtained by single recombination. These clones contained either the wild-type ptsH (secondrecombination according to 3b) or one of the three desired ptsH alleles (second recombination according to 3c). Amplification of the ptsH gene byPCR and DNA sequencing allowed us to distinguish between these two possibilities and ptsH mutants with the three different ptsH alleles couldthus be obtained.

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and maltose uptake in L. casei. When L. casei wild-type

cells were grown in a medium containing glucose and

either ribose or maltose, a diauxic growth behaviour

similar to that obtained with cells growing in the presence

of glucose and lactose was observed (data not shown).

However, whereas the lag time of the diauxic growth in

the presence of glucose and lactose was not or only partly

reduced in the ptsH mutants (Fig. 4), the diauxic growth

completely disappeared when the ptsH strains were

grown in a medium containing glucose and either maltose

or ribose (data not shown). These results suggest that

phosphorylation of HPr at Ser-46 plays an important role

in regulation of the utilization of these two non-PTS

sugars by L. casei. In order to distinguish whether this

effect was mediated via interaction of the CcpA/P±Ser±

HPr complex with cre sequences or via interaction of P±

Ser±HPr with a sugar permease according to the

proposed mechanism of inducer exclusion ( Ye et al.,

1994a, b; Ye and Saier, 1995a, b), sugar transport

experiments were performed. The uptake of ribose by

ribose-grown L. casei wild-type cells is shown in Fig. 6A.

The addition of glucose to ribose-transporting wild-type

cells caused no inhibition of ribose uptake but instead

increased the transport about fourfold.

In contrast to the stimulatory effect exerted by glucose

on ribose uptake, maltose transport was found to be

instantaneously arrested when glucose was added to L.

casei wild-type cells transporting maltose. Maltose uptake

was also completely abolished when glucose was added

to the cell suspension 10 min before the addition of

maltose (Fig. 6B). The ptsH1 (S46AHPr) mutant showed

a completely different behaviour to the wild-type strain

(Fig. 6C). Maltose uptake in this strain was slightly higher

and the addition of glucose caused a further increase of

the maltose transport rate. A similar stimulatory effect of

glucose on maltose transport was found for the ptsH2

(S46THPr) mutant (Fig. 6D), whereas no change in the

maltose transport rate following glucose addition was

observed for the ptsH3 (I47THPr) mutant (Fig. 6E). In the

ptsI mutant BL126, which is unable to transport glucose

via the PTS, the presence of glucose exerted no inhibitory

effect on maltose uptake (Fig. 6F). We measured glucose

uptake in the ptsI mutant BL126 and found that glucose

was transported 10 times more slowly than the wild-type

strain (data not shown). The slower glucose uptake and

metabolism was probably responsible for the failure of

glucose to elicit inducer exclusion in the ptsI mutant strain.

By contrast, in a ccpA mutant strain, glucose exerted an

inhibitory effect on maltose uptake identical to that

observed with the wild-type strain. This result clearly

established that CcpA is not involved in glucose-triggered

maltose exclusion.

To make sure that growing the cells for 30 min in

glucose-containing medium had no drastic effect on

expression of the maltose genes, we carried out inducer

exclusion experiments with cells that had not been

exposed to glucose. Under these conditions, addition of

glucose to maltose transporting cells exerted a strong

inhibitory effect on maltose uptake in the wild type and

ccpA mutant strains, although maltose continued to be

slowly taken up by these cells after the addition of

glucose. By contrast, the presence of glucose completely

arrested maltose uptake by cells that had been grown on

glucose for 30 min. However, with the ptsH1, ptsH2 and

ptsH3 mutants grown only on maltose, glucose exerted no

Fig. 5. The effect of the various ptsH mutations on CCR of N-acetylglucosaminidase. The N-acetylglucosaminidase activitiesexpressed in nmol of product formed per min and mg of cells (dryweight) and determined in the L. casei wild type (wt) and the ccpA,ptsH1 (S46 A), ptsH2 (S46T) and ptsH3 (I47T) mutant strains grownin MRS basal medium containing 0.5% glucose or ribose arepresented.

Fig. 4. Growth behaviour of L. casei wild type and ccpA and ptsHmutant strains in MRS basal medium containing 0.1% glucose and0.2% lactose. The symbols represent: filled circles, wild-type BL23;filled squares, ccpA mutant BL71; open circles, ptsH1 mutant BL121;filled triangles, ptsH2 mutant BL122; open triangles, ptsH3 mutantBL123.

Regulatory functions of enzyme I and HPr in L. casei 577

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 570±584

inhibitory effect at all on maltose uptake (data not shown),

clearly establishing that the failure of glucose to inhibit

maltose transport in the ptsH mutant strains is not related

to pregrowing the cells in glucose-containing medium.

The observed inhibition of maltose transport could have

been due to elevated secretion of maltose fermentation

products when glucose was added to wild-type cells. In

the ptsH mutants, this glucose effect might have been

less pronounced. To exclude this possibility, we measured

sugar consumption by resting cells that had been grown

on maltose and, for the last 30 min before harvesting the

cells, on maltose and glucose. The results presented in

Fig. 7A confirm that maltose is not utilized in the presence

of glucose by L. casei wild-type cells. Maltose consump-

tion stopped immediately when glucose was added and

the maltose concentration remained constant in the

medium as long as glucose was present. Maltose

consumption restarted only when glucose had completely

disappeared from the medium. By contrast, the addition of

glucose to ptsH1 mutant cells taking up maltose caused

only a short transient inhibition of maltose consumption,

which was followed by the simultaneous utilization of both

sugars (Fig. 7B). Reduced uptake of glucose by the ptsH1

mutant does not seem to be responsible for the absence

of the inhibitory effect of glucose, as in this strain glucose

was utilized slightly faster compared with the wild-type

strain. These results suggest that phosphorylation of Ser-

46 in HPr is necessary for the exclusion of maltose from L.

casei cells by glucose and probably other rapidly

metabolizable carbon sources and that P±Ser±HPr

Fig. 7. Maltose consumption by resting cells of the L. casei wild-typestrain BL23 and the ptsH1 mutant BL121 in the presence or absenceof glucose. The symbols represent: squares, maltose concentration inthe medium in experiments without glucose; diamonds, maltoseconcentration and circles, glucose concentration in the medium whenglucose was added 3 min after the experiment had been started.

Fig. 6. The effect of glucose on maltose and ribose uptake by wildtype and ptsH mutant cells. Ribose transport by the L. casei wild-typestrain BL23 was measured in the absence and presence of glucose(A). Maltose transport in the absence and presence of glucose wasdetermined in the L. casei wild-type strain BL23 (B), the ptsH1 mutantBL121 (C), the ptsH2 mutant BL122 (D), the ptsH3 mutant BL123 (E),the ptsI mutant BL126 (F) and the ccpA mutant BL71 (G). Thesymbols represent: squares, ribose or maltose uptake in the absenceof other sugars; diamonds, ribose or maltose uptake with glucose(10 mM final concentration) added after 10 or 4 min respectively;triangles, the cells were incubated for 10 min in the presence of20 mM glucose before the maltose uptake reaction was started.

578 R. Viana et al.

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 570±584

plays an important role in the regulatory phenomenon

called inducer exclusion (Ye et al., 1994a, b; Ye and

Saier, 1995a, b).

Discussion

The major mechanism of CCR in B. subtilis (Deutscher

et al., 1997) is completely different from the c-AMP-

dependent CCR mechanism operative in Gram-negative

bacteria such as E. coli and Salmonella typhimurium

(Postma et al., 1993). The B. subtilis CCR mechanism

was thought to be widely distributed within low G1C

Gram-positive bacteria, as the components involved in

this mechanism, i.e. HPr kinase±phosphatase, HPr,

CcpA and the operator sites cre, could be found in most

low G1C Gram-positive bacteria. The participation of

CcpA in CCR has been established for several Gram-

positive bacteria. including in addition to bacilli Staphylo-

coccus xylosus (Egeter and BruÈckner, 1996), L. casei

(Monedero et al., 1997), Lactobacillus pentosus (Lokman

et al., 1997), Listeria monocytogenes (Behari and Young-

man, 1998) and L. lactis (Luesink et al., 1998). In addition,

spontaneous Streptococcus salivarius ptsH mutants have

been isolated in which the preferential use of glucose and

fructose over lactose and melibiose was abolished

(Vadeboncoeur et al., 1994; Gauthier et al., 1997). In

these mutants, Ile-47 or Met-48 of HPr were found to be

replaced with threonine or valine, respectively, suggesting

a role of HPr in CCR in this organism. Mutants carrying a

disrupted ptsH gene have been constructed with L. lactis,

which were subsequently transformed with plasmids

carrying the wild-type ptsHI operon or ptsHI operons

containing a ptsH allele encoding either Ser-46±Ala or

Ser-46±Asp HPr under control of a nisin-inducible

promoter. Overexpression of the plasmid-encoded ptsH

alleles in the L. lactis ptsH mutant suggested a role of

P±Ser±HPr in CCR of the galactose operon and in

carbon catabolite activation of the las operon in this

organism (Luesink et al., 1999), as CCR was observed

after transformation with the plasmid carrying the wild

type or the Ser-46±Asp ptsH but not with the Ser-46±Ala

ptsH.

In order to confirm the suggested general role of

P±Ser±HPr in CCR in Gram-positive bacteria, we cloned

the ptsH and ptsI genes of L. casei and constructed

chromosomal ptsH mutants altered at the ATP-dependent

phosphorylation site of HPr in a second Gram-positive

organism. As in most other bacteria, ptsH and ptsI seem

to be organized in an operon in L. casei, as a major

transcript corresponding to the size of the combined

genes could be detected. The hybridization bands

observed in the Northern blots indicated that there is

some glucose induction in the wild type and ccpA mutant

and all the mutants tested showed very low expression of

the operon on ribose, which together with the fact that the

ptsI (BL126) mutant does not express this operon is

suggesting that PTS activity may be required for induc-

tion. CcpA did not seem to be implicated in regulation of

ptsHI expression, although the man mutation had some

effect. ptsI mutants were constructed that had lost the

capacity to ferment a large variety of carbohydrates

including fructose, mannose, mannitol, sorbose, sorbitol,

amygdaline, arbutine, salicine, cellobiose, lactose, taga-

tose, trehalose and turanose. Some of these carbohy-

drates might not be taken up by the PTS, but their

transport or metabolism might rather depend on a

functional PTS, as has been observed for glycerol uptake

and metabolism in Gram-positive and Gram-negative

bacteria (Postma et al., 1993).

In order to study the importance of phosphorylation of

HPr at Ser-46 for CCR, diauxic growth and inducer

exclusion, we tried to construct four different L. casei ptsH

mutants in which HPr was expected to be phosphorylated

at Ser-46 to various degrees. Unfortunately, we did not

succeed in obtaining a strain producing the Ser-46±Asp

mutant HPr, which has been shown to resemble

structurally P±Ser±HPr and which would have mimicked

the presence of completely phosphorylated HPr in the

cell. This mutation seems to be toxic in L. casei cells, as

some of the clones potentially containing the Ser-46±Asp

mutation were found to have either undergone a second

mutation, changing, for example, the Asp in position 46 of

HPr to Tyr, or to have went through an abnormal second

recombination deleting the erythromycin resistance cas-

sette, but retaining both the wild type and the Ser-46±Asp

ptsH alleles. The other three L. casei ptsH mutants could

be constructed. The Ser-46±Ala ptsH mutation com-

pletely prevents ATP-dependent phosphorylation of HPr

(Eisermann et al., 1988), whereas the B. subtilis Ser-46±

Thr mutant HPr was found to be slowly phosphorylated by

ATP and the HPr kinase±phosphatase (Reizer et al.,

1989). By contrast the Ile-47±Thr mutant HPr was found

to be normally phosphorylated in S. salivarius cells

(Gauthier et al., 1997). Nevertheless, the Ile-47±Thr

ptsH mutation abolishes the preferential use of glucose

and fructose over lactose and melibiose and causes

derepression of a- and b-galactosidase, suggesting that

this mutation prevents the interaction of P±Ser±HPr with

CcpA in S. salivarius.

When introduced into L. casei, all three ptsH mutations

were found to diminish the repressive effect of glucose on

the activity of N-acetylglucosaminidase (Fig. 5). As with

the ccpA mutant strain BL71 complete relief from CCR

was observed for the ptsH1 mutant BL121 (Ser-46±Ala),

suggesting that CCR of N-acetylglucosaminidase in L.

casei is mediated via the CcpA/P±Ser±HPr-dependent

mechanism. The Ile-47±Thr mutation caused a less

pronounced relief from CCR, which was further weakened

Regulatory functions of enzyme I and HPr in L. casei 579

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 570±584

in the Ser-46±Thr mutant. The amount of ATP-phos-

phorylated Ser-46±Thr mutant HPr present in the cell is

difficult to predict. Although probably only slowly phos-

phorylated by the HPr kinase±phosphatase (Reizer et al.,

1989), the threonyl-phosphorylated form of the mutant

HPr might accumulate to relatively high concentrations in

the cell if the kinetic parameters for the dephosphorylation

reaction of the phosphorylated mutant HPr have changed

as well. This might explain why the Ser-46±Thr mutation

always had the weakest releasing effect on CCR and

diauxic growth. Nevertheless, the above results clearly

confirm that phosphorylation of HPr at Ser-46 is impli-

cated in CCR in L. casei and support the concept that the

CcpA/P±Ser±HPr-dependent CCR mechanism is widely

distributed within low G1C Gram-positive bacteria.

The ptsH1 and ptsH3 mutations also diminished the

lag time of the diauxic growth in glucose- and lactose-

containing medium, whereas the ptsH2 mutation (Ser-46±

Thr) had no such effect. A second mechanism involved in

CCR and regulation of diauxie of the L. casei lac operon

seems to be operative as even with a ccpA mutant strain

no complete relief from diauxic growth could be observed

(Fig. 4). This second mechanism probably depends on

P±His±HPr-catalysed phosphorylation of LacT, the tran-

scriptional antiterminator regulating the expression of the

lac operon in L. casei (Gosalbes et al., 1997; Gosalbes

et al., 1999). In B. subtilis, a similar P±His±HPr-

dependent control of the transcriptional regulators LicT

and LevR has been suggested to depend on phosphor-

ylation of HPr at Ser-46, as this regulation disappeared in

ptsH1 mutants (KruÈger et al., 1996; Martin-Verstraete

et al., 1998). The finding that the lag phase during diauxic

growth of a L. casei ptsH1 mutant is longer than in a ccpA

mutant might indicate that a third CcpA-dependent CCR

mechanism for the L. casei lac operon exists. Possibly as

a result of this complex regulation of the lactose system,

the effect of the ptsH mutations on diauxic growth was

clearer when the experiments were carried out with media

containing glucose and either maltose or ribose. With both

sugars, the diauxic growth observed with the wild-type

strain was completely abolished in the three ptsH

mutants.

Nevertheless, the repressive effect of glucose on the

metabolism of ribose and maltose seemed to be mediated

by two different mechanisms. The diauxic growth

observed with glucose and ribose is probably due to the

CcpA/P±Ser±HPr dependent CCR mechanism and not to

inducer exclusion, as the presence of glucose in the

transport buffer exerted a stimulatory effect on ribose

transport (Fig. 6A). A ptsI mutation was also found to

exert a stimulatory effect on ribose or arabinose uptake

and metabolism in L. sakei (Stentz et al., 1997). It is

possible that the presence of dephosphorylated PTS

proteins caused either by ptsI disruption or by the uptake

of a rapidly metabolizable PTS sugar leads to elevated

ribose uptake via an as yet unknown regulatory mechan-

ism. By contrast, maltose uptake was found to be

instantaneously arrested when glucose was added to

the transport buffer, suggesting that maltose metabolism

is regulated by an inducer exclusion mechanism (Fig. 6B),

although it cannot be excluded that expression of the

maltose operon is additionally regulated by the CcpA/P±

Ser±HPr dependent CCR mechanism. Whereas inducer

exclusion is mediated via enzyme IIAGlc in Gram-negative

bacteria, P±Ser±HPr has been suggested to carry out

this function in L. brevis ( Ye et al., 1994a, b; Ye and

Saier, 1995a, b). However, this assumption was based

only on in vitro experiments, which have been carried out

using the heterologous B. subtilis Ser-46±Ala mutant HPr

instead of L. brevis P±Ser±HPr. As a consequence, the

involvement of P±Ser±HPr in inducer exclusion in Gram-

positive bacteria remained questionable. The finding that

the repressive effect of glucose on maltose transport had

completely disappeared in the ptsH1 and ptsH2 mutants

provided for the first time in vivo evidence that P±Ser±

HPr is involved in inducer exclusion in Gram-positive

bacteria. This concept was further supported by the

recent construction of a L. casei hprK mutant unable to

phosphorylate wild-type HPr at Ser-46. As observed with

the ptsH1 and ptsH2 mutant strains, no inhibitory effect of

glucose on maltose uptake could be detected with the

hprk mutant (Dossonnet et al., 2000).

By contrast, this novel inducer exclusion mechanism is

independent of CcpA, as glucose was found to arrest

maltose transport in a ccpA mutant in a way similar to that

observed with the wild-type strain. Surprisingly, although

glucose exerted a short transient stop of maltose uptake

in the Ile-47±Thr ptsH mutant, maltose was subsequently

taken up at a rate similar to that observed in the

experiment carried out in the absence of glucose

(Fig. 6E). This suggests that the Ile-47±Thr ptsH mutation

affects not only the interaction of P±Ser±HPr with CcpA,

but that it also prevents the interaction with proteins of

non-PTS transport systems, which are subject to regula-

tion by inducer exclusion. In HPr of Gram-positive

bacteria, the highly conserved Ile-47, Met-48 and Met-

51 form a surface-exposed hydrophobic patch located

between the two phosphorylation sites Ser-46 and His-15

(Jia et al., 1994). To explain the loss of the repressive

effect of glucose and fructose on the metabolism of

lactose and melibiose in the Ile-47±Thr and Met-48±Val

S. salivarius ptsH mutants, it has been proposed that this

hydrophobic patch might be important for the interaction

of P±Ser±HPr with CcpA (Gauthier et al., 1997), which

was experimentally confirmed by NMR measurements

(Jones et al., 1997). As inducer exclusion is also

abolished in the Ile-47±Thr ptsH mutant, this hydrophobic

patch might as well be important for the interaction of

580 R. Viana et al.

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 570±584

P±Ser±HPr with non-PTS permeases regulated by inducer

exclusion.

Experimental procedures

Strains, plasmids and culture conditions

The L. casei strains and plasmids used in this study are listedin Table 1. L. casei cells were grown at 378C under staticconditions in MRS medium (Oxoid) or MRS fermentationmedium (Adsa-Micro) containing 0.5% of the indicatedcarbohydrates. For diauxic growth experiments, L. caseistrains were grown in MRS basal medium containing in 1 l;polypeptone (Difco), 10 g; meat extract (Difco), 10 g; yeastextract (Difco), 5 g; K2HPO4.3H2O, 2 g; sodium acetate, 5 g;di-ammonium citrate, 2 g; MgSO4, 0.1 g; MnSO4, 0.05 g andTween 80, 1 ml. The basal medium was supplemented withdifferent sugars at a final concentration of 0.5%, but for thediauxic growth experiments the sugar concentrations werechanged as indicated in the text. E. coli DH5a was grown withshaking at 378C in Luria±Bertani (LB) medium. Transformedbacteria were plated on the respective solid media containing1.5% agar. The concentrations of antibiotics used for theselection of E. coli transformants were 100 mg ml21 ampi-cillin, and 300 mg ml21 erythromycin and for the selection ofL. casei integrants 5 mg ml21 erythromycin. The sugarutilization pattern of certain strains was determined with theAPI50-CH galeries (Biomerieux).

Recombinant DNA and transformation procedures

Genomic DNA from L. casei strains was purified using thePurogene DNA isolation Kit (Gentra Systems). Restrictionand modifying enzymes were used according to therecommendations of the manufacturers. For DNA sequen-cing, a Perkin Elmer ABI310 automatic sequencer was usedand the reaction conditions and reagents were as recom-mended by the manufacturer. General cloning procedures inE. coli were performed according to Sambrook et al. (1989).L. casei strains were transformed by electroporation with aGene-pulser apparatus (Bio-Rad Laboratories,) as describedbefore (Posno et al., 1991).

PCR amplification, cloning of PCR fragments and reverse

PCR

For PCR amplification of the two fragments comprising part ofthe ptsHI operon, we used a Progene thermocycler (Real)programmed for 30 cycles, including the following threesteps: 30 s at 958C, 30 s at 508C and 1 min at 728C, followedby a final extension cycle at 728C for 5 min. The followingdegenerate oligonucleotides were used for the amplificationof DNA fragments containing part of ptsHI from L. casei:PTS-H2 (5 0-ATG GAA AAR CGN GAR TTY AAY-3 0)(MEKREFN); PTS-I3 (5 0-GCC ATN GTR TAY TGR ATYARR TCR TT-3 0) (NDLIQYTMA); PTS-I4 (5 0-CCR TCN SANGCN GCR ATN CC-3 0) (GIAASDG); where R stands for A orG, Y for C or T, S for C or G and N for any nucleotide.Underlined in parentheses are the N-terminal amino acid

sequence of HPr and the conserved enzyme I sequences,which served to design the primers.

Cloning of PCR fragments was achieved with the Sure-Clone Ligation Kit (Pharmacia Biotech). Cloning of theregions flanking the insertion site of plasmid pRV300 wasperformed as follows. DNA (10 mg) from L. casei BL124(obtained after integration of plasmid pVME800 into thechromosome of BL23) was digested with SacI or HindIII,diluted 500-fold, religated with T4 DNA ligase and differentaliquots were used to transform E. coli DH5a. Plasmid DNAwas isolated from several transformants and subsequentlysequenced. The region upstream of ptsH containing thecomplete promoter could not be obtained with this technique.Reverse PCR was therefore used to get this missing part ofthe ptsHI operon. For this purpose, DNA isolated from the L.casei wild-type strain BL23 was cut with Pst I and religatedwith T4 DNA ligase (Gibco-BRL). Twenty nanograms of theligated DNA and two primers derived from the 5 0 part of ptsHand oriented in opposite directions were used to specificallyamplify using PCR a 2.3 kb fragment containing the upstreamregion of ptsH. The sequence comprising 560 bp upstreamfrom the ptsHI promoter has been determined in this fragment.

RNA isolation and Northern blot analysis

L. casei strains were grown in MRS fermentation mediumsupplemented with 0.5% of the different sugars to anOD550 of between 0.8 and 1. Cells from a 10 ml culturewere collected by centrifugation, washed with 50 mM EDTAand resuspended in 1 ml of Trizol (Gibco BRL). One gram ofglass beads (diameter 0.1 mm) was added and the cells werebroken by shaking the cell suspension in a Fastprep apparatus(Biospec) twice for 45 s. RNA was isolated according to theprocedure recommended by the manufacturer of Trizol andquantified with the RiboGreen kit (Molecular Probes) using aVersaFluorTM fluorometer (Bio-Rad) prior to being separated byformaldehyde-agarose gel electrophoresis and transferred toHybond-N membranes (Amersham).

The RNA probes used in the Northern blots were obtainedeither with the EcoRI fragment of ptsI present in pVME800 orwith the 205 bp HindIII±EcoRV fragment of ptsH cloned inpRV300. As these plasmids are pBluescriptII SK2 deriva-tives, antisense RNA could be synthesized in vitro fromlinearized plasmids with T3 or T7 RNA polymerase using thereagents from the Boehringer Mannheim digoxigenin-RNAlabelling kit. Hybridization (558C), washing and staining wereperformed as suggested by the supplier. For approximativequantification of the relative intensity of the hybridizing bandsin the Northern blots, rRNA bands observed after the transferin the methylene blue-stained membranes were used as aninternal standard for each sample. For this purpose, bands inthe films after the Northern blots and the stained 23S rRNAbands in the membranes were scanned with an HP ScanJet5100C and quantified by densitometry with the SCION IMAGE

software (Scion). Then, the relative band intensities in thefilms were calculated for each sample.

Primer extension experiments

Using 20 ml of reverse transcriptase buffer (Amersham)

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Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 570±584

containing 0.5 mM dNTP, 15 mg of RNA was annealed for5 min at 658C with 0.2 pmol of a [32P]-5 0-labelled oligonu-cleotide with the sequence 5 0-ACT TGC TTG CCT GTA CC-3 0 (see PTS-P.E. in Fig. 1). The subsequent extensionreaction was carried out at 428C for 1 h using 10 U of AMVreverse transcriptase (Amersham). The cDNA product andthe products of sequencing reactions performed with thesame primer were loaded on a 6% polyacrylamide26 M ureagel and labelled bands were detected by autoradiography.

Enzymatic assays

For the N-acetylglucosaminidase assays, permeabilized L.casei cells were prepared following a previously describedmethod (Chassy and Thompson, 1983). The N-acetyl-glucosaminidase assays were carried out at 378C in a volumeof 250 ml, containing 10 mM potassium phosphate pH 6.8,1 mM MgCl2, 5 mM p-nitrophenyl N-acetyl-b-D-glucosami-nide (Sigma) and 5 ml of permeabilized cells. The reactionwas stopped with 250 ml of 5% Na2CO3 and the OD420 wasmeasured. Protein concentrations were determined with theBio-Rad dye-binding assay.

Protein purification

Cells from an overnight culture (1 l of MRS medium) werecentrifuged and washed twice with 20 mM Tris-HCl, pH 7.4.The cells were resuspended in 20 mM ammonium bicarbo-nate buffer, pH 8 (2 ml per gram of cell pellet), sonicated(Branson Sonifier 250) and then centrifuged to remove thecell debris. As HPr resists heat treatment, the supernatantwas kept at 708C for 5 min to precipitate most of the otherproteins. An additionnal centrifugation step was performed toremove the heat-denatured proteins. The supernatant wasloaded on a Sephadex G-75 column (42 cm 1 1.6 cm)equilibrated with 20 mM ammonium bicarbonate, pH 8,which was eluted with the same buffer, and fractions of1.5 ml were collected. To test for the presence of HPr inthese fractions, a mutant complementation assay with theStaphylococcus aureus ptsH mutant strain S797A wascarried out (Hengstenberg et al., 1969). HPr activity wasdetected in fractions 48±56. These fractions were pooled andconcentrated to a final volume of 500 ml.

Half of the partially purified HPr was separated by reversephase chromatography on a Vydac C-18 HPLC column(300 AÊ , 250 mm 1 4.6 mm; Touzart et Matignon). Solvent Awas an aqueous solution of 0.1% (v/v) trifluoroacetic acid andsolvent B contained 80% acetonitrile and 0.04% trifluoroace-tic acid. Proteins were eluted with a linear gradient from 5% to100% of solvent B in 60 min at a flow rate of 500 ml min21.

Fractions with a volume of about 500 ml were collectedmanually. The presence of HPr in the fractions was tested bya PEP-dependent phosphorylation assay containing 10 mMMgCl2, 50 mM Tris-HCl, pH 7.4, 10 ml aliquots of thefractions, 10 mM [32P]-PEP and 1.5 mg of B. subtilis enzymeI(His)6. Enzyme I(His)6 and HPr(His)6 of B. subtilis werepurified by ion chelate chromatography on a Ni±NTASepharose column (Qiagen) after expression from plasmidspAG3 and pAG2 respectively (Galinier et al., 1997).HPr(His)6 from B. subtilis was used as a standard in the

phosphorylation reactions. [32P]-PEP was prepared from[g-32P]-ATP via the pyruvate kinase exchange reaction(Roossien et al., 1983). The assay mixtures were incubatedfor 10 min at 378C and separated on 15% polyacrylamidegels containing 1% SDS (Laemmli, 1970). After drying thegels, radiolabelled proteins were detected by autoradiogra-phy. HPr was found to elute at 60% acetonitrile in fractions44±46. These fractions were pooled, lyophilized and aliquotscorresponding to < 0.5 nmol of HPr were used to determinethe first 21 N-terminal amino acids of HPr by automatedEdman degradation on a 473-A Applied Biosystems micro-sequencer.

Construction of ptsH mutants

Site-directed mutagenesis was performed in order to replacethe codon for Ser-46 of L. casei ptsH with a codon for Ala,Asp or Thr and the codon for Ile-47 with a codon for Thr. Forthis purpose, PCR amplification was carried out, using as atemplate the plasmid pVMH1 containing the L. casei wild-type pstH gene as well as the 5 0 part of the ptsI gene and asprimers the reverse primer of pBluescript (Stratagene) andone of the following oligonucleotides: 5 0ptsHS46A (5 0-AAGAGC GTT AAC TTG AAG GCT ATC ATG GGC G-3 0);5 0ptsHS46T (5 0-AAG AGC GTT AAC TTG AAG ACT ATCATG GGC G-3 0); 5 0ptsHS46D (5 0-AAG AGC GTT AAC TTGAAG GAT ATC ATG GGC G-3 0); 5 0ptsHI47T (5 0-AAG AGCGTT AAC TTG AAG TCT ACC ATG GGC G-3 0). In theseoligonucleotides, the codons for Ser-46 or Ile-47 werereplaced by the indicated codon (underlined). The resulting1.4 kb PCR fragments containing the ptsH alleles (fromcodon 40) and the 5 0 part of ptsI were digested with HpaI (theHpaI site present in ptsH before codon 46 is indicated initalics in the above primers) and SacI and used to replace thewild-type 1.4 kb HpaI±SacI fragment in pVMH1.

In order to confirm the presence of the mutations, thesequence of the ptsH alleles was determined in the fourconstructed plasmids. To eliminate mutations possiblyintroduced in the ptsI gene by the PCR amplification, the1.35 kb BalI±SacI fragment from pVMH1 was used toreplace the corresponding fragment in each of the fourplasmids containing the various ptsH alleles. A unique BalIsite is present 27 bp behind codon 46 of L. casei ptsH inpVMH1 and the pVMH1 derivatives carrying the differentptsH alleles.

Sugar transport and sugar consumption by resting cells

Cells were grown to mid-exponential phase in MRS fermen-tation broth containing 0.5% of the indicated sugars. Thirtyminutes before harvesting the cells, glucose was added to afinal concentration of 0.5% to fully induce the synthesis of theglucose-specific PTS transport proteins. Experiments werealso carried out without pregrowing the cells in the presenceof glucose. Cells were washed twice with 50 mM sodiumphosphate buffer, pH 7, containing 10 mM MgCl2 andresuspended in 50 mM Tris-maleate buffer, pH 7.2, contain-ing 5 mM MgCl2. Transport assays were carried out in 1 ml ofthis latter buffer containing 1% peptone and 0.6 mg of cells(dry weight). Samples were preincubated for 5 min at 378C

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prior to adding [14C]-labelled sugars (0.5 mCi mmol21,Isotopchim, Ganagobie-Peyruis) to a final concentration of1 mM. Samples of 100 ml were withdrawn at different timeintervals, rapidly filtered through 0.45 mm pore-size filters,washed twice with 5 ml of cold Tris-maleate buffer and theradioactivity retained was determined by liquid scintillationcounting.

In order to follow the sugar consumption by the L. caseiwild type and ptsH1 mutant strains, cells were grown andharvested as described for the transport studies and 18 mg ofcells (dry weight) were resuspended in 5 ml of 50 mM sodiumphosphate buffer, pH 7. After 5 min incubation at 378C,maltose and glucose were added to a final concentration of0.04% and 0.2% respectively. Samples of 300 ml werewithdrawn at different time intervals, boiled for 10 min andclarified by centrifugation. The sugar content in the super-natant was determined with a coupled spectrophotometrictest using a-glucosidase and hexokinase/glucose-6-P dehy-drogenase as recommended by the supplier (Boehringer).

Acknowledgements

We are thankful to M.-M. Boutillon for amino acid sequencing, to

A. Lepingle for DNA sequencing and to M. Zagorec for supplying

plasmid pRV300. A. Galinier is acknowledged for valuable

discussions. This research was supported by the Spanish

CICYT project No. ALI98-714, the CNRS, the INRA, the INA-

PG and the European Community Biotech Programme contract

No. BIO4-CT96-0380 and by the Medical Research Council of

Canada. R.V. and V.M. were supported by a grant from the

Spanish Secretariat of Universities and Research. During his

postdoctoral stay in the Laboratoire de GeÂneÂtique des Micro-

organismes at the INRA-CNRS, V.M. received a grant from the

Spanish Ministry of Education and Culture.

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