FEMS Microbiology Letters 180 (1999) 297^304

Relationships between arginine degradation, pH and survival in

Lactobacillus sakei
Marie-Christine Champomier Verge©s a; *, Manuel Zun¬iga b ,
Franc°oise Morel-Deville a;1 , Gaspar Përez-Mart|¨nez b , Monique Zagorec a ,
S. Dusko Ehrlich c
a
Laboratoire de Recherches sur la Viande, Institut National de la Recherche Agronomique, Domaine de Vilvert,
78352 Jouy en Josas Cedex, France
b
Departamento de Biotecnologia, Instituto de Agroquimica y Technologia de Alimentos (C.S.I.C.), Poligono de la coma s/n,
Apartado de Correos 73, 46100 Burjassot, Valencia, Spain
c
Laboratoire de Gënëtique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert,
78352 Jouy en Josas Cedex, France

Received 8 August 1999; received in revised form 20 September 1999; accepted 21 September 1999

Abstract

Lactobacillus sakei is one of the most important lactic acid bacteria of meat and fermented meat products. It is able to
degrade arginine with ammonia and ATP production by the arginine deiminase pathway (ADI). This pathway is composed of
three enzymes: arginine deiminase, ornithine transcarbamoylase and carbamate kinase, and an arginine transport system. The
transcription of the ADI pathway is induced by arginine and subjected to catabolite repression. In order to understand the
physiological role of the degradation of this amino acid we investigated the growth of bacteria under various conditions. We
show that arginine degradation is responsible for an enhanced viability during the stationary phase when cells are grown under
anaerobiosis. Arginine is necessary for the induction of the ADI pathway but in association with another environmental signal.
Using a mutant of the L-lactate dehydrogenase unable to lower the pH we could clearly demonstrate that (i) low pH is not
responsible for cell death during the stationary phase, so survival is due to another factor than elevated pH, (ii) neither low pH
nor oxygen limitation is responsible for the induction of the ADI pathway together with arginine since the ldhL mutant is able
to degrade arginine under aerobiosis. ß 1999 Federation of European Microbiological Societies. Published by Elsevier
Science B.V. All rights reserved.

Keywords : Lactic acid bacteria; L-LDH ; arc operon; arcA (ADI) pathway ; Phosphoenol pyruvate phosphotransferase system;
Catabolyte repression; Acid stress; Meat

* Corresponding author; 1. Introduction

E-mail: verges@biotec.jouy.inra.fr
1
The arginine deiminase pathway of arginine deg-
Present address: Laboratoire de reproduction et dëvel-
oppement des plantes ENS-Lyon, 46 allëe d'Italie, 69364 radation (Fig. 1) is composed of three enzymes: ar-
Lyon cedex 07, France. ginine deiminase (ADI, EC 3.5.3.6), catabolic orni-

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 9 2 - 9

FEMSLE 9073 28-10-99

298 M.-C. Champomier Verge©s et al. / FEMS Microbiology Letters 180 (1999) 297^304
Fig. 1. Schematic representation of the arginine deiminase pathway (ADI). ADI: arginine deiminase; cOTC : catabolic ornithine transcar-
bamoylase ; CK: carbamate kinase.
thine transcarbamoylase (cOTC, EC 2.1.3.3) and car-
bamate kinase (CK, EC 2.7.2.2). A fourth protein
important for this pathway is a membrane arginine
transport system (arginine/ornithine antiporter). This
pathway, leading to ATP and ammonia production,
is widely present among bacterial and archaebacteri-
al species such as Bacillus, Streptococcus, Clostridi-
um, Lactococcus, Pseudomonas, Mycoplasma and
Halobacterium [1^3] and can ful¢l various roles. In
Pseudomonas aeruginosa it is used exclusively under
anaerobiosis as sole energy source for growth [1]. In
Halobacterium salinarum it is involved in the fermen-
tative degradation of arginine [3] whereas in Bacillus
licheniformis it is involved in nitrogen regulation
under anaerobiosis [4].
Several types of complex regulations of the argi-
nine degradation pathway have been observed
among bacteria. In P. aeruginosa and B. licheniformis
oxygen is the main factor controlling its expression.
In some lactic acid bacteria such as Carnobacterium
and Streptococcus, relationships with sugar metabo-
lism and pH have been investigated, and repression
by glucose was observed [5^8].
The genes encoding the enzymes of the ADI path-
way have been most extensively studied in P. aeru-
ginosa [9], H. salinarum [3] and Rhizobium etli [10].
FEMSLE 9073 28-10-99
In Gram-positive bacteria the genetic organisation is
known for Clostridium perfringens [2] and B. lichen-
iformis [4]. The genes from Streptococcus sanguis
have also been cloned [11] but their sequence has
not yet been reported.
The four genes involved in arginine degradation:
arcA (ADI), arcB (cOTC), arcC (CK) and arcD (the
antiporter), usually form an operon, the organisation
and transcription of which vary in di¡erent species.
In P. aeruginosa the genes are cotranscribed in a
single polycistronic transcript arcDABC, which is
then processed by a RNase [12]. The transcription
of the arc operon is under the control of an Anr-
dependent promoter belonging to the Fnr-dependent
promoter family [9]. In H. salinarum the gene organ-
isation is arcRACB. Oxygen limitation yields di¡er-
ential induction of transcripts controlled by ArcR
[3]. In B. licheniformis, the gene cluster is arc-
RABDC. arcR encodes an ArgR protein with func-
tions similar to the Escherichia coli ArgR [4]
Among lactic acid bacteria Lactobacillus sakei,
formerly called L. sake [13], is one of the main spe-
cies belonging to the natural £ora of fresh vacuum-
packed meat. It is also used as starter in the meat
fermentation process mainly for its ability to rapidly
metabolise sugars resulting in a pH drop that pre-
 M.-C. Champomier Verge©s et al. / FEMS Microbiology Letters 180 (1999) 297^304 299

vents the development of spoilage micro-organisms with erythromycin 5 Wg ml31 for the propagation of
and ensures a right ¢rmness of the products. The the ptsI and arc mutant strains. Aerobiosis was ob-
ability of L. sakei to degrade arginine by the ADI tained by shaking £asks at 150 rpm and anaerobiosis
pathway was reported many years ago but this prop- was obtained by blowing nitrogen according to Hun-
erty was mainly used as a di¡erentiating feature be- gate [19]. Growth was followed by measuring optical
tween L. sakei and Lactobacillus curvatus, the closely density at 600 nm and by the determination of col-
related species also found in meat and meat products ony forming units after plating diluted aliquots on
[14]. The presence of the ¢rst two enzymes (ADI and MRS agar plates. The initial pH of the MCD me-
cOTC) in L. sakei was shown [15] but the physiolog- dium is 6.50. Values obtained after growth and re-
ical role of this pathway has not been extensively ported in the ¢gures represent the mean þ S.D. of at
studied yet. least three independent experiments.
In a previous work we have cloned and sequenced
the arc operon of L. sakei [16]. The gene order is 2.2. Arginine deiminase activities
arcABCTD. Mutants in the four genes involved in
the ADI pathway were constructed [16]. Upstream of These were determined as previously described
the arcA gene a CRE (catabolite responsive element) [16].
sequence was found indicating a regulation by catab-
olite repression. However, neither Fnr-like nor Anr-
like boxes were found upstream of arcA. In the 3. Results
present study we investigated the physiological role
of arginine degradation by the ADI pathway. In or- Previous studies have shown that L. sakei is aux-
der to explain the role of this pathway and its regu- otrophic for arginine and is unable to grow on argi-
lations we used mutants in the arc operon along with nine as sole carbon source [15]. Thus the degradation
a ptsI mutant and an ldhL mutant. of arginine needs the presence of glucose as a carbon
source but high glucose concentrations repress argi-
nine degradation. Arginine degradation can be ob-
2. Materials and methods served only when arginine is present in the growth
medium at 3 g l31 [15]. However, these studies made
2.1. Strains and growth conditions use of complex media, making detailed interpreta-
tions di¤cult.
The origin of strains is listed in Table 1. All L. In the present study we used a chemically de¢ned
sakei strains were routinely grown at 30³C on MRS medium [18] supplemented with two carbon sources
medium [17]. MCD medium [18] containing 0.05 or present in meat (glucose and ribose), at two arginine
3 g l31 arginine and 1 g l31 sugar was used for concentrations (0.05 g l31 is the minimal concentra-
physiological studies. The media were supplemented tion required for growth; 3 g l31 is the concentration
that fully induces the ADI pathway). In addition, the
Table 1 e¡ects of aerobiosis and anaerobiosis were investi-
Origin of L. sakei strains gated. We also tested the behaviour of RV2000, a
Strain Description Reference mutant of the L-lactate dehydrogenase (L-LDH),
23K Wild-type strain Laboratory collection which is unable to lower the pH of the medium
plasmid-cured [23] [20]. The growth and the viability of bacteria were
followed as well as the pH variations re£ecting argi-
RV2000 23K ldhL: :Cma [20]
nine degradation via the ADI pathway.
RV1000 23K ptsI: :pRV10 [22]
RV4000 23K acaA: :pRV401 [16] We tested di¡erent growth conditions regarding
RV4010 23K arcB: :pRV402 [16] arginine concentration (0.05 g l31 and 3 g l31 ), aero-
RV4020 23K arcC: :pRV403 [16] biosis or anaerobiosis, with glucose or ribose as the
RV4030 23K arcD: :pRV404 [16] carbon source. The results are presented in Fig. 2.
a
Cm: chloramphenicol resistance gene of pC194. Under aerobiosis on glucose (Fig. 2A) viable cells

FEMSLE 9073 28-10-99

300 M.-C. Champomier Verge©s et al. / FEMS Microbiology Letters 180 (1999) 297^304

Fig. 2. Growth of L. sakei strain 23K in MCD medium containing glucose 1 g l31 (circles) or ribose 1 g l31 (triangles) as carbon sources.
A low concentration of arginine (0.05 g l31 , ¢lled symbols) or an inducing concentration (3 g l31 , open symbols) was added. Bacteria
were grown under aerobiosis (A) or anaerobiosis (B). Experiments were repeated several times and a representative experiment is shown.
Standard deviation was less than 5% of the mean for experiments under anaerobiosis and less than 10% under aerobiosis. The ¢nal pH
measured after 48 h cultivation is indicated.

diminished soon after growth stopped, whatever the
arginine concentration. The ¢nal pH was 5.50 re£ect-
ing the production of acid from glucose fermenta-
tion. On ribose under aerobiosis, the growth rate
was slower and survival was observed during station-
ary phase. The ¢nal pH reached 6.88, indicating that
after the pH drop due to the ribose degradation,
ammonia production led to a pH rise.
Under anaerobiosis (Fig. 2B), both on glucose and
on ribose, the e¡ect of inducing concentration of
arginine was more pronounced. Survival was better
both on ribose and on glucose when arginine 3 g l31

FEMSLE 9073 28-10-99

 M.-C. Champomier Verge©s et al. / FEMS Microbiology Letters 180 (1999) 297^304 301

Fig. 3. Growth of L. sakei strain RV4000 in MCD medium containing ribose 1 g l31 under anaerobiosis. A low arginine (0.05 g l31 , ¢lled
symbols) or an inducing concentration (3 g l31 , open symbols) was added. Experiments were repeated several times and a representative
experiment is shown. Standard deviation was less than 5%. The ¢nal pH measured after 48 h cultivation is indicated.

was added. In both cases, the ¢nal pH was higher than on glucose and was correlated with the higher
than the initial pH of the medium, suggesting ammo- ¢nal pH (7.38 on ribose versus 6.73 on glucose).
nia production resulting from arginine degradation. These results suggest that a better survival in the
As under aerobiosis, survival was better on ribose stationary phase is linked to the degradation of ar-

Fig. 4. Growth of L. sakei strain RV1000 in MCD medium containing glucose 1 g l31 under anaerobiosis. A low concentration of argi-
nine (0.05 g l31 , ¢lled symbols) or an inducing concentration (3 g l31 , open symbols) was added. Experiments were repeated several times
and a representative experiment is shown. Standard deviation was less than 5% of the mean. The ¢nal pH measured after 48 h cultivation
is indicated.

FEMSLE 9073 28-10-99

302 M.-C. Champomier Verge©s et al. / FEMS Microbiology Letters 180 (1999) 297^304

ginine correlated with an increased pH value. Indeed
all the mutants in the arc operon were impaired in
survival during the stationary phase whether arginine
was added or not as shown for the arcA mutant
(RV4000) in Fig. 3.
A better survival on ribose than on glucose might
re£ect a repression of the ADI pathway on glucose.
The presence of a CRE element upstream of arcA,
responsible for the catabolite repression of the tran-
scription of the arc operon, might explain why on
glucose the ADI pathway is not active [16]. Carbon
catabolite repression requires the CcpA protein and
the phosphoenol pyruvate phosphotransferase sys-
tem (PTS) [21]. A mutant of enzyme I of the PTS
was previously constructed. Although this mutant
(RV1000) does not possess a functional PTS, it is
able to grow on glucose by a PTS-independent trans-
port system [18,22]. RV1000 was then tested for its
growth and survival on glucose. We could expect, as
was shown in several other bacteria, that in RV1000
catabolite repression is abolished. Indeed, RV1000
seemed to have a higher arginine degradation level
than the wild-type strain in the presence of glucose
(Fig. 4). The ¢nal pH in the medium with arginine
was 7.20, which suggests that arginine degradation is
higher than in the wild-type strain, for which pH was
6.73 when grown in the same conditions. These re-
sults support a catabolite repression e¡ect of glucose
which is abolished in the ptsI mutant. Furthermore,
no drop in population with arginine 0.05 g l31 was
observed but the pH was still low (5.50), suggesting a
di¡erent metabolism of glucose in the ptsI mutant.
All these results clearly indicate that the degrada-
tion of arginine via the ADI pathway is correlated
with an enhanced viability under anaerobiosis. This
role has been mentioned previously for other species
but it was not clearly demonstrated whether elevated
pH per se (due to ammonia production by the ADI
pathway) was responsible for this enhanced viability.
In order to investigate the putative role of pH in the
regulation of the ADI pathway and the survival of
L. sakei during stationary phase, we used a mutant
de¢cient in L-LDH activity. This mutant does not
produce lactate and does not lower the pH of the
medium below 6.20 [20]. As shown in Fig. 5, when
an inducing arginine concentration was omitted, the
cfu observed during stationary phase strongly de-
creased although the pH was high (6.90). This sug-
gests that the pH drop leading to the low pH value
after glucose consumption is not the factor respon-

Fig. 5. Growth of L. sakei strain RV2000 in MCD medium containing glucose 1 g l31 under aerobiosis. A low concentration of arginine
(0.05 g l31 , ¢lled symbols) or an inducing concentration (3 g l31 , open symbols) was added. Experiments were repeated several times and
a representative experiment is shown. Standard deviation was less than 10% of the mean. The ¢nal pH measured after 48 h cultivation is
indicated.

FEMSLE 9073 28-10-99

 M.-C. Champomier Verge©s et al. / FEMS Microbiology Letters 180 (1999) 297^304
sible for cell death after exponential growth. More-
over, when an inducing arginine concentration was
added, the ldhL mutant, like the wild-type, has an
increased cell viability during the stationary phase
certainly linked to arginine degradation as can be
deduced from the high pH value (8.35). These data
seem to indicate that the low pH reached after ex-
ponential growth is not responsible per se for cell
death. The protective e¡ect of arginine degradation
is not due to elevated pH since this strain, which
does not lower the pH, has a low viability during
the stationary phase and needs to degrade arginine
to survive. Moreover, in mutant RV2000, this argi-
nine degradation also took place during growth
under aerobiosis revealing that lack of oxygen is
not the factor responsible for the induction of the
ADI pathway, but rather another signal that might
be linked to carbohydrate metabolism. The behav-
iour of this strain indicates that the pH drop might
not be this signal. Furthermore, ADI measurements
con¢rmed that arginine degradation was associated
with survival. For the wild-type, maximum activities
were obtained when cells were grown under anaero-
biosis with arginine 3.19 þ 0.03 U under anaerobiosis
versus 0 under aerobiosis, whereas RV2000 had ac-
tivity under both aerobiosis (2.55 þ 0.31 U) and
anaerobiosis (8.88 þ 0.88 U).
4. Discussion
We have previously shown that transcription of
the arc genes encoding the ADI pathway is induced
by arginine and is repressed by carbon catabolite
repression [16]. In this work we show that arginine
degradation by L. sakei is clearly associated with a
higher survival during the stationary phase since (i)
the survival is correlated with the conditions of ex-
pression of the ADI pathway and (ii) survival is
abolished in the arc mutants. This degradation oc-
curs through the ADI pathway and no other arginine
degradation route could be detected, since arc mu-
tants could no longer degrade arginine and survive.
The presence of arginine is necessary to induce this
survival but another environmental factor is neces-
sary to promote this degradation. Catabolite repres-
sion of the arc operon was con¢rmed by the use of a
ptsI mutant in which arginine catabolism was no
FEMSLE 9073 28-10-99
303
longer repressed by glucose. Such a repression by
glucose has long been reported for various species
except for some Carnobacterium strains which are
reported to degrade arginine by the ADI pathway
even at high glucose concentrations [6]. In L. sakei
it lightens the role of the PTS in the catabolite re-
pression mechanism. Concerning anaerobiosis we
could demonstrate that the lack of oxygen per se is
not responsible for induction of the arc operon
which is di¡erent from what was observed in P. aer-
uginosa [9], R. etli [10] or B. licheniformis [4]. In H.
salinarum, the induction by oxygen limitation was
di¡erent for the di¡erent transcripts [3]. In L. sakei
arginine degradation could take place under aerobio-
sis but only when cells were grown in the presence of
ribose as the carbon source or in a ldhL mutant. In
this mutant, the end products resulting from glucose
catabolism are di¡erent than in the wild-type strain
[20]. Furthermore, ribose metabolism also leads to
di¡erent intracellular compounds and end products
since ribose is catabolised through the phosphoceto-
lase pathway. The signal responsible for ADI induc-
tion might thus be linked rather to the metabolic
state of the cells. Intracellular ATP, NADH or other
intermediate metabolites pools might be good candi-
date signals.
The enhanced viability might be attributed to the
pH rise due to ammonia production by the ADI
pathway as mentioned for S. sanguis [8]. These au-
thors showed that the enzymes of the ADI pathway
have their optimal pH of activity at lower values
than other intracellular enzymes. The activity of
the ADI catabolic pathway was supposed to protect
streptococci of the oral cavity from an acid environ-
ment. Nevertheless, these authors could demonstrate
that the beginning of arginine degradation was not
part of the acid-adaptive response [5]. In L. sakei,
this protective role against cell death cannot be re-
lated only to pH protection, since the ldhL mutant
that was unable to lower the pH below 6.20 also
needs arginine degradation to survive. Moreover, ar-
ginine degradation can take place even when the pH
is still high and no clear-cut correlation between low
pH and cell death was observed. Since ATP is pro-
duced from arginine degradation this indicates that
the energy level is the triggering factor for survival
rather than the pH.
The environmental conditions leading to the ex-
304 M.-C. Champomier Verge©s et al. / FEMS Microbiology Letters 180 (1999) 297^304
pression of the ADI pathway in L. sakei are: low
oxygen concentration, low glucose concentration or
presence of ribose, and arginine. They all seem to be
compatible with its natural meat environment and it
is likely that L. sakei has developed regulatory mech-
anisms particularly well adapted to this environment.
Acknowledgements
This work was supported by a Spanish-French co-
operation project PICASSO Action No. 96026. We
thank V. Davant for her technical assistance.
References
[1] Cunin, R., Glandsdor¡, N., Pierard, A. and Stalon, V. (1986)
Biosynthesis and metabolism of arginine in bacteria. Micro-
biol. Rev. 50, 314^352.
[2] Ohtani, K., Bando, M., Swe, T., Banu, S., Oe, M., Hayashi,
H. and Shimizu, T. (1997) Collagenase gene (colA) is located
in the 3P-£anking region of the perfringolysin O (pfo) locus in
Clostridium perfringens. FEMS Microbiol. Lett. 146, 155^
159.+.
[3] Ruepp, A. and Soppa, J. (1996) Fermentative arginine degra-
dation in Halobacterium salinarum (formerly Halobacterium
halobium) : genes, gene products and transcripts of the arc-
RACB gene cluster. J. Bacteriol. 178, 4942^4947.
[4] Maghnouj, A., Sousa de Cabral, T.G., Stalon, V. and Van der
Wauven, C. (1998) The arc RABDC gene cluster encoding the
arginine deiminase pathway of Bacillus licheniformis and its
activation by the arginine repressor argR. J. Bacteriol. 180,
6468^6475.
[5] Curran, T.M., Lieou, J. and Marquis, R.E. (1995) Arginine
deiminase system and acid adaptation of oral streptococci.
Appl. Environ. Microbiol. 61, 4494^4496.
[6] Leisner, J.G., Tidemand, J. and Larsen, L.M. (1994) Catabo-
lism of arginine by Carnobacterium spp. isolated from vacuum
packed sugar-salted ¢sh. Curr. Microbiol. 29, 95^99.
[7] Liu, S.Q. and Pilone, G.J. (1998) A review: arginine metabo-
lism in wine lactic acid bacteria and its practical signi¢cance.
J. Appl. Microbiol. 84, 319^327.
[8] Marquis, R.E., Bender, G.R., Murray, D.E. and Wong, A.
(1987) Arginine deiminase system and bacterial adaptation
to acid environments. Appl. Environ. Microbiol. 53, 198^200.
[9] Gallimand, M., Gamper, M., Zimmerman, A. and Haas, D.
(1991) Positive FNR-like control of anaerobic arginine degra-
dation and nitrate respiration in Pseudomonas aeruginosa.
J. Bacteriol. 173, 1598^1606.
FEMSLE 9073 28-10-99
[10] D'Hooghe, I., Vander Wauven, C., Michiels, J., Tricot, C., de
Wilde, P., Vanderleyden, J. and Stalon, V. (1997) The arginine
deiminase pathway in Rhizobium etli: DNA sequence analysis
and functionnal study of the arcABC genes. J. Bacteriol. 179,
7403^7409.
[11] Burne, R.A., Parsons, D.T. and Marquis, R.E. (1989) Cloning
and expression in Escherichia coli of the genes of the arginine
deiminase system of Streptococcus sanguis NCTC 10904. In-
fect. Immun. 57, 3540^3548.
[12] Gamper, M., Ganter, M., Polito, B. and Haas, D. (1992)
RNA processing modulates the expression of the arcDABC
operon in Pseudomonas aeruginosa. J. Mol. Biol. 226, 943^
957.
[13] Tru«pper, H.G. and de Clari, L. (1997) Taxonomic note: nec-
essary correction of speci¢c epithets formed as substantive
(nouns) in apposition. Int. J. Syst. Bacteriol. 47, 908^909.
[14] Montel, M.C., Talon, R., Fournaud, J. and Champomier,
M.C. (1991) A simpli¢ed key proved by DNA-DNA hybrid-
ization for identifying Lactobacillus and Carnobacterium of
meat and meat products. J. Appl. Bacteriol. 70, 469^472.
[15] Montel, M.C. and Champomier, M.C. (1987) Arginine catab-
olism in Lactobacillus sake isolated from meat. Appl. Environ.
Microbiol. 53, 2683^2685.
[16] Zun¬iga, M., Champomier-Verge©s, M.C., Zagorec, M. and Për-
ez-Martinez, G. (1998) Structural and functional analysis of
the arginine deiminase gene cluster of Lactobacillus sake.
J. Bacteriol. 180, 4154^4159.
[17] de Man, J.C., Rogosa, M. and Sharpe, M.E. (1960) A medium
for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130^
135.
[18] Lauret, R., Morel-Deville, F., Berthier, F., Champomier-
Verge©s, M.C., Postma, P., Ehrlich, S.D. and Zagorec, M.
(1996) Carbohydrate utilisation in Lactobacillus sake. Appl.
Environ. Microbiol. 62, 1922^1927.
[19] Hungate, R.E. (1969) A roll tube method for the cultivation
of strict anaerobes. In: Methods in Microbiology (Norris, J.R.
and Ribbons, D.W., Eds.), Vol. 3B, pp. 117^132. Academic
Press, London.
[20] Malleret, C., Lauret, R., Ehrlich, S.D., Morel-Deville, F. and
Zagorec, M. (1998) Disruption of the sole ldhL gene in Lac-
tobacillus sakei prevents the production of both L- and D-lac-
tate. Microbiology 144, 3327^3333.
[21] Saier Jr, M.H., Chauvaux, S., Cook, G.M., Deutscher, J.,
Paulsen, I.T., Reizer, J. and Ye, J.J. (1996) Catabolite repres-
sion and inducer control in Gram positive bacteria. Micro-
biology 142, 217^230.
[22] Stentz, R., Lauret, R., Ehrlich, S.D., Morel-Deville, F. and
Zagorec, M. (1997) Molecular cloning and analysis of the
ptsHI operon in Lactobacillus sake. Appl. Environ. Microbiol.
63, 2111^2116.
[23] Berthier, F., Zagorec, M., Champomier-Verge©s, M.C. and
Morel-Deville, F. (1996) High frequency transformation of
Lactobacillus sake by electroporation. Microbiology 142,
1273^1279.