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Journal of Bacteriology, July 1999, p. 3928-3934, Vol. 181, No. 13
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Elements Involved in Catabolite Repression and
Substrate Induction of the Lactose Operon in Lactobacillus
casei
María José
Gosalbes,
Vicente
Monedero, and
Gaspar
Pérez-Martínez*
Departamento de Biotecnología,
Instituto de Agroquímica y Tecnología de Alimentos,
46100 Burjassot, Valencia, Spain
Received 28 December 1998/Accepted 19 April 1999
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ABSTRACT |
In Lactobacillus casei ATCC 393, the chromosomally
encoded lactose operon, lacTEGF, encodes an antiterminator
protein (LacT), lactose-specific phosphoenolpyruvate-dependent
phosphotransferase system (PTS) elements (LacE and LacF), and a
phospho-
-galactosidase. lacT, lacE, and
lacF mutant strains were constructed by double crossover.
The lacT strain displayed constitutive termination at a
ribonucleic antiterminator (RAT) site, whereas lacE and
lacF mutants showed an inducer-independent antiterminator
activity, as shown analysis of enzyme activity obtained from
transcriptional fusions of lac promoter (lacp)
and lacp
RAT with the Escherichia coli gusA
gene in the different lac mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis
detected a 100-nucleotide transcript starting at the transcription
start site and ending a consensus RAT sequence and terminator region.
In a ccpA mutant, transcription initiation was derepressed
but no elongation through the terminator was observed in the presence
of glucose and the inducing sugar, lactose. Full expression of
lacTEGF was found only in a man ccpA double
mutant, indicating that PTS elements are involved in the
CcpA-independent catabolite repression mechanism probably via LacT.
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INTRODUCTION |
Lactobacillus casei is a
lactic acid bacterium (LAB) found in many food products, such as
fermented vegetables, milk, and meat, as well as in the human body and
other natural environments. Recently, L. casei has been used
in new fermented milk products with original flavors for which certain
health benefits are claimed.
During milk fermentation, lactose is fermented by LAB through different
pathways that differ in intermediary metabolites and their
bioenergetics. However, it is the transport and phosphorylation mechanism that will determine the metabolism of the translocated disaccharide. Three lactose transport mechanisms have been identified in LAB: lactose-galactose antiporters, lactose-H+ symport
systems, and the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) (19). The lactose-specific
PTS (Lac-PTS) is bioenergetically the most efficient one since the sugar is translocated and phosphorylated in a single step. This system
has been described only for Streptococcus mutans,
Lactococcus lactis, L. casei, and the non-LAB
Staphylococcus aureus (1-3, 11, 12, 18, 19, 23, 31,
37).
L. casei ATCC 393 has two lactose assimilation mechanisms,
the chromosomal Lac-PTS and a permease/-galactosidase system encoded by plasmid pLZ15 (13, 21). In L. casei ATCC
393[pLZ15
], the genetic structure and nucleotide
sequence of lactose assimilation genes differs from that in S. mutans, L. lactis, and Staphylococcus aureus
(22). In L. casei, the lactose genes are
transcribed as an operon, where the genes of the tagatose-6-phosphate
pathway are not included, as they are in other lac operons
described (19). The cluster lacTEGF encodes for a
regulatory protein (lacT), lactose-specific PTS proteins
(lacE and lacF), and a phospho--galactosidase
(P--Gal) (lacG). The promoter region contains a
cre element overlapping the 
35 region, which is followed
by a highly conserved sequence, the ribonucleic antiterminator (RAT)
sequence, and a terminator structure. It has previously been reported
(22, 34) that the expression of the lac operon in
L. casei ATCC 393[pLZ15] is subject to dual
regulation: carbon catabolite repression (CR) and induction by lactose
through transcriptional antitermination. Most CR was shown to be
mediated by the general regulatory protein CcpA that regulates
lac operon expression, possibly by binding to the
cre element at the lactose promoter (lacp).
However, an additional CcpA-independent CR effect was observed, which
was related to a functional glucose-specific PTS (EIIMan).
The second regulatory mechanism involves induction by lactose and is
mediated by LacT, a protein that belongs to the BglG family of
transcriptional antiterminators (3, 22). This later
mechanism is remarkably different from the induction system found in
the lac operon in L. lactis, where gene
expression is controlled by LacR with tagatose-6-phosphate the likely
inducer (18, 19). There is only one other antiterminator
protein described in LAB, BglR from L. lactis
(10).
Antitermination activity has been extensively studied in homologous
proteins, such as BglG from Escherichia coli, which
regulates -glucoside utilization genes (24, 32, 33, 43,
45). However, antiterminators seem to be more frequently found in
gram-positive bacteria. In Bacillus subtilis, four different
antiterminators have been described: SacT, SacY, LicT, and GlcT, which
control the expression of genes related to sucrose, -glucan, or
glucose assimilation (7, 17, 40, 42, 46, 47, 50). The
antitermination activity of all of these proteins is dependent on their
binding to a RAT sequence, resulting in the unwinding of a neighboring terminator structure in their respective mRNAs (9). BglG
from E. coli has been found to be phosphorylated by the
-glucoside PTS transporter, BglF (EIIBgl), which is
encoded in the bgl operon. Phosphorylated BglG is monomeric
and has no antitermination activity. However, in the presence of
-glucosides, BglG is dephosphorylated, which in turn promotes dimer
formation and subsequently full antitermination activity (4-6,
43, 44).
The antiterminator protein SacY controls expression of the
sacB gene in B. subtilis; it functions similarly
to BglG, and the PTS component involved in this case is SacX
(EIIScr) (15, 26). Tortosa et al.
(48) found two conserved domains (P1 and P2) common to a
number of transcriptional regulators, and through elegant in vitro
experiments they demonstrated phosphorylation of SacY by HPr-His-P.
Also, SacT and LicT were shown to be activated by phosphorylation by
the general components of the PTS (HPr and EI) in B. subtilis (8, 16, 27-29). Recently, Stülke et al. (47) have described the conserved domains common to
PTS-controlled transcriptional regulators as the PTS regulation domains
(PRDs). They proposed that the PRD closer to the N terminus (PRD-I) is related to the negative control played by the specific sugar permeases, whereas the PRD closer to the C terminus (PRD-II) shows a positive regulation by HPr.
To establish the role of the lac genes in the regulation of
the lac operon in L. casei ATCC
393[pLZ15], different mutants (lacE,
lacT, and lacF) were obtained by double crossover
and then used to monitor the expression of E. coli
-glucuronidase gene (gusA) as reporter under the control
of lacp. Transcriptional analysis was also performed in the
three lac mutants, in a man (encoding
EIIMan) mutant and in the ccpA man double
mutant. These experiments confirmed that the RAT-terminator/LacT
interaction is involved in the CcpA-independent CR mechanism and
demonstrated that the antiterminator activity of LacT is also
negatively regulated by the lactose-specific enzymes,
EIILac.
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MATERIALS AND METHODS |
Plasmids, bacterial strains, and growth conditions.
The
L. casei strains and plasmids used in this work are listed
in Table 1. L. casei cells
were grown in MRS medium (Oxoid) and MRS fermentation broth
(Adsa-Micro; Scharlau S.A., Barcelona, Spain) plus 0.5% carbohydrate
at 37°C under static conditions. E. coli DH5
was grown
with shaking at 37°C in Luria-Bertani (LB) medium. Bacteria were
plated on media solidified with 1.5% agar. When required, the
concentrations of antibiotics used were 100 µg of ampicillin, 300 µg of erythromycin, or 10 µg of chloramphenicol per ml to select
E. coli transformants and 5 µg of erythromycin or 5 µg
of chloramphenicol per ml for L. casei.
Recombinant DNA procedures.
Genomic DNA from L. casei strains was purified by using a Purogene DNA isolation kit
(Gentra Systems, Inc., Minneapolis, Minn.) as described by the
manufacturer. Restriction and modifying enzymes were used according to
the recommendations of manufacturers. General cloning procedures were
performed as described by Sambrook et al. (41).
To obtain plasmid pNZRAT, the promoter
lacp was amplified
with primers lac11 (5'-TAGCACTGATCATTAAA-3') and lac33
(5'-TTGCACTGGGAGGGGAT-3'),
using
L. casei DNA as
the template, and the PCR product was cloned
into the
SmaI
site of pUC18. The orientation of the insert was
checked by PCR. A
clone with the appropriate orientation was digested
with
EcoRI and
PstI, and the resulting fragment was
cloned into
PstI/
EcoRI-digested pNZ272 vector
(
36). The plasmid obtained,
pNZRAT, carries a
transcriptional fusion of the
L. casei lacTEGF promoter,
including the RAT sequence and terminator structure,
with the
gusA gene of
E. coli. pNZlac (
34)
carries a transcriptional
fusion of the
lac promoter,
lacking the RAT-terminator region,
with the
gusA gene.
L. casei strains were transformed by electroporation
with a
Gene-Pulser apparatus (Bio-Rad Laboratories, Richmond,
Calif.) as
described elsewhere (
38).
RNA isolation and Northern blot analysis.
L. casei
strains were grown in MRS fermentation medium with different sugars to
an optical density at 550 nm of 0.8 to 1. Cells from a 10-ml culture
were collected by centrifugation, washed with 50 mM EDTA, and
resuspended in 1 ml of Trizol (Gibco BRL). One gram of 0.1-mm-diameter
glass beads was added, and the cells were broken by shaking in a
Fastprep apparatus (Biospec, Bartlesville, Okla.) two times for 45 s. RNA was isolated as described by Gibco BRL, separated by
formaldehyde-agarose gel electrophoresis, and transferred to Hybond-N
membranes (Amersham).
RNA probes were obtained as follows. Probe Ppt was obtained from
plasmid pMJ64. A fragment from nucleotides (nt)
162 to +125
with
respect to the transcriptional start site of the
lac operon
was cloned between the
EcoRV and
BamHI sites of
pBluescriptII
SK
. Probe PIIgal was derived from pMJ33
carrying an internal fragment
of the
lac operon cloned in
the
EcoRV site of pT7Blue-T-vector
(Novagen). Antisense RNAs
were synthesized in vitro from linearized
plasmids with T3 and T7 RNA
polymerase, respectively, using the
reagents from the Boehringer
digoxigenin-RNA labeling kit as recommended
by the manufacturer.
Hybridization, washing, and staining were
done as described by the
supplier.
Enzymatic assays.
P--Gal and -glucuronidase activities
were assayed as previously described (14, 36) in
permeabilized L. casei cells (49).
Construction of L. casei lac mutants.
Mutations
in lacT, lacE, and lacF genes were
obtained by Campbell-like recombination using integrative plasmid
pRV300 (30).
Three plasmids, pMJ41, pMJ39, and pMJ45, were constructed by cloning a
fragment of the genes
lacT,
lacE, and
lacF, respectively,
in the integration vector. pMJ41
contained a PCR product obtained
by using primers lac11
(5'-TAGCACTGATCATTAAA-3') and lac2
(5'-CAACGATATAAGCGCAGATC-3').
The fragment was made blunt
ended and digested with
XbaI and then
cloned in pRV300
digested with
SpeI and
EcoRV, and an internal
PstI site was made blunt ended with the Klenow fragment of
E. coli DNA polymerase, thus generating a frameshift
mutation in
the cloned fragment. A 1-kb
HindIII/
BglII fragment from pLZ613
(
1) was introduced into pRV300, and the
SphI site
was treated
as was the
PstI site described above to give
pMJ45. pMJ39 carried
a 1.7-kb insert that had a 965-bp deletion in
lacE. For its construction,
two PCR fragments spanning the
regions upstream and downstream
of the desired deletion, which also had
newly created
SacI sites
(underlined regions below), were
digested with
SacI and ligated.
The oligonucleotides used as
primers in the PCRs were lac11 (5'-TAGCACTGATCATTAAA-3')
and
lac25 (5'-CGATAT
GAGCTCAGATC-3') for one fragment
and lac26
(5'-CAAC
GAGCTCAACAAAC-3') and lac6
(5'-CTTGCTGTCTAAATAGCC-3')
for the other fragment. The
ligation product was cloned as a
KpnI-digested
blunt-end
fragment into
KpnI/
EcoRV-digested pRV300. These
three
plasmids were used to transform
L. casei to
erythromycin resistance
(Erm
r), as integration of the DNA
fragments into
L. casei chromosome
occurred through a single
crossover by Campbell-like recombination.
For each transformation, one
Erm
r colony was grown for 200 generations without
antibiotic. Strains
that had undergone a second recombination event due
to the excision
of the vector could be detected as Erm
s.
The proper first and second recombination events were confirmed
by
Southern blot hybridization of the chromosomal DNA, and the
phenotype
of the appropriate mutants was
analyzed.
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RESULTS |
Transcriptional analysis of lac operon in
EIIMan and CcpA-deficient mutants.
In a previous study
(22), P--Gal was measured in BL23, BL23D
(man), BL71 (ccpA), and BL72 (man
ccpA) grown on glucose, lactose, and glucose plus lactose. Only
the double mutant, BL72, showed full derepression of P--Gal activity
when grown on the two latter sugars, whereas BL23D (man) had
24% of the activity found during growth on lactose. To investigate
this behavior at the molecular level, Northern blot experiments were
performed. With probe PIIgal (Fig. 1A), a
signal corresponding to a transcript of 4.5 kb was obtained in all
strains when the cells were grown on lactose (Fig. 1A, lanes 2, 6, 10, and 14). This size is in agreement with the expected length (4.8 kb) of
a transcript which runs from the transcriptional start site of
lac operon to the rho-independent terminator
t2 (3, 22). Additional bands
corresponding to the 23S and 16S rRNAs were also noticed, which is
sometimes the case in Northern blot experiments (25, 35,
39). In the wild-type strain, the 4.5-kb transcript was detected
only on lactose-grown cells. When a mixture of glucose and lactose was
used to grow all strains, transcription of the complete lac
operon occurred only in the man mutant and ccpA
man double mutant, although the intensity of the signal was lower
in BL23D (Fig. 1A, lanes 4 and 12). However, the 4.5-kb mRNA was never
found in glucose- or ribose-grown cells. These results correlate
perfectly with the P--Gal activities described elsewhere
(22).
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FIG. 1.
Northern blots prepared with RNA from different L. casei mutants impaired in the catabolite repression signal
transduction. The probes used were PIIgal (A) and Ppt (B); the strains
used were BL23D (man) (lanes 1 to 4), BL71 (ccpA)
(lanes 5 to 8), BL72 (man ccpA) (lanes 9 to 12), and BL23
(wild type) (lanes 13 to 16). Cells were grown on glucose (lanes 1, 5, 9, and 13), lactose (lanes 2, 6, 10, and 14), ribose (lanes 3, 7, 11, and 15) or glucose plus lactose (lanes 4, 8, 12, and 16). The diagram
shows the structure of the lac operon and the relative
positions of the RNA probes, PIIgal and Ppt, used in this experiment.
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RNAs from the same sources were used in another Northern blot with the
Ppt probe from the region between nt
162 and +125
with respect to the
transcriptional start site of
lac operon (Fig.
1). A
transcript of about 100 nt was detected in all samples where
large mRNA
species were not found. This indicates that in the
absence of inducer
(e.g., ribose-grown cells) transcription stopped
at the terminator
structure downstream of the promoter (Fig.
1B,
lanes 3, 7, 11, and 15).
In the wild type, the amount of this
100-nt transcript clearly
decreased under repressing conditions
(Fig.
1B; compare lanes 13, 15, and 16). However, in BL71 (
ccpA),
the intensities of the
100-nt RNA obtained under repressing and
nonrepressing conditions were
nearly identical (Fig.
1B, lanes
5, 7, and 8). This result corroborates
previous data (
22,
34)
and further suggests that CcpA
protein mediates catabolite repression
at the level of transcription
initiation, by binding to the
cre site of
lacp.
However, the absence of a functional CcpA protein
is not enough to
overcome glucose repression, as full derepression
of
lac
operon was found only in glucose-plus-lactose-grown cells
of the BL72
(
man ccpA) mutant. Possibly there is an additional
CcpA-independent catabolite repression mechanism that involves
the
transport of glucose by the PTS and also probably the LacT
protein.
Construction of L. casei lac mutants and expression
studies using the gusA gene as reporter.
The lactose
operon, lacTEGF, of L. casei BL23 encodes the
regulatory protein LacT, lactose-specific EIIA and EIICB PTS elements (LacF and LacE), and P--Gal (LacG) (22). To establish the
role of lac gene products in induction of the
lacTEGF operon, mutants BL154 (lacT), BL153
(lacE), and BL155 (lacF) were obtained by a
double-crossover event (Fig. 2). Although
all mutants turned out to be impaired in lactose fermentation,
P--Gal activity, encoded by lacG, could be used to report
the expression of the gene cluster. Consequently, lactose induction of
this activity was determined in the lac mutants and in the
wild-type strain grown on ribose and ribose plus lactose. In BL23,
expression of the lac operon was induced by lactose (9 and
17.9 nmol/min/mg [dry weight], respectively), whereas BL155
(lacF) showed higher P--Gal activity under both
conditions (27.7 and 29.5 nmol/min/mg [dry weight], respectively).
These results indicate that LacF is involved in the induction of the
lac operon. No activity was detected in BL154
(lacT), which would be impaired in the antiterminator protein, indicating a lack of induction in the presence of lactose. Surprisingly, no P--Gal activity was detected in BL153.
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FIG. 2.
Construction of lacT (A), lacE
(B), and lacF (C) mutants of L. casei. Plasmids
pMJ41, pMJ39, and pMJ45 carrying a frameshift in the PstI
site, a 0.965-kb deletion in lacE, and a frameshift in the
SphI site, respectively, were used to transform the
wild-type strain. The different mutants were selected after
double-crossover events.
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The effect of the mutations in
lacE,
lacT, and
lacF was also studied with the
-glucuronidase reporter
system in plasmids
pNZlac and pNZRAT (Table
2). In plasmid pNZlac, which lacks the
RAT-terminator area but contains the
cre element in
lacp,
-glucuronidase
activity was detected in similar
amounts in all strains grown
on ribose but was negligible when strains
were grown on glucose.
Moreover, no induction by lactose was found in
the wild type.
With pNZRAT, carrying the whole promoter, a remarkable
decrease
of activity occurred in the
lacT mutant with
respect to the other
strains, consistent with the antiterminator nature
of LacT. The
fact that
-glucuronidase activity in BL23 grown on
ribose was
20-fold higher than that detected in the
lacT
mutant suggests
that antitermination mediated by LacT also occurs to
some extent
in the absence of lactose and without glucose in the
wild-type
strain. The highest
-glucuronidase activity was found in
BL153
(
lacE) and BL155 (
lacF) grown under
noninducing conditions, indicating
a negative effect of
EII
Lac on the antiterminator activity of LacT. No release
of glucose
repression was observed in the different strains transformed
with
pNZlac or pNZRAT, but there was a greater repression when the
RAT-terminator element was present in the fusion used, except
in the
lacT strain, suggesting that there is a glucose repression
effect mediated by the LacT protein. In the case of strain BL153
(
lacE), divergent results were found between the homologous
(P-
-Gal)
and heterologous (
gusA) reporter systems.
However, the disruption
of
lacE might have caused a
translational defect in
lacG, as the
operon seems to be
transcribed at a high rate under nonrepression
conditions (Fig.
3A, and B, lanes 6 and 7).
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FIG. 3.
Northern blot analysis of RNA from the wild-type strain
and different lac mutants. The probes used were PIIgal (A)
and Ppt (B); the strains used were BL23 (wild type) (lanes 1 to 5),
BL153 (lacE) (lanes 6 to 9), BL154 (lacT) (lanes
10 to 13), and BL155 (lacF) (lanes 14 to 17). Cells were
grown on lactose (lane 1), on ribose (lanes 2, 6, 10, and 14), ribose
plus lactose (lanes 3, 7, 11, and 15), glucose (lanes 4, 8, 12, and
16), or glucose plus lactose (lanes 5, 9, 13, and 17).
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Influence of Lac-PTS elements on transcription of the lactose
operon.
We analyzed in vivo the role of lactose-specific proteins
by means of Northern blotting using the same RNA probes (PIIgal and
Ppt) as before. With the PIIgal probe, strong signals were obtained on
ribose- and ribose-plus-lactose-grown cells of BL155 (lacF)
(Fig. 3A, lanes 14 and 15), in agreement with the P--Gal activity
detected (see above). Very intense signals were also observed in BL153
(Fig. 3A, lanes 6 and 7). The remarkable difference between the latter
result and that with the wild-type strain (Fig. 3A, lanes 2 and 3)
indicates that in lacE and lacF mutants, lactose induction is not required for the antitermination activity. This suggests that in vivo, LacE or LacF interacts with the antiterminator, LacT. Again, there is an excellent correlation between the
hybridization patterns obtained with both probes. In the presence of
glucose, no large mRNA was detected, while the Ppt probe showed that
100-nt mRNA was observed in all samples, albeit with slight changes in intensity (Fig. 3). A smaller size of the full transcript was noticed
in ribose-grown cells of BL153, confirming the deletion generated by
recombination (Fig. 3B, lanes 6 and 7). In BL154 (lacT), no
4.5-kb mRNA or any other transcript larger than 100 nt could be
detected under any conditions (Fig. 3 and B, lanes 10 to 13),
indicating that RNA polymerase always stops at the terminator site when
LacT is absent.
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DISCUSSION |
The lactose operon, lacTEGF, in L. casei
ATCC 393 is located on the chromosome and encodes the transcriptional
antiterminator LacT, lactose-specific PTS proteins, and P--Gal
(22). Previous reports suggested that this operon could have
a regulatory system different from that described for the lactose
operons in L. lactis, S. aureus, and S. mutans (19). In this work, we describe the construction
and analysis of lacE, lacF, and lacT
mutants showing the involvement of EII elements of the Lac-PTS in
modulation of the lac operon of L. casei.
Lactose induction: modulation of LacT activity of
EIILac.
Regulation by antitermination has been
described for several operons in low-GC, gram-positive bacteria, such
as sacPA, sacB, bgl, licTS,
and glc of B. subtilis (7, 17, 40, 42, 46, 47, 50). In gram-negative bacteria, this system has been found in
the bgl operon of E. coli and the arb
operon of Erwinia chrysanthemi (20, 24, 32, 33, 43,
45). Antiterminator proteins of these systems have been assigned
to the BglG family on the basis of sequence homology,
cross-complementation, and the finding that their DNA targets share a
consensus RAT sequence (40). LacT from L. casei
shows homology to proteins of this family and has been shown to
complement the sacB system of B. subtilis
(3). Indeed, the His residues which are potentially
phosphorylated in PRD-I (H101 and H159) and PRD-II (H210 and H273) of
SacY are conserved in LacT (47). In wild-type L. casei ATCC 393, a transcript corresponding to the whole operon
lacTEGF was detectable only in the presence of lactose.
Taking into account the evidence shown here and knowledge gained from
homologous systems, the general mechanism by which LacT controls
transcription of the lac operon would be as follows. LacT
binds to the RAT sequence and prevents formation of the terminator in
the presence of lactose. In the absence of the inducer, the
antiterminator would be inactive, and transcription would start and
proceed only until the RNA polymerase reaches the
rho-independent terminator. Hence, only a small transcript spanning a region of about 100 nt (from the transcription start site to
the terminator) could be detected. As expected, in a lacT mutant constitutive termination (the presence of 100-nt transcript in
all conditions) was observed, consistent with the model for LacT.
The antiterminator proteins BglG in
E. coli and SacY in
B. subtilis are regulated through phosphorylation by
sugar-specific
PTS components or Hpr (
40,
47,
48). We
examined the role
of LacE (EIICB) and LacF (EIIA) in the regulation of
LacT antiterminator
activity, by inactivation of the
lacE
and
lacF genes. Transcriptional
studies showed an
inducer-independent antiterminator activity
in the
lacE and
lacF mutants, indicating that their corresponding
proteins
in the wild type may be involved in the inactivation
of the
antiterminator LacT. The model described for
B. subtilis antiterminators (
40,
47) could apply here, as in the absence
of functional II
Lac elements, the antiterminator LacT was
active, possibly because
it cannot be phosphorylated at one of the
conserved PTS regulation
domains (PRD-I) (
47). In the wild
type, the phosphoryl group
would be transferred to the incoming
inducing sugar, with the
same effect. This is the first report in which
sugar-specific
PTS elements from a lactic acid bacterium have been
conclusively
shown to be involved in the induction mechanism via an
antiterminator
protein.
Involvement of LacT in catabolite repression.
Preliminary
reports showed that lactose utilization was repressed by glucose, since
L. casei ATCC 393 displayed a likely diauxie (plateau
lasting 15 to 20 h) when grown on both sugars (49). It
was then shown that transcription of the lac operon was
subject to CcpA-mediated CR (22, 34). However, an additional
CcpA-independent CR mechanism was suggested when P--Gal activity was
monitored in mutants lacking CcpA and EIIMan individually
and in a ccpA man double mutant (22, 34). This additional CR mechanism was dependent on a functional glucose-PTS transporter. In the bgl and lev operons of
B. subtilis, HPr-dependent phosphorylation of the regulator
proteins controls a CcpA-independent CR effect (27, 28, 40).
However, the experiments with lacp-gusA fusions suggest that
the RAT sequence and LacT are involved in the proposed CcpA-independent
catabolite repression mechanism. In L. casei, Northern blot
analysis showed that transcription initiation of lacTEGF
operon is fully derepressed in the ccpA mutant, but that
there was no elongation beyond the terminator in the presence of
glucose. The double mutant BL72 (man ccpA) exhibits full
expression of the lactose operon when grown on glucose plus lactose;
therefore, LacT is active. This strain is impaired in the glucose-PTS
transporter, which probably links the EIIMan or other PTS
elements with the glucose repression mediated by LacT, which was
observed in the ccpA mutant.
The model of PTS-mediated control of PRD-containing regulators
described by Stülke et al. (
47), which also includes
the
mentioned antiterminators, could explain the regulation of LacT
activity. In this model, PRD-I would be phosphorylated by
inducer-specific
EII components of the PTS in the absence of inducer
and PRD-II
would be phosphorylated by HPr in absence of glucose (or
repressing
carbohydrates). Consequently, the antiterminator LacT would
exist
in three forms: (i) active, when dephosphorylated in PRD-I and
phosphorylated in PRD-II; (ii) inactive (noninduced), when
phosphorylated
in both domains; and (iii) inactive (CR), when PRD-II is
dephosphorylated
by HPr when grown on glucose. The presence
(nonphosphorylated
PRD-I) or absence (phosphorylated PRD-I) of the
inducer, lactose,
would not affect this later form. However, we cannot
exclude the
possibility that phosphorylation of PRD-II can be carried
out
by the sugar-specific PTS transporter, EII
Man. Clearly
the analysis of defined
L. casei mutants lacking the
HPr
gene (
ptsH) or defective
ptsH and either
lacF or
ccpA could
lead to a further
understanding of this regulation system, as
could studies on the
phosphorylation of
LacT.
| |
ACKNOWLEDGMENTS |
We thank B. M. Chassy for providing L. casei ATCC
393[pLZ15] and F. Morel-Deville for supplying the
pRV300 vector.
This work was financed by the EU project BIO4-CT96-0380 and by funds of
the Spanish CICyT (Interministerial Commission for Science and
Technology) (ref. ALI 95-0038). V.M. was supported by a grant of the
Consellería de Educación y Ciencia de la Generalitat Valenciana.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Biotecnología, Instituto de Agroquímica y
Tecnología de los Alimentos (CSIC), Polígono de la Coma
s/n, Apartado de correos 73, 46100 Burjassot, Valencia, Spain. Phone:
34 96 3900022. Fax: 34 96 3636301. E-mail:
gaspar.perez{at}iata.csic.es.
| |
REFERENCES |
| 1.
|
Alpert, C.-A., and B. M. Chassy.
1988.
Molecular cloning and nucleotide sequence of the factor IIIlac gene of Lactobacillus casei.
Gene
62:277-288[Medline].
|
| 2.
|
Alpert, C.-A., and B. M. Chassy.
1990.
Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei.
J. Biol. Chem.
265:22561-22568[Abstract/Free Full Text].
|
| 3.
|
Alpert, C.-A., and U. Siebers.
1997.
The lac operon of Lactobacillus casei contains lacT, a gene coding for a protein of BglG family of transcriptional antiterminators.
J. Bacteriol.
179:1555-1562[Abstract/Free Full Text].
|
| 4.
|
Amster-Choder, O.,
F. Houman, and A. Wright.
1989.
Protein phosphorylation regulates transcription of the -glucoside utilization operon in E. coli.
Cell
58:847-855[Medline].
|
| 5.
|
Amster-Choder, O., and A. Wright.
1990.
Regulation of activity of a transcriptional antiterminator in E. coli by phosphorylation.
Science
249:540-542[Abstract/Free Full Text].
|
| 6.
|
Amster-Choder, O., and A. Wright.
1993.
Transcriptional regulation of bgl operon of Escherichia coli involves phosphotransferase system-mediated phosphorylation of a transcriptional antiterminator.
J. Cell. Biochem.
51:83-90[Medline].
|
| 7.
|
Arnaud, M.,
M. Débarbouillé,
G. Rapoport,
M. H. Saier, Jr., and J. Reizer.
1996.
In vitro reconstitution of transcriptional antiterminator by the SacT and SacY proteins of Bacillus subtilis.
J. Biol. Chem.
271:18966-18972[Abstract/Free Full Text].
|
| 8.
|
Arnaud, M.,
P. Vary,
M. Zagorec,
A. Klier,
M. Débarbouillé,
P. Postma, and G. Rapoport.
1992.
Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity.
J. Bacteriol.
174:3161-3170[Abstract/Free Full Text].
|
| 9.
|
Aymerich, S., and M. Steinmetz.
1992.
Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family.
Proc. Natl. Acad. Sci. USA
89:10410-10414[Abstract/Free Full Text].
|
| 10.
|
Bardowski, J.,
S. D. Ehrlich, and A. Chopin.
1994.
BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in -glucoside utilization in Lactococcus lactis.
J. Bacteriol.
176:5681-5685[Abstract/Free Full Text].
|
| 11.
|
Breidt, F. J.,
W. Hengstenberg,
U. Finkeldei, and G. C. Stewart.
1987.
Identification of genes for lactose-specific components of the phosphotransferase system in lac-operon of Staphylococcus aureus.
J. Biol. Chem.
262:16444-16449[Abstract/Free Full Text].
|
| 12.
|
Breidt, F. J., and G. C. Stewart.
1987.
Nucleotide and deduced amino acid sequences of Staphylococcus aureus phospho--galactosidase gene.
Appl. Environ. Microbiol.
53:969-973[Abstract/Free Full Text].
|
| 13.
|
Chassy, B. M.,
E. Gibson, and A. Giuffrida.
1976.
Evidence for extrachromosomal elements in Lactobacillus.
J. Bacteriol.
127:1576-1578[Abstract/Free Full Text].
|
| 14.
|
Chassy, B. M., and J. Thompson.
1983.
Regulation of lactose-phosphoenolpyruvate-dependent-phosphotransferase system and -D-phosphogalactosidase galactohydrolase activities in Lactobacillus casei.
J. Bacteriol.
154:1195-1203[Abstract/Free Full Text].
|
| 15.
|
Crutz, A. M., and M. Steinmetz.
1992.
Transcription of the Bacillus subtilis sacX and sacY genes, encoding regulators of sucrose metabolism, is both inducible by sucrose and controlled by the DegS-DegU signalling system.
J. Bacteriol.
174:6087-6095[Abstract/Free Full Text].
|
| 16.
|
Crutz, A. M.,
M. Steinmetz,
S. Aymerich,
R. Richter, and D. Le Coq.
1990.
Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system.
J. Bacteriol.
172:1043-1050[Abstract/Free Full Text].
|
| 17.
|
Débarbouillé, M.,
M. Arnaud,
A. Fouet,
A. Klier, and G. Rapoport.
1990.
The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators.
J. Bacteriol.
172:3966-3973[Abstract/Free Full Text].
|
| 18.
|
de Vos, W. M.,
I. Boerrigter,
R. J. Van Rooyen,
B. Reiche, and W. Hengstenberg.
1990.
Characterization of lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis.
J. Biol. Chem.
265:22554-22560[Abstract/Free Full Text].
|
| 19.
|
de Vos, W. M., and E. E. Vaughan.
1994.
Genetics of lactose utilization in lactic acid bacteria.
FEMS Microbiol. Rev.
15:217-237[Medline].
|
| 20.
|
El Hassouni, M.,
B. Henrissat,
M. Chippaux, and F. Barras.
1992.
Nucleotide sequences of arb genes, which control -glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new -glycohydrolase family including enzymes from eubacteria, archeabacteria, and humans.
J. Bacteriol.
174:765-777[Abstract/Free Full Text].
|
| 21.
|
Flickinger, J. L.,
E. V. Porter, and B. M. Chassy.
1986.
The characterization of a plasmid isolated from Treponema hyodysenteriae and Treponoma innocens, abstr. H-174, p. 156.
In
Abstracts of the 86th Annual Meeting of the American Society for Microbiology. American Society for Microbiology, Washington, D.C.
|
| 22.
|
Gosalbes, M. J.,
V. Monedero,
C.-A. Alpert, and G. Pérez-Martínez.
1997.
Establishing a model to study the regulation of the lactose operon in Lactobacillus casei.
FEMS Microbiol. Lett.
148:83-89[Medline].
|
| 23.
|
Honeyman, A. L., and R. I. Curtiss.
1993.
Isolation, characterization and nucleotide sequence of Streptococcus mutans lactose-specific enzyme II (lacE) gene of the PTS and the phospho--galactosidase (lacG) gene.
J. Gen. Microbiol.
139:2685-2694[Abstract/Free Full Text].
|
| 24.
|
Houman, F.,
M. R. Diaz-Torres, and A. Wright.
1990.
Transcriptional antitermination in bgl operon of E. coli is modulated by a specific RNA binding protein.
Cell
62:1153-1163[Medline].
|
| 25.
|
Huang, F.,
G. Coppola, and D. H. Calhoun.
1992.
Multiple transcripts encoded by the ilvGMEDA gene cluster of Escherichia coli K-12.
J. Bacteriol.
174:4871-4877[Abstract/Free Full Text].
|
| 26.
|
Idelson, M., and O. Amster-Choder.
1998.
SacY, a transcriptional antiterminator from Bacillus subtilis, is regulated by phosphorylation in vivo.
J. Bacteriol.
180:660-666[Abstract/Free Full Text].
|
| 27.
|
Krüger, S., and M. Hecker.
1995.
Regulation of the putative bglPH operon for aryl--glucoside utilization in Bacillus subtilis.
J. Bacteriol.
177:5590-5597[Abstract/Free Full Text].
|
| 28.
|
Krüger, S.,
S. Gertz, and M. Hecker.
1996.
Transcriptional analysis of bglPH expression in Bacillus subtilis: evidence for two distinct pathways mediating carbon catabolite repression.
J. Bacteriol.
178:2637-2644[Abstract/Free Full Text].
|
| 29.
|
Le Coq, D.,
C. Lindner,
S. Krüger,
M. Steinmetz, and J. Stülke.
1995.
New -glucoside (bgl) genes in Bacillus subtilis: the bglP gene product has both transport and regulatory functions similar to those of BglF, its Escherichia coli homolog.
J. Bacteriol.
177:1527-1535[Abstract/Free Full Text].
|
| 30.
|
Leloup, L.,
S. D. Ehrlich,
M. Zagorec, and F. Morel-Deville.
1997.
Single crossing-over integration in the Lactobacillus sake chromosome and insertional inactivation of the pts and the lacI genes.
Appl. Environ. Microbiol.
63:2117-2123[Abstract].
|
| 31.
|
Maeda, S., and M. J. Gasson.
1986.
Cloning, expression and location of Streptococcus lactis gene for phospho--galactosidase.
J. Gen. Microbiol.
132:331-340[Abstract/Free Full Text].
|
| 32.
|
Mahadevan, S.,
A. E. Reynolds, and A. Wright.
1987.
Positive and negative regulation of bgl operon in Escherichia coli.
J. Bacteriol.
169:2570-2578[Abstract/Free Full Text].
|
| 33.
|
Mahadevan, S., and A. Wright.
1987.
A bacterial gene involved in transcription antitermination: regulation at a Rho-independent terminator in bgl operon of E. coli.
Cell
50:485-494[Medline].
|
| 34.
|
Monedero, V.,
M. J. Gosalbes, and G. Pérez-Martínez.
1997.
Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.
J. Bacteriol.
179:6657-6664[Abstract/Free Full Text].
|
| 35.
|
Murakawa, G. J.,
C. Kwan,
J. Yamashita, and D. P. Nierlich.
1991.
Transcription and decay of the lac messenger: role of an intergenic terminator.
J. Bacteriol.
173:28-36[Abstract/Free Full Text].
|
| 36.
|
Platteeuw, C.,
G. Simons, and W. M. de Vos.
1994.
Use of the Escherichia coli -glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria.
Appl. Environ. Microbiol.
60:587-593[Abstract/Free Full Text].
|
| 37.
|
Porter, E. V., and B. M. Chassy.
1988.
Nucleotide sequence of the -D-phospho-galactosidase gene of Lactobacillus casei: comparison to analogous pbg genes of other Gram-positive organisms.
Gene
62:263-276[Medline].
|
| 38.
|
Posno, M.,
R. J. Leer,
N. van Luijk,
M. J. F. van Giezen,
P. T. H. M. Heuvelmans,
B. C. Lokman, and P. H. Pouwels.
1991.
Incompatibility of Lactobacillus vectors with replicons derived from small cryptic Lactobacillus plasmids and segregational instability of the introduced vectors.
Appl. Environ. Microbiol.
57:1822-1828[Abstract/Free Full Text].
|
| 39.
|
Raya, R.,
J. Bardowski,
P. S. Andersen,
S. D. Ehrlich, and A. Chopin.
1998.
Multiple transcriptional control of the Lactococcus lactis trp operon.
J. Bacteriol.
180:3174-3180[Abstract].
|
| 40.
|
Rutberg, B.
1997.
Antitermination of transcription of catabolic operons.
Mol. Microbiol.
23:413-421[Medline].
|
| 41.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 42.
|
Schentz, K.,
J. Stülke,
S. Gertz,
S. Krüger,
M. Krieg,
M. Hecker, and B. Rak.
1996.
LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family.
J. Bacteriol.
178:1971-1979[Abstract/Free Full Text].
|
| 43.
|
Schnetz, K., and B. Rak.
1988.
Regulation of bgl operon of Escherichia coli by transcriptional antitermination.
EMBO J.
7:3271-3277[Medline].
|
| 44.
|
Schentz, K., and B. Rak.
1990.
-Glucoside permease represses the bgl operon of E. coli by phosphorylation of the antiterminator protein and also interacts with glucose-specific enzyme II, the key element in catabolic control.
Proc. Natl. Acad. Sci. USA
87:5074-5078[Abstract/Free Full Text].
|
| 45.
|
Schnetz, K.,
C. Toloczki, and B. Rak.
1987.
-Glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes.
J. Bacteriol.
169:2579-2590[Abstract/Free Full Text].
|
| 46.
|
Stülke, J.,
I. Martin-Verstraete,
M. Zagorec,
M. Rose,
A. Klier, and G. Rapoport.
1997.
Induction of Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT.
Mol. Microbiol.
25:65-78[Medline].
|
| 47.
|
Stülke, J.,
M. Arnaud,
G. Rapoport, and I. Martin-Verstraete.
1998.
PRD-a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria.
Mol. Microbiol.
28:865-874[Medline].
|
| 48.
|
Tortosa, P.,
S. Aymerich,
C. Lindner,
M. H. Saier, Jr.,
J. Reizer, and D. Le Coq.
1997.
Multiple phosphorylation of SacY, a Bacillus subtilis antiterminator negatively controlled by the phosphotransferase system.
J. Biol. Chem.
272:17230-17237[Abstract/Free Full Text].
|
| 49.
|
Veyrat, A.,
V. Monedero, and G. Pérez-Martínez.
1994.
Glucose transport by the phosphoenolpyruvate: mannose phosphotransferase system in Lactobacillus casei ATCC 393 and its role in carbon catabolite repression.
Microbiology
140:1141-1149[Abstract/Free Full Text].
|
| 50.
|
Zukovski, M. M.,
L. Miller,
P. Cosgwell,
K. Chen,
S. Aymerich, and M. Steinmetz.
1990.
Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory genes.
Gene
90:153-155[Medline].
|
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Abstract
Introduction
