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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.
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 |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
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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'-CGATATGAGCTCAGATC-3') for one fragment and lac26 (5'-CAACGAGCTCAACAAAC-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 (Ermr), as integration of the DNA fragments into L. casei chromosome occurred through a single crossover by Campbell-like recombination. For each transformation, one Ermr 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 Erms. 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.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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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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.
|
-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
EIILac 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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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 |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 IILac 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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* 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.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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