From Useful Bacterial Surface Proteins,
Unité de Recherches Laitières et Génétique
Appliquée, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France and the § Laboratoire de
Microbiologie et Génétique, CNRS, UMR 5122, Université Lyon I, 10 avenue R. Dubois,
69622 Villeurbanne Cedex, France
Received for publication, December 6, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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The specific oligopeptide transport system Opp is
essential for growth of Lactococcus lactis in milk. We
examined the biodiversity of oligopeptide transport specificity in the
L. lactis species. Six strains were tested for (i)
consumption of peptides during growth in a chemically defined medium
and (ii) their ability to transport these peptides. Each strain
demonstrated some specific preferences for peptide utilization, which
matched the specificity of peptide transport. Sequencing of the binding
protein OppA in some strains revealed minor differences at the amino
acid level. The differences in specificity were used as a tool to
unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in
L. lactis MG1363 deleted for its oppA gene. The
substrate specificity of these engineered strains was found to be
similar to that of the L. lactis MG1363 parental strain,
whichever oppA gene was expressed. In situ
binding experiments demonstrated the ability of OppA to interact with
non-transported peptides. Taken together, these results provide
evidence for a new concept. Despite that fact that OppA is essential
for peptide transport, it is not the (main) determinant of peptide
transport specificity in L. lactis.
The oligopeptide transport system Opp has been described in many
bacteria. This transport system may be involved in (i) nutrient acquisition in Lactococcus lactis (1, 2) or
Streptococcus thermophilus (3); (ii) recycling of cell wall
peptides for peptidoglycan synthesis in Salmonella
typhimurium and Escherichia coli (4); (iii) sensing of
extracellular signaling molecules (pheromones) required for the
initiation of competence and sporulation in Bacillus
subtilis (5-8), for the induction of conjugation in
Enterococcus faecalis (9, 10), and for the induction of virulence in several pathogenic bacteria (11-14); and (iv) growth at
low temperatures and intracellular survival in macrophages of
Listeria monocytogenes (15).
Opp is a member of a superfamily of highly conserved ATP-binding
cassette transporters. In Gram-negative bacteria, the transporter comprises a periplasmic solute-binding protein (OppA) and a translocon consisting of two integral membrane proteins (OppB and OppC) and two
membrane-bound cytoplasmic ATP-binding proteins (OppD and OppF). In
Gram-positive bacteria, OppA proteins are lipoproteins anchored to the
cell membrane by their N-terminal lipid moiety. Although several copies
of the gene encoding the binding protein might be present in
Gram-positive bacteria (3, 16), only one seems to be functional in
L. lactis (17, 18). OppA serves as an initial receptor. It
binds the substrate and delivers it to the transmembrane complex. It is
generally considered as the specificity determinant of the system,
whereas the rate of peptide transport is imposed by the rate of peptide
donation from OppA to the OppBC complex (19). The ATP-binding proteins
couple ATP hydrolysis to the transport process.
The substrate specificity of Opp from S. typhimurium has
been well established. S. typhimurium Opp transports
peptides from two to five amino acids with a broad range of sequences
(20). S. typhimurium OppA has a higher affinity for
tripeptides than for dipeptides (21). The Gram-positive bacterium
L. lactis shows significant differences in peptide uptake
and affinity compared with S. typhimurium. First of all,
L. lactis MG1363 is able to transport oligopeptides
containing up to 18 amino acids (22). Its binding protein
preferentially interacts with nonameric peptides, but is able to
bind peptides containing up to 35 amino acid residues (23).
Nevertheless, the strain preferentially uses hydrophobic basic peptides
with molecular masses ranging between 600 and 1100 Da (24), whereas di-
and tripeptides are not transported by the L. lactis Opp
system (16, 25).
In this work, we compared the ability of different strains of L. lactis to transport oligopeptides. In the L. lactis
species, we demonstrate the existence of variability in both the
specificity of peptide transport and the amino acid sequence of
L. lactis OppA
(OppALl).1 In an
attempt to correlate these diversities, we expressed the different
OppALl proteins in an oppA mutant with
its native oppDFBC operon still functional. The
ability of these engineered strains to transport peptides was compared
with that of the corresponding wild-type strains. We reveal that,
although OppALl was essential for peptide transport
function, there was no correlation between the sequence of the binding
protein and the specificity of peptide transport. These results suggest
a role for the OppBCDF component in imposing the specificity of peptide
transport in L. lactis.
Bacterial Strains, Plasmids, and Growth Conditions--
The
bacterial strains used in this study are listed in Table
I. L. lactis strains were
stored at
E. coli strains were grown with aeration at 37 °C in LB
broth supplemented with the appropriate antibiotics when necessary. L. lactis wild-type strains were grown at 30 °C in M17
broth or in chemically defined medium (CDM) (33). Engineered strains (SL5145, SL5146, SL5147, SL5152, SL5174, and SL5175) were grown in
similar media containing both 0.5% (w/v) glucose and 0.25% (w/v)
lactose and supplemented with erythromycin (2.5 mg/liter) and
chloramphenicol (2.5 mg/liter). In some growth experiments, one of the
essential amino acids (methionine, valine, histidine, glutamine,
leucine, or isoleucine) was omitted from CDM and replaced with a
peptide containing the omitted amino acid (final concentrations of
0.08, 0.28, 0.03, 0.27, 0.36, and 0.16 mmol/liter, respectively).
Growth experiments were performed with an ultramicroplate reader
(Bio-Tek Instruments, Inc., Winooski, VT) using 96-well sterile microplates. Each well contained 200 µl of culture medium and was
inoculated with ~107 colony-forming units/ml of an
overnight culture. Prior to inoculation, the strain was washed twice
with 50 mmol/liter
KH2PO4/K2HPO4 (pH 6.9).
The A600 was measured over 10 h every 30 min after gentle shaking. To prevent evaporation of the culture medium,
each well was overlaid with sterile paraffin oil. The apparent growth
rate is defined as the maximal slope of the semilogarithmic plot
against time of A600 measurements.
DNA Manipulations and Sequencing--
Total DNA of L. lactis strains was isolated from a 2-ml culture grown overnight in
M17 broth. Cells were harvested by centrifugation at 8000 × g for 10 min and resuspended for 2 h at 37 °C in 0.1 mol/liter Tris, 0.1 mol/liter EDTA, 25% (v/v) glucose, and 0.1 g/liter
mutanolysin (pH 7.0). Cells were lysed by incubation for 30 min at
37 °C in 0.1 mol/liter Tris, 0.01 mol/liter EDTA, 0.5% (v/v)
sarcosyl, 1 g/liter proteinase K, and 1.25 g/liter RNase. Proteins were removed by two successive 25:24:1 (v/v/v)
phenol/chloroform/isoamyl alcohol extractions and one 24:1 (v/v)
chloroform/isoamyl alcohol extraction. The last supernatant volume was
adjusted to 400 µl with Tris/EDTA/sarcosyl solution, and 50 µl of 3 mol/liter potassium acetate (pH 4.8) was added. DNA was precipitated
with 1.2 ml of ice-cold ethanol and finally resuspended in 200 µl of
10 mmol/liter Tris buffer.
The oppA genes from several lactococcal strains were
amplified by PCR using primers oppstart
(5'-ACACGCATGGACAAATTAAAAGTAACT-3') and oppstop
(5'-CGGGATCCAACTATTTGGTGGC-3'), designed according to the L. lactis SSL135 oppA sequence (17). In the case of
L. lactis IL1403, the primers were oppAstart*
(5'-GGGCATGCAAAAATTAAAAGTAACT-3') and oppAstop*
(5'-GGATCCCTATTTGGTTGCCATCTTAT-3') (16). PCR products were restricted
with SphI plus BamHI and cloned into expression plasmid pLET5 treated with the same restriction enzymes. The resulting hybrid plasmids (pLEM1 to pLEM4) were transferred into E. coli MC1022 (32), and structure was confirmed by restriction
digestion and DNA sequencing. After extraction from E. coli
(34), the pLEM derivatives were transferred into L. lactis SL5145 by electroporation as previously described (35).
Transformants were selected on M17 agar medium supplemented with 0.5%
(w/v) lactose, 5 mg/liter erythromycin, and 5 mg/liter chloramphenicol.
Purification of L. lactis Wg2 OppA-His6
Recombinant Protein and of anti-OppA Antibodies--
The
oppA open reading frame (not including the signal sequence
codons) from L. lactis Wg2 was PCR-amplified using
primers oppAsensHis2 (5'-CGCGGATCCAATCAAAGCTCAAGTACAAGTACA-3')
and oppArevHis1 (5'-CGGGGTACCCTATTTGGTGGCCAACTTAGC-3'). The
PCR product was restricted with BamHI plus KpnI
and cloned into the QIAexpress vector pQE30 (QIAGEN S. A.,
Courtaboeuf, France) restricted with the same enzymes. The
resulting plasmid (pQEW) was then introduced into E. coli NM522, yielding E. coli 8163.
Expression of the OppA-His6 protein was carried out in 500 ml of culture essentially as described by QIAGEN S. A. After removal of the culture medium, cells were resuspended in 10 mmol/liter Tris,
100 mmol/liter NaH2PO4, 8 mol/liter urea, 0.1%
(v/v) Triton X-100, 20 mmol/liter
Anti-OppA antibodies were purified from the serum of an immunized
rabbit (Valbex-Université Claude Bernard Lyon I,
Villeurbanne, France) as described above, except that the resin was
additionally washed with 40 ml of 50 mmol/liter Tris and 150 mmol/liter
NaCl (pH 7.4) and then with 40 ml of 50 mmol/liter Tris and 1 mol/liter NaCl (pH 7.4). Antibodies were eluted during a 30-min incubation with 4 mol/liter MgCl2. The elution fractions were
collected, dialyzed against sterile water, and lyophilized.
Immunoblotting--
L. lactis protein extracts were
first separated by SDS-12% polyacrylamide gel electrophoresis and then
electrotransferred onto nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany). The OppA protein was detected by
the method of Harlow and Lane (36) using anti-OppA polyclonal
antibodies (diluted 1:220), peroxidase-conjugated anti-rabbit IgG
(diluted 1:4000; Sigma, Saint-Quentin Fallavier, France), and the
BM chemiluminescence blotting substrate kit (Roche Molecular
Biochemicals, Meylan, France).
Milk Peptide Purification--
Milk proteins were precipitated
with 1% (v/v) trifluoroacetic acid. After removal of the proteins by
centrifugation at 10,000 × g for 10 min at 4 °C,
the supernatant was ultrafiltered through a 3000-Da cutoff membrane
(YM3, Amicon, Inc., Beverly, MA). Peptides were isolated by solid-phase
extraction using reverse-phase cartridges (Sep-Pak C18,
Waters Associates, Milford, MA). The peptides were separated at
40 °C by HPLC on a reverse-phase C18 column (Nucleosil (250 × 4.6 mm), Colochrom, Gagny, France) at a flow rate
of 1 ml/min. Solvents A and B were 0.115% (v/v) trifluoroacetic acid and 0.1% (v/v) trifluoroacetic acid and 60% (v/v) acetonitrile in
MilliQ water, respectively. A 5-min isocratic phase in solvent A was
followed by a linear gradient of solvent B (0-60% within 40 min). The
collected fractions were submitted to a second separation using 5 mmol/liter
KH2PO4/K2HPO4 (pH 6.9)
and 60% (v/v) acetonitrile in 5 mmol/liter
KH2PO4/K2HPO4 (pH 6.9)
as the solvents. The eluted peptides were collected, dried in a
SpeedVac concentrator (Savant Instruments, Inc., Farmingdale,
NY), resuspended in MilliQ water, and desalted using the first
HPLC separation system (trifluoroacetic acid/acetonitrile). Purified
peptides were identified by mass spectrometric analysis and N-terminal microsequencing.
Peptide Transport--
The transport assays were adapted from
previously described procedures (2, 37). Cells were grown to
A650 ~ 0.8 in CDM containing free amino acids
as the nitrogen source. Prior to transport assays, cells were washed
twice with 50 mmol/liter
KH2PO4/K2HPO4 (pH
6.9) and then de-energized for 30 min at 30 °C with 10 mmol/liter 2-deoxy-D-glucose (38). For each transport assay, cells
(A650 = 1; corresponding to 0.2 g of
cell protein/liter) were incubated for 5 min at 22 °C in the
presence of 25 mmol/liter glucose and 2 mmol/liter MgSO4.
When required, the serine proteinase inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (1 mmol/liter; Interchim, Montluçon, France) was added to the incubation mixture. We first verified that this inhibitor had no effect on transport process. Uptake
was initiated by adding the peptide at a final concentration of 50 µmol/liter, unless otherwise stated. One-ml samples were taken, and
cells were separated from the incubation medium by filtration using
cellulose acetate filters (0.45-µm pore size; Schleicher & Schüll). Cells were subsequently washed twice with 2 ml of
ice-cold KH2PO4/K2HPO4
(50 mmol/liter) at pH 6.9. Peptide uptake was monitored by determining
the intracellular concentration of free amino acids constituting the
peptide under study, as previously described (2). It is worth
mentioning that intact peptide could not be detected inside the cells
due to the high rate of peptide hydrolysis by internal peptidases (37).
The amino acids were first derivatized with o-phthalaldehyde
and then separated at 37 °C on a reverse-phase HPLC C18
column (UptiSelect (250 × 4.6 mm), Interchim) at a flow rate of 1 ml/min. Solvent A was 50 mmol/liter sodium acetate (pH 5.7) and 3%
(v/v) tetrahydrofuran, and solvent B was 95% (v/v) methanol and 5%
(v/v) tetrahydrofuran. A 5-min isocratic phase in 18% (v/v) solvent B
was followed by a linear gradient of solvent B (18-100% within 35 min). For detection of fluorescence, the excitation and emission
wavelengths were 340 and 455 nm, respectively.
Binding Assays--
The ability of lactococcal strains to bind
peptide VGDE was estimated at 30 °C as follows. Concentrated
de-energized cells (A650 ~ 15) were incubated
for 2 min (23) in 50 mmol/liter
KH2PO4/K2HPO4 (pH 6.5)
containing 500 µmol/liter VGDE. Cells were collected on a 0.22-µm
pore size filter (Schleicher & Schüll) and washed three times
with ice-cold potassium phosphate buffer. The filter was then coated
for 2 min with a solution of peptide YGGFL (500 µmol/liter) in
potassium phosphate buffer. After removing cells by filtration, the
peptides contained in the buffer were concentrated by solid-phase
extraction using an anion cartridge exchanger (Accell Plus QMA, Waters
Associates) and analyzed by HPLC as described above.
Peptide Utilization by Different Strains of L. lactis--
Six
strains of L. lactis were grown in CDM lacking an essential
amino acid in the free form and supplied by a pure peptide. None of the
strains was able to grow when the omitted amino acid was not replaced
by a peptide. Twenty-five different peptides were selected on the basis
of their various biochemical characteristics and their origin (Table
II). Six of them were purified from milk. They were initially chosen because of their disappearance from milk
after growth of some of the strains (data not shown).
Most peptides (18 of 25) were able to sustain growth of the six strains
at a maximal rate in CDM deprived of one essential amino acid in the
free form and provided in peptide form (Fig. 1). Growth systematically corresponded to
consumption of the peptide, as revealed by HPLC analysis of the culture
medium (data not shown). Nevertheless, except for CNRZ261, none of the
strains was able to use all the tested peptides as a source of amino
acids. Five peptides (VGDE, DRVYIHPFHL, RPKPQQFFGLM, ISQRYQK, and LPQY)
were differently consumed by the six L. lactis strains,
suggesting that the strains under study do not share the same
preferences for peptide utilization. Moreover, L. lactis Wg2
grew very poorly in the presence of the basic heptapeptide ISQRYQK as
the source of Gln or Ile, whereas this strain grew at a maximal rate in
the presence of the basic heptapeptide YPFPGPI (source of Ile) or TVYQHQK (source of Gln). This indicates that previous observations made
with L. lactis MG1363, which indicated a preference for
peptide utilization related to both the mass and the charge of the
peptide (24), cannot be extended to the L. lactis
species.
Variability in Peptide Utilization Corresponds to Variability in
Peptide Transport--
The peptides used for growth experiments were
incubated with cell-free extracts. They were all cleaved at the amino
acid level. The lack of growth of some strains in CDM was therefore not
due to an inability of the cells to cleave the peptide intracellularly. The peptides were also incubated either in the presence of the PI-type proteinase PrtP released from L. lactis
E8, CNRZ261, and Wg2 by incubation in a Ca2+-free buffer
(39) or in the presence of PIII-type PrtP anchored to
resting L. lactis CNRZ437 cells (note that autoproteolysis of PIII-type PrtP affects its specificity) (40). Only one
of them (RPKPQQFFGLM) was cleaved by PrtP. Other peptides were not hydrolyzed. This suggests that most (if not all) of the differences observed during growth experiments were not due to a difference in
extracellular cleavage of the peptide by PrtP. Consequently, the most
convenient hypothesis to explain the differences in growth is that the
strains under study do not have identical oligopeptide transport capabilities.
To ascertain this hypothesis, we tested the ability of four of the
strains to transport three peptides that revealed a difference in
preferences for peptide utilization between strains (VGDE, DRVYIHPFHL,
and RPKPQQFFGLM). To prevent extracellular cleavage of RPKPQQFFGLM by
PrtP, uptake was performed in the presence of 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride. As expected, the ability of
the strains to grow correlated with their ability to transport the
peptides (Table III). By analyzing the
presence of free Val or free Met in the external medium, we have
experimentally excluded the possibility that differences in peptide
uptake rates could be due to differences in amino acid efflux rates
between strains. Despite the fact that VGDE ensured a maximal growth
rate of L. lactis Wg2 or CNRZ437, a large difference in the
initial rate of uptake was observed between the two strains. On the
other hand, IL1403 did not grow at a maximal rate in the presence of VGDE, although its initial rate of VGDE uptake was higher than that of
Wg2. This indicates that the rate of peptide transport did not
determine the growth rate, at least under our experimental conditions.
The Binding Protein OppA Is Not Responsible for Variability in
Transport Specificity--
The binding protein is generally considered
to be responsible for the specificity of peptide transport in
ATP-binding cassette transporters (19). The oppA genes from
L. lactis strains MG1363, Wg2, and CNRZ437 were therefore
cloned and sequenced. Comparison of the deduced amino acid sequences,
including those available in the GenBankTM/EBI Data Bank
(SSL135, IL1403, and SK11), revealed some differences at the amino acid
level (Fig. 2). The OppA sequence of
MG1363 was identical to that of SSL135, but differed from those of Wg2, CNRZ437, IL1403, and SK11 by 9, 10, 72, and 2 residues,
respectively.
In an attempt to assign the differences in peptide transport pattern to
the variations in the OppA amino acid sequence, the oppA
genes from four different strains were introduced into the OppA-defective strain AMP15 (28). The oppA genes were cloned under the control of the T7 promoter into plasmid pLET5 (29). To ensure
the expression of oppA, the two plasmids pILpOL and pMG820
were also introduced in the recipient strain, yielding L. lactis strains SL5147, SL5152, SL5174, and SL5175, carrying the
oppA genes from MG1363, Wg2, IL1403, and CNRZ437,
respectively (Table I). As controls, plasmid pLET5 free of cloned
oppA plus pILpOL and pMG820 were introduced in MG1363 and
AMP15, yielding MG3+ and SL5146, respectively. The growth
rates and maximal bacterial populations in milk of strains MG1363,
MG3+, and SL5147 were similar (1.6 ± 0.1 h
As expected, the properties of peptide transport by SL5147 exactly
matched those of MG1363. Surprisingly, L. lactis SL5152, SL5174, and SL5175, carrying the oppA genes from Wg2,
IL1403, and CNRZ437, respectively, had the same preferences for peptide utilization as MG1363 (Fig. 4). For
example, strains SL5152, SL5174, and SL5175 were unable to grow in CDM
containing VGDE as the source of Val, whereas the corresponding
L. lactis wild-type strains Wg2, IL1403, and CNRZ437,
respectively, grew. Moreover, the growth rates of SL5152, SL5174,
SL5175, and MG1363 in CDM containing RPKPQQFFGLM as the source of Met
were comparable, although Wg2, IL1403, and CNRZ437 were unable to use
this peptide as the source of Met (Fig. 1). It is worth mentioning that
L. lactis strains SL5152, SL5147, SL5174, and SL5175 are
Prt
Uptake experiments confirmed that L. lactis strains SL5152,
SL5174, and SL5175 transported the same peptides despite their expression of different OppA proteins (Table
IV). The ability of the engineered
strains to transport specific peptides was identical to that of MG1363.
Initial rates of uptake were slightly lower than those obtained with
the wild-type strain. This was presumably due to a lower expression
level of the binding protein in the engineered strains compared with
MG1363 (Fig. 3). The complementation of MG163 OppALl Is Able to Bind a Non-transported
Peptide--
The ability of the binding protein OppA from L. lactis strain MG1363 to bind in situ non-transported
peptides was estimated. De-energized cells were first incubated in the
presence of the non-transported peptide VGDE (loading step). After
extensive washing, cells were then incubated in the presence of YGGFL
(chase step). The removal of VGDE from OppA by YGGFL was estimated by
submitting the chase buffer to HPLC analysis after concentrating its
peptide content by solid-phase extraction. No VGDE could be detected
when YGGFL was omitted from the chase buffer. Similarly, only traces of
VGDE were detected when using L. lactis AMP15, indicating
that this strain did not bind a significant amount of VGDE during the loading step. In contrast, L. lactis MG1363 released ~0.35
nmol of VGDE/mg of protein in the presence of YGGFL (mean of three determinations, S.D. = 0.02) (Fig. 5).
These results demonstrate the ability of L. lactis MG1363
OppA to interact in situ with a non-transported peptide.
Previous studies of substrate specificity in peptide transport
and/or utilization by L. lactis were performed using only
one strain, MG1363 (22, 24). The results demonstrated that the substrate specificity of L. lactis strain MG1363 is rather
atypical compared with other model Gram-negative bacteria such as
E. coli and S. typhimurium (20, 21). The present
study provides evidence for variability in the ability of L. lactis strains to consume peptides as a source of amino acids
during growth. For instance, MG1363 was unable to grow using the
tetrapeptide VGDE as the source of Val. This result is in agreement
with the established preferences for peptide utilization by this
strain, i.e. rejection of acidic peptides with low molecular
mass (24). However, this peptide was utilized by all the other strains
under study. Consequently, the MG1363 preferences are not
representative of the genus Lactococcus. We were able to
distinguish four strains on the basis of their capabilities to use
several peptides. This variability in peptide consumption resulted from
a difference in the ability of the strains to transport peptides. The
specificity of peptide transport by L. lactis strain MG1363
did not mirror that of all the lactococcal strains. Our first original
conclusion is that the L. lactis genus shows a
biodiversity in oligopeptide transport specificity.
Oligopeptide utilization by L. lactis requires the presence
of the functional oligopeptide transport system Opp. Previous studies
indicated that the binding protein OppA dictates the specificity of the
Opp system (28, 41). As proof, a The biodiversity in peptide transport specificity in lactococci was
used as a tool to unravel the role of the binding protein OppALl in determining the substrate specificity of L. lactis. Comparison of the OppALl sequences, including
those of the four strains studied, showed some amino acid
substitutions. To determine to what extent these substitutions were
responsible for the specificity, we cloned and expressed the
oppA genes of the four strains in the recipient strain
L. lactis MG1363 These intriguing results question the role of OppALl in the
specificity of peptide transport. If a peptide is not bound by OppALl, it will not be transported by the Opp system. In
this respect, OppALl might be considered as one determinant
of peptide transport specificity. Nevertheless, the specificity of
peptide binding by OppALl is very broad (23). The use of
recombinant strains allowed us to establish that the inability of
lactococci to transport specific peptides was not due to peptide
exclusion by OppALl. In other words, the specificity of
peptide transport (i.e. the ability to ultimately transport
specific peptides or not) is not solely dependent on
OppALl. The broad binding specificity of OppALl
excludes it from being the major specificity determinant of
oligopeptide transport. Our second original conclusion is that, although OppALl is absolutely necessary for peptide
transport, it does not (exclusively) determine its specificity.
Complementary results corroborate this interesting observation. Binding
experiments indicated that the tetrapeptide VGDE was able to interact
in situ with the oligopeptide-binding protein of L. lactis MG1363. This observation was in agreement with the reported
competitive inhibition exerted by VGDE on the transport of a reporter
peptide by L. lactis
MG1363.2 This indicates that
a peptide could bind to OppALl even if it is not
transported by the Opp system. These results further support our
conclusion, i.e. the specificity of peptide uptake by
L. lactis is not exclusively dictated by
OppALl.
The proposed model for oligopeptide transport by L. lactis
is a four-step process: (i) reversible binding of the substrate to the
open form of the binding protein; (ii) conformational change of the
binding protein, resulting in the partial entrapment of the substrate;
(iii) transfer of the partially entrapped substrate from the binding
protein to the transmembrane complex; and (iv) translocation of the
substrate across the membrane (23, 43). The peptide VGDE was
translocated by the three L. lactis strains Wg2, IL1403, and
CNRZ437 at different rates (Table III), although its
Km value was in the same range for the three strains (250 µmol/liter). This result indicates that the first step of transport, peptide binding by OppA, is not a limiting step for the
transport process. It is in agreement with previous kinetic analyses
that demonstrated that the rate of transport is determined by the
kinetics of peptide donation from the binding protein to the
translocator complex (19). In this kinetic model, the binding protein
would act as a plug that blocks the ligand from returning to the
external medium (44). A consequence of the peptide binding would be to
transmit a signal via the transmembrane complex to the ATP subunits
that results in an increase in the transporter affinity for ATP and
subsequently leads to the opening of the translocation pore and the
concomitant release of the substrate from the binding protein.
Transmembrane proteins OppB and OppC have been described as important
actors in several bacterial phenomena. For example, bacterial adherence
can be affected by mutations in the binding protein and other domains
of the permease complex (42, 45). Indeed, the adherence of
Streptococcus gordonii is affected by mutating either
the binding protein (SarA) or OppC (42). One possibility is that the
transmembrane complex OppBC acts as a filter. It is worth mentioning
that the three available lactococcal opp sequences display
variability in the OppB and OppC sequences (16-18). This sequence
variability might explain the transport variability among L. lactis strains. If this hypothesis is correct, it remains to be
shown how the transmembrane complex participates in the specificity of
the oligopeptide transport process (e.g. exclusion of
specific peptides from the channel or impairment of the interaction of
the transmembrane complex with the binding protein liganded with
specific peptides).
Our results show that the specificity of oligopeptide transport in
L. lactis is determined by at least two successive filters. The first filter, OppA, captures oligopeptides and initiates the transport process, but is rather aspecific. Our further work will focus
on the second filter, presumably the transmembrane channel, and will
aim at identifying the ensemble of determinants involved in
oligopeptide transport specificity in L. lactis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C in M17 broth (31) containing 0.5% (w/v)
glucose or lactose. E. coli strains were stored at
80 °C in LB broth (32) containing 10% (v/v) glycerol and
supplemented with chloramphenicol (5 mg/liter), erythromycin (2.5 mg/liter), or ampicillin (100 mg/liter) when required.
Strains and plasmids used in this study
-mercaptoethanol, and 1 mg/liter
lysozyme (pH 8.0). The cell suspension was incubated for 1 h at
25 °C with gentle agitation (200 rpm), and a clear cell-free extract
was obtained by centrifugation at 12,000 × g for 15 min. Purification of the OppA-His6 protein was carried out
under denaturing conditions by applying the cell-free extract to
nickel-nitrilotriacetic acid resin (QIAGEN S. A.) and using an elution
system of 10 mmol/liter Tris, 100 mmol/liter
NaH2PO4, and 8 mol/liter urea according to the
manufacturer's instructions. The OppA-His6-containing
fractions, as determined by SDS-PAGE analysis, were dialyzed against
sterile water and lyophilized.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Biochemical features of peptides used in this study
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Fig. 1.
Growth rates of L. lactis
wild-type strains in CDM in which one essential amino acid is
provided in peptide form. Bars (from left to right,
decreasing gray intensity) indicate L. lactis
MG1363, Wg2, IL1403, CNRZ437, CNRZ261, and E8, respectively. The mean
of two independent determinations is presented. Growth rates are
expressed as the percentage of the growth rate obtained in CDM
containing all the amino acids in the free form, i.e. 1.3, 0.9, 1.5, 0.9, 1.0, and 1.0 h 1 for L. lactis
MG1363, Wg2, IL1403, CNRZ437, CNRZ261, and E8, respectively. The amino
acid provided by each peptide is indicated in Table II. Peptide
concentration was adjusted to the corresponding free amino acid
concentration in CDM (33) as described under "Experimental
Procedures."
Initial rate of peptide transport by wild-type L. lactis strains
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Fig. 2.
Alignment of OppALl
sequences. The OppA sequences of L. lactis strains
SSL135, IL1403, and SK11 were obtained from Refs. 17, 16 and 18,
respectively. The OppA sequences from L. lactis strains
MG1363, Wg2, and CNRZ437 were determined in this work. The
corresponding GenBankTM/EBI accession numbers are AY189900,
AY189901, and AY189902, respectively. The histidine in position 2 of
OppA sequences from MG1363, Wg2, and CNRZ437 resulted from the presence
of an SphI site in primer oppstart used for gene
amplification.
1 and (1.0 ± 0.1) × 108
colony-forming units/ml, respectively). The amounts of OppA expressed by SL5147, SL5152, SL5174, and SL5175 grown in the appropriate medium
(CDM containing 5 g/liter glucose as the carbon source, 2.5 g/liter
lactose as the inducer (29), and 2.5 mg/liter each erythromycin and
chloramphenicol) were in the same range and slightly lower in each case
than that produced by L. lactis MG1363 as revealed by
Western blot analyses (Fig. 3). As a last
control, no significant differences in the uptake of the control
peptide YGGFL by MG1363 and MG3+ or SL5147 could be
detected, whereas SL5146 was unable to transport peptides.
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Fig. 3.
OppA expression by engineered and wild-type
strains of L. lactis revealed by immunoblot
analysis. Lane 1, L. lactis MG1363
(wild-type strain); lane 2, L. lactis SL5147
(L. lactis AMP15 expressing the oppA gene from
L. lactis MG1363); lane 3, L. lactis
SL5152 (L. lactis AMP15 expressing the oppA gene
from L. lactis Wg2); lane 4, L. lactis
SL5174 (L. lactis AMP15 expressing the oppA gene
from L. lactis IL1403); lane 5, L. lactis SL5175 (L. lactis AMP15 expressing the
oppA gene from L. lactis CNRZ437); lane
6, L. lactis SL5146 (negative control); lane
7, purified OppA-His6.
strains and therefore unable to cleave peptides in
the external medium prior to transport.
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Fig. 4.
Growth rates of engineered strains of
L. lactis in CDM in which one essential amino acid is
provided in peptide form. Bars (from left to right,
decreasing gray intensity) indicate L. lactis
SL5147, SL5152, SL5174, and SL5175, respectively. The mean of two
independent determinations is presented. Growth rates are expressed as
the percentage of the growth rate obtained in CDM containing all the
amino acids in the free form, i.e. 1.1 ± 0.1 h 1 for the four strains. The amino acid provided by each
peptide is indicated in Table II. Peptide concentration was adjusted to
the corresponding free amino acid concentration in CDM (33) as
described under "Experimental Procedures."
oppA with
the binding protein isolated from L. lactis strain Wg2,
IL1403, or CNRZ437 restored the substrate specificity of L. lactis MG1363 rather than that of the OppA donor strain.
Initial rate of peptide transport by engineered L. lactis strains
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Fig. 5.
Binding of VGDE by L. lactis
strain MG1363. L. lactis strain MG1363 was loaded with
VGDE (500 µM) and then chased with YGGFL (500 µM). Lower trace, L. lactis AMP15
(OppA mutant); upper trace, L. lactis MG1363 (wild-type strain). The peptide content of the chase
buffer was concentrated by solid-phase extraction prior to HPLC
analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
oppA mutant was
shown to be unable to transport peptides, and mutagenesis of OppA
affected the overall specificity of the protein for peptides (28).
oppA (strain AMP15) (28). Complementation by different oppA genes restored all four
functional Opp hybrid transporters. This suggests that none of the
substitutions in the different OppALl proteins impaired
donation of the substrate from the binding protein to the transmembrane
channel of MG1363. The substrate specificity of the four engineered
strains was found to be identical to that of L. lactis
strain MG1363. The substrate specificity of the complemented L. lactis strains SL5152, SL5174, and SL5175 did not match that of
the corresponding wild-type strains (Wg2, IL1403, and CNRZ437, respectively).
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ACKNOWLEDGEMENT |
---|
We thank Dr. A. Gruss for helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY189900, AY189901, and AY189902.
¶ To whom correspondence should be addressed. Tel.: 33-134-652-068; Fax: 33-134-652-065; E-mail: juillard@jouy.inra.fr.
Published, JBC Papers in Press, February 16, 2003, DOI 10.1074/jbc.M212454200
2 Helinck, S., Charbonnel, P., Foucaud, C., Piard, J.-C., and Juillard, V., J. Appl. Microbiol., in press.
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ABBREVIATIONS |
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The abbreviations used are: OppALl, L. lactis OppA; CDM, chemically defined medium; HPLC, high pressure liquid chromatography.
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