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(Received for publication, July 28, 1994; and in revised form, November 11, 1994) From the
In the proteolytic pathway of Lactococcus lactis, milk
proteins (caseins) are hydrolyzed extracellularly to oligopeptides by
the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or
transport followed by intracellular hydrolysis, has been addressed.
Mutants have been constructed that lack a functional di-tripeptide
transport system (DtpT) and/or oligopeptide transport system (Opp) but
do express the P Lactic acid bacteria possess an active proteolytic system that
is involved in the degradation of milk proteins ( On the basis of
the size of the majority of the fragments formed from the hydrolysis of
caseins by the proteinase PrtP, peptidases have been implicated in the
further hydrolysis of the peptides outside the cell (Smid et
al., 1991; Pritchard and Coolbear, 1993). Extracellular peptidases
would allow the cells to utilize caseins more efficiently and
completely. The apparent discrepancy between the need for extracellular
peptidases and the experimental data supporting an intracellular
location of the enzymes has led us to investigate the
Figure 1:
The proteinase activity (A)
and growth on milk (B) of L. lactis wild type and
peptide transport mutants. The proteinase activity of L. lactis MI1 (
Fig. 2shows the time course of the intracellular amino acid
pools(1, 2, 3, 4, 5, 6, 7) for the
Opp
Figure 2:
Time course of intracellular amino acid
accumulation (1-7) for the wild type (A), the
oligopeptide transport mutant (B), the di-tripeptide transport
mutant (C), and the double mutant (D) upon the
addition of
A number of
observations regarding the accumulation of amino acids upon hydrolysis
of
The experiments described in this study have revealed a
number of important properties of the proteolytic pathway of L.
lactis. First, all of the essential and growth-stimulating amino
acids for L. lactis can be released from A large number of peptides are released
from In future studies, we aim to identify the time course of peptide
product formation and the size restriction and substrate specificity of
the oligopeptide transport system for its natural substrates. It is
always assumed that peptide transport systems do not transport
fragments longer than 5 or 6 residues (Payne and Smith, 1994). An
intriguing aspect is the observation that the Opp system of L.
lactis transports peptides with a size of at least 8 residues
(Tynkkynen et al., 1993), but perhaps the transportable
species can even be longer.
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1569-1574
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Casein-derived Peptides by the Oligopeptide Transport System Is a
Crucial Step in the Proteolytic Pathway of Lactococcus lactis(*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-type proteinase (specific for hydrolysis
of 
- and to a lesser extent 
-casein). The wild type strain
and the DtpT mutant accumulate all
casein-derived amino acids in the presence of -casein as
protein substrate and glucose as a source of metabolic energy. The
amino acids are not accumulated significantly inside the cells by the
Opp and DtpT
Opp
mutants. When cells are incubated with a mixture of amino acids
mimicking the composition of
-casein, the amino acids are taken up
to the same extent in all four strains. Analysis of the extracellular
peptide fraction, formed by the action of PrtP on -casein,
indicates that distinct peptides disappear only when the cells express
an active Opp system. These and other experiments indicate that (i)
oligopeptide transport is essential for the accumulation of all
-casein-derived amino acids, (ii) the activity of the Opp system
is sufficiently high to support high growth rates on -casein
provided leucine and histidine are present as free amino acids, and
(iii) extracellular peptidase activity is not present in L.
lactis.
-,
-,
-, and -casein). Most of the amino acids
released from the hydrolysis of caseins are essential or growth
stimulating. The proteolytic system of Lactococcus lactis consists of a proteinase, several peptidases, amino acid
transporters, and two peptide transport systems. The extracellularly
located proteinase (PrtP) performs the first step in the degradation of
caseins and is essential for growth on milk to high cell densities. The
total peptide formation resulting from the action of the
P-type proteinase on -casein has been analyzed in
vitro using purified proteinase. ()This study has
indicated that -casein is degraded to fragments of 4-30
residues, of which 17% is smaller than 9 residues. None of the
peptidases studied to date possesses an amino-terminal signal that
could target the protein to the outside of the cell (Pritchard and
Coolbear, 1993; Kok and De Vos, 1993). Furthermore, biochemical and
immunological data indicate that the aminopeptidases (PepN and PepC),
the X-prolyl-dipeptidyl aminopeptidase (PepX), the
endopeptidase (PepO), the tripeptidase (PepT), and the glutamyl
aminopeptidase (PepA) are present inside the cell (Tan et al.,
1992; Baankreis, 1992). In view of these observations, it would seem
that the casein-derived peptides have to be taken up by the cells
before further hydrolysis can take place. Two peptide transport systems
have been identified in L. lactis: (i) a proton motive
force-driven di-tripeptide carrier (DtpT) (Smid et al., 1989a;
Kunji et al., 1993; Hagting et al., 1994) and (ii) an
ATP-driven oligopeptide transport system (Opp) that is capable of
transporting peptides of 4 and up to at least 8 residues (Kunji et
al., 1993; Tynkkynen et al., 1993).-casein
utilization in genetically well defined peptide transport mutants
expressing the P-type proteinase of L. lactis NCDO712. The analysis of the intracellular and extracellular amino
acid and peptide pools has revealed that all essential and
growth-stimulating amino acids can be taken up by the cells via the Opp
system. Remarkably, none of the amino acids accumulated significantly
inside the cell when the Opp system was inactivated. These observations
provide direct evidence that peptidases are not involved in
extracellular degradation and subsequent utilization of -casein by L. lactis.
Bacterial Strains, Plasmids, and Growth
Conditions
The bacterial strains and plasmids used in this
study are listed in Table 1. Lactococcal strains were grown at 28
°C in M17 broth (Difco) or in a chemically defined medium (CDM)
(Poolman and Konings, 1988) at pH 6.6, supplemented with 0.5% (w/v)
glucose or lactose and when appropriate with erythromycin (5
µg/ml). The strains were stored at -20 °C in M17 broth
with 10% glycerol.
General DNA Techniques
Plasmid DNA and
chromosomal DNA from L. lactis were isolated by the methods of
Anderson and McKay(1983). L. lactis was transformed by
electroporation as described before (Holo and Nes, 1989). DNA
modification enzymes were obtained from Boehringer GmbH (Mannheim,
Germany). Southern hybridizations were performed using the digoxygenin
DNA labeling and detection kit according to the instructions of the
manufacturer (Boehringer).Di-tripeptide and Oligopeptide Transport-deficient
Mutants of L. lactis
Using L. lactis AG300 as
parent strain (Table 1), plasmid pLS19A, containing a 1,130-base
pair internal fragment of oppA ligated into pLS19 (Leenhouts et al., 1990), was used to inactivate the oppA gene (oppA encodes the oligopeptide binding protein). Disruption of
the oppA gene was confirmed by Southern analysis. The double
mutant 
dtpT oppA::pLS19A was designated L. lactis CV4. The wild type strain (MG1363) and the various peptide
transport mutants (AG300, VS772, and CV4) were conjugated with plasmid
pLP712 (carrying the proteinase and lactose utilization genes) as
described (Gasson and Davies, 1980).Enzyme Assays
The proteinase activity of
exponentially growing acceptor and conjugated strains was assayed with
MeO-Suc-Arg-Pro-Tyr-pNA as substrate (final concentration of 0.5
mM) (Chromogenix, Mölndal, Sweden) in 80
mM Tris-HCl, pH 7.0, containing 10 mM CaCl
, essentially as described (Exterkate, 1990).Growth Experiments
To analyze growth in
milk, exponentially growing cells were inoculated (at A 0.05) in 10% (w/v) skimmed milk (Oxoid,
Basingstoke, United Kingdom) supplemented with 80 mM phosphoglycerate, pH 6.7. To prevent breakdown of caseins due to
extensive heating, milk (and other casein-containing media) were heated
to 100 °C followed by rapid cooling to 4 °C. Growth rates in
milk were determined by diluting samples 10-fold in an EDTA-borate
buffer and measuring the absorbance at 660 nm after 5 min (Hugenholtz
and Veldkamp, 1985). Growth of L. lactis on peptides was
tested in CDM (Poolman and Konings, 1988), containing all essential
amino acids except for one, which was supplied in the form of a di- or
pentapeptide. The limiting factors for growth on
-casein were
identified by adding individual or combinations of essential or
growth-stimulating amino acids (2 mM) to CDM containing 0.3
mM -casein as sole source of protein. Growth was
monitored by measuring changes in absorbance at 660 nm.Transport Assays
Prior to transport,
cells were washed with 100 mM potassium-MES, ()pH
6.5, containing 2 mM CaCl to prevent
autoproteolysis and release of the proteinase (Laan and Konings, 1989).
To inhibit protein synthesis, chloramphenicol (50 µg/ml) was
present in all further steps. Cells (A ±
25) were de-energized with 2-deoxyglucose (10 mM) for 20 min
at 30 °C, washed twice with MES/CaCl
, and resuspended
in 100 mM MES, pH 6.5, without CaCl. For transport
assays, cells (A ± 10) were preincubated
for 3 min in the presence of 25 mM glucose, after which 0.3
mM
-casein, peptides (0.5 mM), or a mixture of
amino acids mimicking the concentration of 0.3 mM -casein
were added. Transport was monitored by determining the intracellular
amino acid pools at various time intervals as described (Kunji et
al., 1993).Cell Lysis and Viability
Samples were
taken in parallel with the transport assays, cells were removed by
filtration (0.45 µm of cellulose-nitrate), and the filtrate was
incubated with lysyl-pNa (10 mM) for several hours at 30
°C. Lysyl-pNa is a chromogenic substrate that is specific for
aminopeptidases, and its hydrolysis was measured as described (Tan and
Konings, 1990). The aminopeptidase activities were related to those of
sonicated cell samples (3 
15 s at an amplitude of 6 µm, on
ice and under N atmosphere). Propidium iodide (Fluorescent
Probes, Eugene, OR) fluorescence was used to measure the viability of
cells during the experiments. The probe interacts with DNA only when
the permeability barrier of the cytoplasmic membrane is disrupted.
Samples were taken during the course of the experiments, propidium
iodide was added (1 µg/ml), and the fluorescence before and after
sonication was compared (excitation and emission wavelengths of 290 and
605 nm, respectively). Since sonication does not result in 100% lysis,
it can be expected that quantification of lysis determined in this way
is an overestimation.Miscellaneous
The P-type
proteinase of L. lactis was purified from strain MG611
according to Juillard et al. Protein was
determined by the method of Lowry et al.(1951), using bovine
serum albumin as standard. Growth experiments, peptidase, and
proteinase activity determinations were performed in low protein
binding ELISA plates (Greiner). Changes in absorption were measured at
the appropriate wavelengths in a Titertek MC600 ELISA spectrophotometer
(Flow Laboratories). To prevent evaporation during the incubations at
30 °C, the incubation mixtures (200 µl) were covered with 50
µl of silicon oil (1.01 mg/ml) (Wacker).Chemicals
Peptides were obtained from
Bachem Feinchemikalien AG (Switzerland), and -casein was from
Sigma. All amino acids were in the L configuration. Milli-Q
water (Millipore Corp.) was used in all experiments.
Characterization of the Peptide Transport
Mutants
The phenotypes of the wild type and peptide
transport mutants (MI2, EG110, EG135, EG165) (Table 1) were
checked by different criteria. First, growth of the organisms on
specific peptides was measured using a chemically defined medium
containing all essential amino acids except for one, which was supplied
in the form of Ala-Glu or Tyr-Gly-Gly-Phe-Leu (Table 2) (amino
acids omitted from the medium are underlined). The strains lacking a
functional DtpT system failed to grow in the presence of Ala-Glu as
sole source of glutamate, while strains in which the Opp system was
inactivated did not grow on Tyr-Gly-Gly-Phe-Leu as sole source of
leucine. All strains grew equally well in the presence of amino acids
as essential and growth-stimulating nutrients. Second, the uptake of
substrates that are specific for the Ala/Gly, the di-tripeptide, and
oligopeptide transport systems was studied (Table 2). In
accordance with the growth experiments, the di-alanine was taken up
when DtpT was functional, and tetra-alanine was transported when the
Opp system was functional, whereas high uptake rates of alanine were
observed in all four strains. Third, the presence or absence of a
functional di-tripeptide transport system was inferred from the
sensitivity of the strains for the toxic dipeptide Ala--chloro-Ala
(250 µM) (Table 2). Strains containing the
di-tripeptide transport system were sensitive to the toxic dipeptide,
while strains in which the system was absent were resistant. Fourth,
the proteinase activity of the acceptor and conjugated strains was
assayed by monitoring the hydrolysis of the chromogenic substrate
MeO-Suc-Arg-Pro-Tyr-pNA (Fig. 1A). High proteinase
activities were present in all conjugated strains, while no activity
was observed in the acceptor strains. The specific proteinase
activities corresponded to 1.9, 2.1, 3.8, and 4.8 nmol/min
mg of
protein for the Opp DtpT
,
Opp
DtpT
, Opp
DtpT
, and Opp
DtpT
strains, respectively. Fifth, the growth
properties of the conjugated strains in milk were studied by monitoring
the change in optical density and pH (Fig. 1B). The
results clearly indicate that oligopeptide transport is essential for
growth on milk. Strains lacking a functional di-tripeptide transport
system, on the other hand, grew equally well as the wild type. Slow
growth of the oligopeptide transport mutants in milk was observed after
24 h, possibly due to cell lysis followed by release of peptidases into
the medium.
), VS772 (
), AG300 (
), and CV4 (
)
and the corresponding conjugants carrying pLP712 (closedsymbols) was assayed with the chromogenic substrate
MeO-Suc-Arg-Pro-Tyr-pNA by measuring the change in absorption at 414 nm
upon hydrolysis of the substrate. Growth on skimmed milk was determined
as indicated under ``Materials and
Methods.''
The effect of the various peptide
transport mutations on the utilization of -Casein Utilization by the Wild Type and Peptide
Transport Mutants-casein was studied in vivo by incubating chloramphenicol-treated cells with 0.3
mM -casein in the presence of glucose as source of
metabolic energy. At given time intervals, cells were separated from
the medium by filtration, and the cell fraction was extracted with
perchloric acid. Both the extra- and intracellular fractions were
analyzed by reverse-phase HPLC for the presence of amino acids and
peptides after derivatization with dansylchloride. DtpT
(A),
Opp
DtpT
(B),
Opp
DtpT
(C), and
Opp
DtpT
(D) strains upon
the addition of
-casein. The wild type and di-tripeptide transport
mutant rapidly accumulated almost all of the amino acids present in
-casein within minutes after the addition of the protein substrate (Fig. 2, A and C). The rates of amino acid
accumulation were in case of the di-tripeptide transport mutant on
average 1.6 times higher as compared with the wild type, which might
reflect the higher proteinase activity (Fig. 1A) and/or
the higher oligopeptide transport activity of the strain (Table 2). Remarkably, none of the amino acids from -casein
accumulated to significant levels in the strains lacking a functional
oligopeptide transport system, despite the presence of functional amino
acid and/or di-tripeptide transport systems (Fig. 2, B and D). The observation that a single mutation,
abolishing oligopeptide transport activity, resulted in a defect to
accumulate amino acids argues strongly against degradation of peptides
by extracellularly located peptidases. In the analysis of the elution
profiles from the intracellular fraction, almost all of the peaks could
be attributed to amino acids, whereas significant amounts of peptides
could not be detected (see also Kunji et al., 1993; Tynkkynen et al., 1993; Hagting et al., 1994).
-casein. De-energized and chloramphenicol-treated
cells were incubated with 0.3 mM -casein after 3 min of
pre-energization with 25 mM glucose as source of metabolic
energy. The amino acid pools were determined as described under
``Materials and Methods'' and are indicated by their one
letter denomination. The increase in amino acid concentration compared
with the concentration at t = 0 min is depicted. The
time 0 concentrations for the amino acids were less than 5 nmol/mg for
Asn, Gln, Ser, Arg, Thr, Gly, Pro, Met, Val, Phe, Leu, Ile, His, Lys,
and Tyr and 19 and 52 nmol/mg for Ala and Glu,
respectively.
-casein deserve further attention. Due to the high
intracellular pools of Glu, Asp could not be separated well from Glu,
and the sum of both pools is represented by Glu in Fig. 2, A1-D1. Furthermore L. lactis converts Gln into
Glu and Asn into Asp (Poolman et al., 1987a), and therefore,
the ``Glu'' peak forms an indication of the accumulation of
Glu, Gln, Asp, as well as Asn. His might also be converted since the
pools initially drop upon energization in all four strains (Fig. 2, A6-D6). With the exception of Gln to
Glu, Asn to Asp, and Arg to ornithine and citrulline (Poolman et
al., 1987b), L. lactis has no possibilities to convert
amino acids into other compounds under the conditions employed.
Furthermore, using amino acid-depleted resting L. lactis cells, we have never detected significant synthesis of amino acids
from precursors of the glycolytic pathway (Poolman et al.,
1987a; this study). Therefore, we conclude that, with the possible
exception of Gly and Thr (see Fig. 2, B4 and D4), the increases in amino acid pools in the Opp strains (Fig. 2, A and C) result from
the uptake of the corresponding amino acids in the form of
oligopeptides.
Quantification of Cell Lysis
The amount
of cell lysis occurring during the course of the transport assays was
estimated from the aminopeptidase activities and viability of the
culture. Aminopeptidases PepC and PepN have been characterized as
highly active intracellular enzymes (Tan et al., 1992), and
their activities could therefore be used as markers for cell lysis. No
detectable lysis was observed at the start of the experiment, probably
due to extensive washing prior to the transport assays. After 15 min of
incubation, some peptidase activity could be detected corresponding to
at most 1% cell lysis; significant differences between the wild type
and peptide transport mutants could not be detected. The viability of
the cell suspensions remained constant during the course of the
experiments (data not shown). All together, these experiments show that
the observed differences in -casein utilization between strains
with and without a functional Opp system are not a consequence of
differences in cell lysis (and subsequent release of intracellular
peptidases).Growth of the Wild Type and Peptide Transport Mutants
on
It is worthwhile noting that at least six
amino acids (Glu/Gln, Leu, Val, Ile, Met, His) are essential for L.
lactis MG1363, whereas another four (Asn, Pro, Phe, and Ala) are
needed to support reasonable rates of growth. (-Casein
)The wild
type and DtpT mutant of L. lactis grew
slowly and only to low final optical densities on 0.3 mM
-casein as sole source of amino acids, indicating that one or
more essential amino acids are not liberated or transported
sufficiently fast to support optimal growth (data not shown). By adding
individual or combinations of essential and growth-stimulating amino
acids to media containing -casein as protein source of amino
acids, the limiting factors for optimal growth on -casein alone
could be identified. These experiments showed that the addition of His
and Leu (2 mM each) was sufficient for growth of the wild type
and di-tripeptide transport mutant on 0.3 mM -casein; the
maximal growth rates (µ
) were 0.63 h for both strains. Subsequent additions of Gln, Val, and Met (2
mM) were found to be growth stimulatory. The µ
values increased to 0.83, 0.86, and 0.89 h,
respectively. On the contrary, the oligopeptide transport mutants did
not grow under any of the conditions (i.e. media containing
-casein plus indicated amino acids). Only when a complete mixture
of amino acids was added, cells grew with a high rate (µ of 0.93 h). This again confirms that the total
package of amino acids derived from
-casein is exclusively taken
up as oligopeptides.Amino Acid Transport in the Wild Type and Peptide
Transport Mutant Strains
The various strains were fed with
a mixture of amino acids mimicking the composition of -casein (Tyr
was omitted). The pool sizes of individual amino acids increased to
similar extents, and the rates of uptake were not significantly
different in all four strains (data not shown). These results
demonstrate that the inability of the oligopeptide transport mutants to
accumulate -casein-derived amino acids is not due to some general
defect of the organisms to transport solutes.The External Accumulation of Peptides and Amino
Acids
To obtain further information about the role of the
Opp system in -casein utilization, the hydrolysis products of
casein generated by the purified proteinase PrtP and by cells
expressing PrtP have been compared. The peptide product formation from
the hydrolysis of -casein in vivo has been studied by
analyzing the external media of the uptake experiments described in Fig. 2. The -casein degradation patterns obtained with the
purified proteinase were similar to those of the in vivo experiments, in which the Opp strains EG110 and
EG165 were used (data not shown). Pronounced differences in peptide
pools can be observed when strains with a functional oligopeptide
transport system are compared with those lacking a functional
oligopeptide transport system. At least 18 peaks were observed in the
media of Opp mutants, which were virtually absent in the media of
strains with an active Opp (data not shown). These peptides are likely
to be substrates of the oligopeptide transport system. Furthermore,
only when the oligopeptide transport system was functional, several
peaks increased rapidly, of which most could be assigned to individual
amino acids. Notice that peptides, taken up by L. lactis, are
rapidly hydrolyzed by intracellular peptidases, which results in large
outwardly directed concentration gradients for the various amino acids.
As a result, amino acids will leak from the cells and appear in the
medium. The estimates are that less than 5% of the internally
accumulated amino acids had effluxed from the cells at the end of the
experiment.
-casein by the
action of the proteinase PrtP in a form that can be transported by the
cells. Second, these peptides are taken up by the oligopeptide
transport system exclusively. When a functional oligopeptide transport
system is absent, no significant intracellular accumulation of amino
acids is observed. Under those circumstances, several peptides
accumulate extracellularly, which do not accumulate when the
oligopeptide transport system is functionally present. Third,
consistent with the observation that PrtP does not release significant
amounts of di- and tripeptides from -casein, inactivation of the di-tripeptide transport system has no effect
on the utilization of this protein substrate. Since di-tripeptide
transport mutants selected on the basis of resistance toward L-Ala--chloro-L-Ala are affected in their
ability to grow on a mixture of caseins (Smid et al., 1989b),
we speculate that this is due to the inability to transport essential
amino acids (most likely His and/or Leu, see below) in the form of
small peptides that are released from proteins other than -casein.
Fourth, the observation that a single mutation abolishing oligopeptide
transport activity results in a defect to accumulate amino acids argues
strongly against the involvement of extracellular peptidases in the
degradation of -casein. If peptidases would have been present
externally, amino acids, dipeptides, and tripeptides would have been
formed and subsequently taken up by the corresponding transport
systems. Biochemical and genetic studies on a number of peptidases have
already suggested that these enzymes are present intracellularly (Tan et al., 1992; Kok and De Vos, 1993). The present studies
indicate that also other not yet identified or poorly characterized
peptidases, involved in -casein utilization, are unlikely to be
present extracellularly. Since synthetic peptides such as
Leu-enkephaline, tetra-alanine, Ala-Glu, and others are not degraded
extracellularly irrespective of whether Opp or
Opp
strains are used (Kunji et al.,
1993),
it is unlikely that Opp-mediated regulation of the
function or expression of extracellular proteases/peptidases has
effected the experiments. Fifth, a small fraction of the
intracellularly accumulated amino acids appears in the extracellular
medium of the wild type and di-tripeptide transport mutant, most likely
due to leakage of amino acids from the cells. Sixth, the observation
that the wild type and di-tripeptide transport mutant of L. lactis grow well on a chemically defined medium supplemented with
-casein and histidine plus leucine as sole source of amino acids
indicates that uptake of the oligopeptides occurs at rates high enough
to meet the growth requirements of the organism for most of the
(essential) amino acids. This is quite remarkable given the competition
between the peptides for a single binding protein. Rapid accumulation
of most amino acids is observed within minutes after the addition of
-casein, indicating that proteolysis and oligopeptide transport
are indeed quite effective.-casein by the activity of the proteinase. Both
from the analysis of the extracellular as well as the intracellular
fractions, it can be concluded that not all proteinase-generated
peptides are utilized by L. lactis. On the basis of the
differences in peptide patterns in the external medium of the
Opp and Opp
strains, one-fourth of
all peptide peaks observed in the HPLC analysis of the peptide pools
are likely to contain substrates of the oligopeptide transport system.
The observation that some peptides do accumulate in the medium despite
a functional Opp system may be a consequence of the size exclusion
limits of the oligopeptide transporter. Peptides up to a length of 30
amino acids are formed by PrtP.
Approximately one-fifth of
the casein-derived peptides falls in the range of 4-8
residues, and these are likely to be transported (Kunji et al., 1993; Tynkkynen et al., 1993). Furthermore,
although the lactococcal oligopeptide transport system must have a
broad substrate specificity, certain peptides may not be transported
due to competition of peptides for a single oligopeptide binding
protein. In addition, a part of the peptide pool may also be taken up
with a rate that is lower than the production rate by the proteinase.
)))
We thank Soile Tynkkynen for kindly providing plasmid
pLS19A, Igor Mierau for strains MI1 and MI2, and Peter Fekkes for
valuable suggestions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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E. M. Hebert, R. R. Raya, and G. S. De Giori Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062 Appl. Envir. Microbiol., December 1, 2000; 66(12): 5316 - 5321. [Abstract] [Full Text]
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 F. J. M. Detmers, F. C. Lanfermeijer, R. Abele, R. W. Jack, R. Tampé, W. N. Konings, and B. Poolman Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis PNAS, October 23, 2000; (2000) 220308797. [Abstract] [Full Text]
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P. Le Bourgeois, M.-L. Daveran-Mingot, and P. Ritzenthaler Genome Plasticity among Related Lactococcus Strains: Identification of Genetic Events Associated with Macrorestriction Polymorphisms J. Bacteriol., May 1, 2000; 182(9): 2481 - 2491. [Abstract] [Full Text]
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F. Chavagnat, M. G. Casey, and J. Meyer Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A Appl. Envir. Microbiol., July 1, 1999; 65(7): 3001 - 3007. [Abstract] [Full Text]
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B. Flambard, S. Helinck, J. Richard, and V. Juillard The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase Appl. Envir. Microbiol., June 1, 1998; 64(6): 1991 - 1996. [Abstract] [Full Text]
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H. Wang, W. Yu, T. Coolbear, D. O'Sullivan, and L. L. McKay A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation Appl. Envir. Microbiol., May 1, 1998; 64(5): 1673 - 1679. [Abstract] [Full Text]
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V. Juillard, A. Guillot, D. Le Bars, and J.-C. Gripon Specificity of Milk Peptide Utilization by Lactococcus lactis Appl. Envir. Microbiol., April 1, 1998; 64(4): 1230 - 1236. [Abstract] [Full Text]
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F. J. M. Detmers, F. C. Lanfermeijer, R. Abele, R. W. Jack, R. Tampe, W. N. Konings, and B. Poolman Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis PNAS, November 7, 2000; 97(23): 12487 - 12492. [Abstract] [Full Text] [PDF]
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