(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-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.
Lactic acid bacteria possess an active proteolytic system that
is involved in the degradation of milk proteins (-,
-,
-, 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).
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 -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.
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 (), 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.''
Fig. 2shows the time course of the intracellular amino acid
pools(1, 2, 3, 4, 5, 6, 7) for the
Opp 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).
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 -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.
A number of
observations regarding the accumulation of amino acids upon hydrolysis
of -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.
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 -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.
A large number of peptides are released
from -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.
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.