From the Department of Genetics, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, The Netherlands
Received for publication, September 25, 2002, and in revised form, February 11, 2003
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ABSTRACT |
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Lipid-modified proteins play important roles at
the interface between eubacterial cells and their environment. The
importance of lipoprotein processing by signal peptidase II (SPase II)
is underscored by the fact that this enzyme is essential for viability of the Gram-negative eubacterium Escherichia coli. In
contrast, SPase II is not essential for growth and viability of the
Gram-positive eubacterium Bacillus subtilis. This could be
due to alternative amino-terminal lipoprotein processing, which was
shown previously to occur in SPase II mutants of B. subtilis. Alternatively, uncleaved lipoprotein precursors might
be functional. To explore further the importance of lipoprotein
processing in Gram-positive eubacteria, an SPase II mutant strain of
Lactococcus lactis was constructed. Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are
essential for growth in milk, the growth of SPase II mutant L. lactis cells in this medium was not affected. Furthermore, the
activity of the strictly PrtM-dependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II. Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase
II. Taken together, these findings show for the first time that
authentic lipoprotein precursors retain biological activity.
One of the most common eubacterial protein sorting (retention)
signals is an amino-terminal lipid-modified cysteine residue, which
anchors proteins to the cytoplasmic or outer membranes (see Refs.
1-4). The known lipid-modified proteins (lipoproteins) are involved in
a large variety of processes, which range from the uptake of nutrients,
resistance against antibiotics, protein secretion, cell wall
biogenesis, sporulation and germination, to the targeting of eubacteria
to different substrates, eubacteria, and host tissues (see Ref. 5).
Furthermore, lipoproteins have been implicated as important mediators
of the inflammatory response in human hosts during eubacterial
infections (6-10).
Lipoproteins are directed into the general (Sec) pathway for protein
secretion by their signal peptides, comprising a positively charged
amino terminus, a hydrophobic core region, and a carboxyl-terminal region containing the cleavage site for signal peptidase
(SPase).1 The major
difference between signal peptides of lipoproteins and secretory
proteins is the presence of a well conserved "lipobox" of four
residues in lipoprotein signal peptides. Invariably, the carboxyl-terminal residue of the lipobox is cysteine that, upon lipid
modification, forms the retention signal of the mature lipoprotein (for
details see Refs. 1 and 11). Modification of this cysteine residue by
the diacylglyceryl transferase (Lgt) is a prerequisite for
specific processing of the lipoprotein precursor by a type II SPase. In
most Gram-negative eubacteria the processed lipoprotein is further
modified by amino acylation of the diacylglyceryl-cysteine amino group
(3). The latter lipid modification step seems not to be conserved in
Gram-positive eubacteria (4, 12). In contrast to lipoproteins,
secretory proteins are specifically processed by type I SPases (see
Ref. 13).
In Gram-negative eubacteria, such as Escherichia coli, the
outer membrane confines numerous proteins to the periplasm. In Gram-positive eubacteria, such as Bacillus subtilis and
Lactococcus lactis, which lack an outer membrane, lipid
modification appears to be of major importance for the anchoring of
exported proteins to the cytoplasmic membrane in order to prevent their
release into the environment (14). This may explain why the
Gram-positive homologues of known periplasmic high affinity
substrate-binding proteins of Gram-negative eubacteria appear to be
lipoproteins. In fact, about one-third of the predicted lipoproteins of
B. subtilis (4, 12) and L. lactis (Table
I) are homologues of such binding proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Predicted lipoprotein signal peptides of L. lactis IL1403
3 to +1, forming the lipobox,
are underlined. The SPase II cleavage site is indicated with a gap in
the amino acid sequence. The lipoprotein YbdC, homologous to the
SpoIIIJ lipoprotein of B. subtilis, containing (putative)
transmembrane domains in the mature part of the
protein is indicated by TM Notably, the plasmid
pLP712-encoded lipoprotein PrtM is indicated by P.
Signal peptides of lipoproteins, which are homologues of known
periplasmic high affinity substrate-binding proteins from Gram-negative
eubacteria are indicated by *. The lipoprotein signal peptide indicated
by 555882-556025 is not present in the annotation of the L. lactis IL1403 genome but was identified upon glimmer
analysis (16) of the IL1403 genome sequence. The sequence of the YjgB
lipoprotein signal peptide was obtained, based on the assumption of a
sequencing mistake. Note that the lipoproteins of L. lactis,
like those of other Gram-positive eubacteria (4, 12), seem to lack Asp
residues at the +2 position relative to the SPase II cleavage site. An
Asp residue at this position results in the retention of lipoproteins
in the cytoplasmic membrane of Gram-negative eubacteria, such as
E. coli
(17).
Lipoprotein processing by SPase II is essential for the viability of
E. coli (18, 19). In contrast, the SPase II of B. subtilis was shown not to be essential for cell viability (12). This is a remarkable observation, because at least one lipoprotein of
B. subtilis is essential for life. This is the folding
catalyst PrsA, a potential peptidyl-prolyl
cis/trans-isomerase of the parvulin type (20). Nevertheless,
the processing, stability, and activity of several lipoproteins are
strongly impaired in B. subtilis cells lacking SPase II (12,
14, 21). For example, these cells accumulate the lipid-modified
precursor form of PrsA. In addition to pre-PrsA, amino-terminally
processed and lipid-modified (mature-like) forms of PrsA are present in
SPase II mutant B. subtilis cells. Notably, the
secretion of -amylase and a variety of other non-lipoproteins, which
is PrsA-dependent, is strongly impaired in the absence of SPase II. The latter observation indicates that pre-PrsA and/or the mature-like forms of PrsA have a reduced activity (12, 14). The
alternative processing of PrsA in the absence of SPase II is catalyzed
by as yet unidentified proteases at the membrane-cell wall interface of
B. subtilis (12).
In addition to a total number of 38 predicted chromosomally encoded lipoproteins (Table I), most lactococcal strains contain the plasmid-encoded lipoprotein PrtM, a close homologue of the PrsA protein of B. subtilis. PrtM is indispensable for the maturation and activation of PrtP, a serine protease that is initially synthesized as a pre-proprotein (22). Pre-pro-PrtP is converted to mature PrtP by SPase I-mediated signal peptide (pre-) removal and subsequent self-cleavage (23). Importantly, PrtP is the key enzyme in the release of peptides and amino acids from casein, and consequently, the activity of this protease is critical for growth of L. lactis in milk. In the absence of PrtM, PrtP is exported in its inactive pro-form, showing that PrtM-assisted folding of PrtP is required for the removal of the pro-peptide by self-cleavage (23-25).
In the present studies, we document the serendipitous identification
and functional analysis of the SPase II-encoding lspA gene
of L. lactis MG1363. This gene is not required for the
growth of L. lactis on rich media or milk. Strikingly, no
alternative processing of the lipoproteins PrtM and OppA was observed
in cells lacking SPase II. The present findings unequivocally
show for the first time that the removal of signal peptides from
authentic lipoproteins, such as PrtM and OppA, is not essential for
their biological activity in L. lactis.
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EXPERIMENTAL PROCEDURES |
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Strains, Plasmids, and Media--
The strains and plasmids used
in this study are listed in Table II. TY
medium for E. coli and B. subtilis contained
Bactotryptone (1%), Bactoyeast extract (0.5%), and NaCl (1%). M9
media 1 and 2 for pulse-chase labeling of E. coli were
prepared as described by van Dijl et al. (32). L. lactis was grown at 30 °C in 10% reconstituted skim milk
(Oxoid Ltd.), 2-fold diluted M17 broth (Difco), or in whey-based medium
(33). The latter two media were supplemented with 0.5% glucose and
0.95% -glycerophosphate (Sigma). Plates with M17 medium diluted
2-fold contained 1.5% agar. When required, media for E. coli were supplemented with kanamycin (20 µg/ml) or ampicillin
(40 µg/ml); media for B. subtilis were supplemented with
kanamycin (10 µg/ml) or erythromycin (1 µg/ml); media for
L. lactis were supplemented with erythromycin (5 µg/ml) and/or
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal;
0.008%).
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DNA Techniques-- Procedures for DNA purification, restriction, ligation, agarose gel electrophoresis, and transformation of E. coli were carried out as described by Sambrook et al. (34). B. subtilis was transformed as described by Tjalsma et al. (35). Chromosomal DNA of L. lactis was isolated according to the method of Leenhouts et al. (36). L. lactis was transformed by electroporation according to the protocol of Holo and Nes (37) using a Bio-Rad GenePulser. Restriction enzymes and Expand polymerase were from Roche Molecular Biochemicals. Southern blotting was performed as described by Chomczynski and Qasba (38). DNA probes were labeled using the ECL oligonucleotide labeling and detection system (Amersham Biosciences). DNA sequences were determined using the dideoxy-chain termination procedure (39). Clones were sequenced with universal, reverse, and synthetic primers using 35S-dATP (Amersham Biosciences) and the T7 polymerase sequencing kit (Amersham Biosciences). For sequence assembly and analysis, the programs COMPARE, BESTFIT, PILEUP, PRETTY, and GAP of the University of Wisconsin Genetics Computer Group software package (40) were used. The sequence of the cloned chromosomal fragment containing the lactococcal lspA gene is available under GenBankTM accession number U63724.
To construct plasmid pGDL64, carrying the lspA gene of L. lactis MG1363 (lspA (Lla)), a PCR was performed with the primers Lsp01 (5'-ACG CGT CGA CTA TTT CTG AAA AGG GCT-3') and Lsp02 (5'-TTT GAA TTC TAC TTA CTG TCA CTC GTT-3'; restriction sites used for cloning are underlined and nucleotides complementary to chromosomal DNA of L. lactis MG1363 are in italics). The amplified fragment was cleaved with SalI and EcoRI and ligated into the corresponding sites of pGDL48 (31) in such a way that lspA transcription is driven by the constitutive erythromycin promoter.
To construct the L. lactis lspA mutant strain
MG1363lsp, the plasmid pORI280-based chromosomal
integration-excision system developed by Leenhouts et al.
(29, 41) was used. For this purpose, two fragments of the
lspA region were amplified by PCR. First, a 515-bp fragment
containing the 5'-end of lspA and upstream sequences was
amplified by PCR with primers lspA1 (5'-AAA TTT TCT
AGA GCA AAG AAA GAG AGG TC-3') and lspA2 (5'-CGC
GCG GCC GCT TAT
TTA TTT AAG CAA CAA CCC AAT TTT TGA AAA CTT
GG-3'; nucleotides generating stop codons in all three reading frames are indicated in boldface). This fragment was cloned into the
XbaI and NotI sites of pORI280. Subsequently, a
740-bp fragment containing the 3'-end of lspA was amplified
with primers lspA3 (5'-TAA ATA AAT
AAG CGG CCG CGC
GCA ACT TGG AGA TAC AAA AAA AAT TTG GCC-3') and
lspA4 (5'-AAA TTT AGA TCT GCA
TGT CCT GCC GCA GG-3'). This fragment was cloned into the NotI and BglII sites of the pORI280 derivative
already containing the 5'-sequences of lspA. The resulting
pORI280-
lsp deletion vector was used to transform
L. lactis MG1363 for chromosomal integration into the
lspA gene. Transformants (erythromycin-resistant and blue on
M17 plates with X-gal) were grown in the absence of erythromycin as
described before (29). Subsequently, 21 white colonies were obtained on
M17 plates with X-gal (frequency 5.3 × 10
5), all of
which were erythromycin-sensitive due to the excision of
pORI280-
lsp from the chromosome. As verified by cleavage
of chromosomal DNA with NotI and SspI and
subsequent Southern blotting, the lspA gene of 9 of these 21 colonies (frequency 2.3 × 10
5) had been replaced
with the mutant lspA gene of pORI280-
lsp. Notably, the mutant lspA gene can be distinguished from the
wild-type lspA gene in Southern blotting experiments due to
the replacement of an SspI restriction site with a
NotI site. The frequencies at which LacZ
and
lspA mutant colonies were obtained were well within the
range of those found for the mutation of other non-essential L. lactis genes with this system (29). The resulting L. lactis strain MG1363
lsp contains an lspA
gene that is truncated after the 26th codon due to the replacement of a
6-bp internal fragment with a 22-bp fragment containing stop codons in
all three reading frames.
SDS-PAGE, Western Blotting, and Immunodetection--
Cell
extracts of E. coli, B. subtilis, and L. lactis were prepared as described previously (42, 43). SDS-PAGE
was carried out as described by Laemmli (44), and Tricine SDS-PAGE as
described by Schagger and von Jagow (45). Proteins were transferred
from gels to polyvinylidene difluoride membranes (Roche Molecular
Biochemicals) as described by Khyse-Andersen (46). -Lactamase,
Braun's lipoprotein, PrtM, OppA, and PrsA were visualized with
specific antibodies and horseradish peroxidase-anti-rabbit IgG
conjugates (Amersham Biosciences).
Growth Measurements and Enzyme Activity Assays--
For growth
measurements, overnight cultures of L. lactis strains in
Glucose M17 medium or whey-based permeate were diluted 100-fold in the
respective fresh media. Overnight cultures in milk were diluted 50-fold
in pre-warmed fresh milk. Absorbances at 600 nm were monitored
in time using a Novaspec II spectrophotometer (Amersham Biosciences).
The absorbance of cultures in milk was determined as described
by Mierau et al. (47). Protease activity was determined at
37 °C using the chromogenic peptide
(MeO-Suc-Arg-Pro-Tyr-p-nitroanilide; Chromogenix AB,
Mölndal, Sweden) as described by Mierau et al. (48).
The hybrid precursor pre(A13i)--lactamase (pre-A13i-Bla) was used as
a reporter to monitor SPase I activity both in pulse-chase labeling
experiments and plate assays as described by van Dijl et al.
(30).
Analysis of SPase II Activity in E. coli--
The E. coli strain Y815, which produces a temperature-sensitive SPase II,
displays an IPTG-dependent growth defect at elevated temperatures due to the IPTG-inducible overproduction of lipid-modified precursors of Braun's lipoprotein (19). Overnight cultures of this
strain were diluted 10-fold in M9 medium-1 and grown for 3 h at
30 °C. Next, the cells were washed and resuspended in M9 medium-2
(methionine- and cysteine-free medium), containing 0.6 mM
IPTG for induction of the E. coli lpp gene on the
resident plasmid pHY001. Upon incubation for 30 min at 42 °C to
inactivate the temperature-sensitive SPase II of E. coli
Y815, the cells were used for pulse-chase labeling with
[35S]methionine (5 min, 42 °C). Immunoprecipitation
with serum against Braun's lipoprotein, Tricine SDS-PAGE, and
fluorography were carried out as described by Pragai et
al. (49).
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RESULTS |
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Serendipitous Identification of the lsp Gene of L. lactis--
The
genes for various type I SPases of bacilli have been cloned in
E. coli using a plate assay in which the processing of the hybrid precursor pre(A13i)--lactamase (pre-A13i-Bla) can be
monitored. The assay is based on the fact that this precursor is not
processed by the SPase I of E. coli. Consequently, all pre-A13i-Bla produced remains associated with the cytoplasmic membrane.
On plates, this is reflected by very low levels of
-lactamase activity around E. coli colonies producing pre-A13i-Bla. In
contrast, a zone of
-lactamase activity can be observed around
colonies producing pre-A13i-Bla when a type I SPase capable of
processing this precursor is co-expressed. This is due to leakage of
processed A13i-Bla from the periplasm into the environment (30, 31, 50,
51). The plate assay for SPase I activity is extremely sensitive as
A13i-Bla activity is even observed around colonies of cells expressing
mutant SPases with very low activity
(52).2 In the present
studies, this assay was employed with the goal to characterize type I
SPases from lactic acid bacteria. To this purpose, genomic DNA of
L. lactis MG1363 was partially digested with
Sau3AI and ligated into the BclI site of plasmid
pGDL42 encoding pre-A13i-Bla. Next, E. coli MC1061 was
transformed with the resulting genomic library, and 25,000 transformants (representing ~20 times the genome equivalent) were
screened on plates for the release of A13i-Bla. Seven transformants
showed a significantly increased zone of A13i-Bla activity. As
demonstrated by restriction analysis and Southern hybridization, the
pGDL42-based plasmids extracted from five of these transformants
contain overlapping inserts of L. lactis DNA (data not
shown). Unexpectedly, the sequencing of the insert of one of these five
plasmids, designated pGDL63, revealed the presence of a gene
(lspA), encoding a typical type II SPase. In contrast, none
of the other sequenced genes on this insert specifies a type I SPase
(data not shown).
Analysis of the deduced amino acid sequence of the cloned L. lactis SPase II revealed that this enzyme contains the five
domains (I-V) that are conserved in all known type II SPases (Fig.
1A) (4, 27). Importantly, the
Asn, Asp, and Ala residues in domains III and V (marked with a
star), which are critical for the activity of the SPase II
of B. subtilis, are conserved in the L. lactis SPase II. Furthermore, like all other known type II SPases, the L. lactis SPase II contains four predicted transmembrane
domains (denoted TM-A to TM-D) that position the
potential catalytic Asn and Asp residues of domains III and V at the
extracytoplasmic membrane surface (Fig. 1B). These
observations strongly suggest that the cloned lspA gene from
L. lactis specifies a functional type II SPase.
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To investigate whether the observed release of A13i-Bla by E. coli cells containing pGDL63 is due to the presence of the
L. lactis lspA gene, this gene was amplified
by PCR and cloned without its up- and downstream genes into the plasmid
pGDL48. The resulting plasmid was named pGDL64. Next, the release of
A13i-Bla by E. coli MC1061 cells containing pGDL64 was
tested on plates. As shown in Fig.
2A, colonies of E. coli MC1061 containing pGDL64 release significantly larger amounts
of A13i-Bla into the surrounding medium than colonies of E. coli MC1063 containing pGDL48 (negative control). This shows that
the cloned lspA gene of L. lactis is responsible
for the observed release of A13i-Bla activity. To verify whether this
release is caused by SPase II-mediated processing of pre-A13i-Bla,
pulse-chase labeling experiments were performed with E. coli
MC1061 cells containing pGDL64, pGDL41 (carrying the gene for the type
I SPase SipS of B. subtilis), or pGDL48 (empty vector). The
results show that after a chase of 10 min, significant amounts of
pre-A13i-Bla were processed to the mature form in E. coli
strains producing the pGDL41-encoded type I SPase SipS (Fig.
2B). In contrast, no processing of pre-A13i-Bla was observed
in E. coli cells expressing the pGDL64-encoded SPase II of
L. lactis, not even after an extended chase time of 30 min. These observations suggest that, under the conditions of the plate assay, either unprocessed pre-A13i-Bla or very low amounts of mature
A13i-Bla are liberated from the cells due to the presence of the SPase
II of L. lactis.
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Signal Peptidase Activity of the L. lactis SPase II in E. coli and
B. subtilis--
To investigate whether the L. lactis SPase
II is active in E. coli, processing of Braun's
prolipoprotein was studied in E. coli Y815 containing the
plasmids pGDL64 (carries lspA of L. lactis) or
pGDL48 (empty vector). The expression of the lpp gene for
Braun's lipoprotein was induced by adding IPTG, and the
temperature-sensitive SPase II of E. coli Y815 was
inactivated by increasing the temperature to 42 °C. Processing of
prolipoprotein was monitored by pulse-chase labeling, subsequent
immunoprecipitation, and Tricine SDS-PAGE (Fig.
3A). Mature lipoprotein was
observed in E. coli Y815 containing the cloned
lspA gene of L. lactis but not in E. coli Y815 carrying the empty vector. These results show that the
product of the L. lactis lspA gene has type II
SPase activity.
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To investigate whether the SPase II of L. lactis can suppress the alternative processing of the lipoprotein PrsA in B. subtilis cells with a disrupted lspA gene, B. subtilis 8G5lsp was transformed with plasmids pGDL64 (contains the L. lactis lspA gene) or pGDL48 (empty vector). Next, pre-PrsA processing was analyzed by Western blotting. As documented previously (12), B. subtilis 8G5lsp (pGDL48) accumulated precursor and mature-like forms of PrsA (Fig. 3B). Note that the mature-like forms of PrsA have a slightly lower mobility on SDS-PAGE than the correctly processed mature PrsA. In contrast, only mature PrsA was detected in B. subtilis 8G5lsp cells, containing the pGDL64-encoded lspA gene. This shows that the SPase II activity in these cells was restored to such an extent that alternative pre-PrsA processing was suppressed. Taken together, these observations demonstrate that the L. lactis SPase II is functional when expressed in E. coli or B. subtilis.
The lspA Gene Is Not Essential for Viability and Growth of L. lactis--
To determine whether the lspA gene is required
for viability and growth of L. lactis, an lspA
disruption strain was constructed using the integration-excision vector
pORI280. This resulted in the L. lactis strain
MG1363lsp in which the lspA gene is truncated after the 26th codon (see "Experimental Procedures" for details). The fact that this strain could be obtained shows that SPase II is not
essential for L. lactis cell viability. Furthermore, growth of L. lactis is not affected by the lspA
disruption, as shown by growth experiments using three different media
as follows: glucose M17 medium, glucose whey-based permeate, or milk.
In fact, the lspA mutant strain seems to grow slightly
better in whey-based permeate or milk than the parental strain
MG1363 (Table III). These results
demonstrate that SPase II is not required for viability and growth of
L. lactis. The observation that the
lspA mutation does not interfere with growth in milk, which
requires the activity of the lipoproteins PrtM and OppA (23, 25, 55),
shows that the absence of SPase II does not preclude the function
of these two lipoproteins.
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Activity of the Lipoprotein Precursors Pre-PrtM and
Pre-OppA--
To monitor the effect of the lspA disruption
on lipoprotein processing in L. lactis, processing of the
lipoproteins PrtM, an orthologue of the B. subtilis PrsA
protein, and OppA, an oligopeptide-binding protein, was analyzed by
Western blotting. It has to be noted that the strains MG1363 and
MG1363lsp do not produce PrtM because this protein is
encoded by endogenous plasmids of L. lactis. Therefore, these strains were transformed with the PrtM-encoding plasmid pLP712,
which also encodes the protease PrtP. As shown in Fig. 4, mature PrtM (~30.5 kDa) is
detectable in L. lactis MG1363 (pLP712), whereas only the
precursor form of PrtM (~33.0 kDa) is detectable in the mutant strain
MG1363
lsp (pLP712). Note that (pre-)PrtM is not
detectable in the negative control strains lacking pLP712. Furthermore,
cells of L. lactis MG1363 (with or without pLP712) contain only mature OppA (~64 kDa). In contrast, all OppA in the lspA mutant strains (with or without pLP712) is present in a
precursor form (pre-OppA; ~66 kDa) that migrates slightly slower on
SDS-PAGE than mature OppA (Fig. 4). Importantly, pre-PrtM and pre-OppA processing was found to be blocked completely in lspA mutant
cells, irrespective of the medium in which these cells are grown
(i.e. glucose M17 medium, whey-based permeate, or milk; only
the results for cells grown in M17 medium are documented in Fig. 4).
These results show that lspA encodes the SPase that is
required for pre-PrtM and pre-OppA processing and that no alternative
processing of these precursors occurs in the absence of this type II
SPase. To monitor the activity of the PrtM precursor, the
PrtM-dependent activity of the pLP712-encoded protease PrtP
was assayed with the chromogenic peptide substrate
MeO-Suc-Arg-Pro-Tyr-p-nitroanilide. As shown in Table III,
the activity of PrtP in the medium of cells grown on whey-based
permeate (glucose WP) was not affected by the absence of SPase II.
Taken together, the present studies show that the biological activities
of the unprocessed precursor forms of the lipoproteins PrtM and OppA
are indistinguishable from those of mature PrtM and OppA, respectively.
This suggests that processing by SPase II is dispensable for
lipoprotein function in L. lactis.
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DISCUSSION |
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Type II SPases are conserved in all eubacterial and mycoplasma species in which the genomes have been sequenced. The pre-lipoprotein substrates of these enzymes represent about 1-7% of the respective proteomes of these organisms (4). By computer-assisted analyses, we have identified 38 chromosomally encoded lipoproteins of L. lactis. Interestingly, 13 of these predicted lipoproteins appear to be high affinity substrate-binding proteins, four of which are (putative) oligopeptide-binding proteins (i.e. OppA, OptA, OptS, and YfcG). In contrast, B. subtilis seems to contain only one oligopeptide-binding protein, even though this organism contains about three times more lipoproteins than L. lactis (12). Thus, the adaptation of L. lactis to growth in milk, which requires the degradation of casein to oligopeptides, seems to be paralleled by the multiplication of genes for oligopeptide uptake systems. Despite the essential role of at least one oligopeptide-binding lipoprotein (i.e. OppA) for the growth of L. lactis in milk, the growth of lspA mutant cells of L. lactis on this medium is not impaired. Notably, only the precursor form of OppA can be detected in cells lacking SPase II. This finding is completely consistent with the main conclusion from the present studies that unprocessed lactococcal lipoprotein precursors can retain their biological activity.
The synthesis and activation of the major extracellular proteolytic enzyme PrtP are second prerequisites for lactococcal growth in milk. The activation of this protease requires the presence of PrtM, which is homologous to the B. subtilis PrsA protein (22). PrtM and PrtP are plasmid-encoded in all known natural strains of L. lactis that grow in milk. Whereas the chromosome of L. lactis seems to lack a gene for a PrtP homologue, a chromosomally encoded PrtM homologue denoted PmpA has been identified. Like PrtM, PmpA is a potential lipoprotein (Table I). However, PmpA is unable to facilitate the activation of PrtP (33), showing that the paralogous PrtM and PmpA proteins have distinct substrate specificities. As demonstrated in the present study, PrtP activity in the medium of lspA mutant L. lactis cells is not affected by the absence of SPase II even though these cells exclusively produce the precursor form of PrtM. This finding shows that processing by SPase II is dispensable for the activity of PrtM.
Previous studies have shown that B. subtilis cells lacking
SPase II accumulate not only the lipid-modified precursor form but also
alternatively processed mature-like forms of the lipoproteins PrsA (12)
and OpuAC (14). Especially in the case of PrsA, it is evident that at
least one of these forms must be active, because PrsA is essential for
the viability of B. subtilis (12). Because the alternative
processing of PrsA in the absence of SPase II cannot be prevented so
far, it is not clear whether the uncleaved precursor form of this
protein might be active. The present data show that L. lactis cells lacking SPase II accumulate pre-PrtM and pre-OppA,
which both display biological activity. Thus, it is conceivable that
the precursor form of the homologous PrsA protein in lspA
mutant cells of B. subtilis is active as well. Although the present findings do not exclude the possibility that alternatively processed forms of B. subtilis PrsA are
active, they do suggest that this alternative processing of pre-PrsA is not required for cell viability. In fact, the alternative processing of
uncleaved B. subtilis lipoprotein precursors might even
reduce the activity of these proteins. This would be consistent with the apparently reduced activity of PrsA in lspA mutant cells
of B. subtilis, which is thought to cause the significantly
reduced secretion of the PrsA-dependent -amylase AmyQ
by these cells.
The present data raise the intriguing question why lipoprotein processing by SPase II does occur in L. lactis since this processing enzyme is dispensable for cell viability, growth in milk, and activation of the major extracellular protease PrtP. One possibility could be that lipoprotein processing is more important for lactococcal fitness under conditions that were, so far, not identified. For example, lspA mutant cells of B. subtilis were shown to be both cold- and heat-sensitive, whereas their growth at optimal temperature (37 °C) was unaffected (12). An alternative possibility would be that, during the evolution of L. lactis in milk, lipoproteins such as OppA and PrtM have become particularly robust in order to ensure the competitiveness of the organism in this medium. In this respect, it is relevant to note that, as judged by proteomic studies in B. subtilis, the process of lipid modification is far more important for lipoprotein retention at the membrane-cell wall interface than the subsequent processing step by SPase II. For example, the absence of the diacylglyceryl transferase Lgt resulted in the shedding of many alternatively processed (unmodified) lipoproteins and even some (unmodified) pre-lipoproteins into the growth medium (14, 56). Nevertheless, even in the absence of lipid modification, lipoprotein precursors are at least to some extent retained at the membrane-cell wall interface due to the hydrophobic nature of their signal peptide (14, 57). Importantly, both lipid modification and the subsequent processing of the modified pre-lipoprotein by SPase II may, in some cases, be more important for protein stability and function than for membrane retention. For example, lack of SPase II or Lgt activity in B. subtilis was shown to result in the instability of the integral membrane protein CtaC, which does not require lipid modification for membrane retention as it has two transmembrane domains (21). Moreover, it has been reported that the tumor necrosis factor-inducing properties of the lipoprotein Vmp of Borrelia recurrentis are associated with the lipid moiety of this protein (7). At present, it is not clear which factors determine whether a certain lipoprotein is lipid-modified for membrane retention only or whether the lipid modification is needed for its activity or stability. Our present data suggest, however, that lipid modification in L. lactis is predominantly required to retain proteins at the membrane-cell wall interface.
Finally, one of the key issues that remains to be resolved in future
work is the phenomenon of alternative processing. First, it is
presently not clear how the SPase II of L. lactis could be
identified in an activity assay that was, so far, shown to be highly
specific for type I SPases. The most likely explanation for this
observation is based on the fact that the pre-A13i-Bla reporter
protein, used in the SPase I cloning assay, does contain a Cys residue
in the carboxyl-terminal region of its signal peptide. Even though this
Cys residue is not part of a typical lipobox, it might be
lipid-modified in some pre-A13i-Bla molecules, which would then serve
as a substrate for the SPase II of L. lactis. The resulting
mature molecules would be released from cells of E. coli by
an unknown mechanism. The alternative possibility that the presence of
the L. lactis SPase II results in the release of some
unprocessed pre-A13i-Bla precursor molecules is considered far less
likely, as this would not specifically require the presence of an
active SPase. Interestingly, the unique type I SPase of L. lactis that was recently identified through genome sequencing (53)
seems to be unable to process pre-A13i-Bla, which explains why this
enzyme was not identified in the SPase cloning
assay.3 Second, but more
importantly, it is presently not known which proteases are responsible
for the alternative processing of pre-lipoproteins of B. subtilis cells that lack SPase II. Previous studies (12, 58)
indicated that the five type I SPases of B. subtilis are not
involved in the alternative amino-terminal processing of pre-PrsA, which is consistent with the view that lipid-modified pre-proteins are
not substrates for type I SPases. An attractive strategy to identify
proteases responsible for alternative lipoprotein processing in
B. subtilis would involve comparative genomics. For example, the genome of L. lactis encodes homologues of many of the
(predicted) proteases at the B. subtilis membrane-cell wall
interface (see Ref. 15). In view of the apparent absence of alternative
processing in L. lactis, it seems unlikely that the
equivalent proteases in B. subtilis are responsible for this
process. By contrast, the most likely candidate proteases are those
membrane- or cell wall-associated proteases of B. subtilis
that have no (clear) homologues in L. lactis. This idea is
currently being examined as the characterization of the enzymology of
alternative lipoprotein processing is of major importance for a
detailed understanding of lipoprotein biogenesis and function in
Gram-positive eubacteria.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Albert Bolhuis, Sierd Bron, Alfred Haandrikman, Jan D. H. Jongbloed, Jan Kok, Zoltan Prágai, and Olivera Gajic for technical support and useful discussions.
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FOOTNOTES |
---|
* 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) U63724.
This paper is dedicated to the memory of Roelke Venema. We
recall sunny and cheerful days with Roelke in our midst.
Supported by Genencor International, Inc. (Leiden, The Netherlands).
§ Supported by Grants Bio2-CT93-0254, QLK3-CT-1999-00413 and QLK3-CT-1999-00917 from the European Union. Present address: Dept. of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
¶ Present address: BioMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Performed this work as a part of the STARLAB Project (Contract
BIO4-CT96-0016) of the European Union. To whom correspondence should be
addressed. Tel.: 31-50-3632287; Fax: 31-50-3632348; E-mail:
Buistg@biol.rug.nl.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M209857200
2 R. Venema, H. Tjalsma, J. M. van Dijl, A. de Jong, K. Leenhouts, G. Buist, and G. Venema, unpublished observations.
3 J. D. H. Jongbloed and J. M. van Dijl, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
SPase, signal
peptidase;
IPTG, isopropyl--D-thiogalactopyranoside;
TY, tryptone/yeast extract;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside.
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