Active Lipoprotein Precursors in the Gram-positive Eubacterium Lactococcus lactis*

Roelke Venemadagger, Harold TjalsmaDagger, Jan Maarten van Dijl§, Anne de Jong, Kees Leenhouts, Girbe Buist||, and Gerard Venema

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


                              
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Table I
Predicted lipoprotein signal peptides of L. lactis IL1403
Chromosomally encoded lipoprotein signal peptides were identified as described previously (12, 15). The hydrophobic H-domain is indicated by gray shading. The residues at positions -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 alpha -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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% beta -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-beta -D-galactopyranoside (X-gal; 0.008%).


                              
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Table II
Strains and plasmids used in this study

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 MG1363Delta lsp, 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-Delta 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-Delta 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-Delta 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 MG1363Delta 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). beta -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)-beta -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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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)-beta -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 beta -lactamase activity around E. coli colonies producing pre-A13i-Bla. In contrast, a zone of beta -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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Fig. 1.   Conserved domains and predicted membrane topology of SPase II from L. lactis. A, deduced amino acid sequences of the conserved boxes I-V (27) of type II SPases (LspA) from L. lactis MG1363 (Lla), B. subtilis (Bsu), and E. coli (Eco). The amino acid sequence of the L. lactis MG1363 SPase II is available under GenBankTM accession number U63724. The complete amino acid sequence of the SPase II from L. lactis MG1363 displays 38.7 and 36.8% identity with those of the type II SPases from B. subtilis and E. coli, respectively. Numbers refer to the position of the first amino acid of each conserved domain in the respective type II SPases. Residues are in boldface when present in at least 18 of 31 previously identified type II SPases (4). Consensus sequences of each conserved domain are indicated. Dashes indicate lack of identity; uppercase letters indicate residues that are strictly conserved in all known type II SPases. Residues that are present in at least 18 of the 31 previously identified SPase II sequences are in lowercase letters. Note that LspA from L. lactis IL1403 (53) contains an Ile residue at position 19 in box I, whereas LspA from L. lactis MG1363 contains a Val residue at this position. Residues important for activity (star ) or stability () of the B. subtilis SPase II (27) are indicated. B, predicted membrane topology of SPase II (Lla). The orientation of putative transmembrane regions (A-D) was predicted with the Toppred2 algorithm (54). The five conserved domains I-V (see A) are indicated.

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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Fig. 2.   Processing of pre(A13i)-beta -lactamase. A, the ability of E. coli cells transformed with pGDL48 (no SPase II) or pGDL64 (SPase II (Lla)) to release mature A13i-Bla into the growth medium was analyzed with the plate assay for SPase I activity as described previously (30). Release of A13i-Bla is reflected by a halo of beta -lactamase activity around colonies on plate. B, processing of pre-A13i-Bla in E. coli MC1061 transformed with pGDL48 (no SPase), pGDL41 (SipS (Bsu)), or pGDL64 (SPase II (Lla)) was analyzed by pulse-chase labeling at 37 °C and subsequent immunoprecipitation, SDS-PAGE, and fluorography. Cells were labeled with [35S]methionine for 1 min prior to chase with excess of non-radioactive methionine. Samples were withdrawn at the time of chase (t = 0) and 10 or 30 min after the chase as indicated. Variations in the amounts of A13i-Bla precipitated from different strains relate only to variability in the incorporation of label into cells of different cultures and not to specific effects of the different SPases. p, precursor; m, mature.

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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Fig. 3.   SPase II of L. lactis is active in E. coli and B. subtilis. A, activity of the L. lactis SPase II in E. coli Y815. Proteins were labeled by incubating cells with [35S]methionine for 5 min at 42 °C. After a chase of 15 min with excess of non-labeled methionine, samples were precipitated with ice-cold trichloroacetic acid. Processing of prolipoprotein by SPase II in E. coli Y815 carrying pGDL48 (no SPase II; negative control) or pGDL64 (SPase II (Lla)) was analyzed by immunoprecipitation and Tricine SDS-PAGE (16.5% acrylamide (w/v)). Pre-LP, diacylglyceryl-modified precursor form of Braun's lipoprotein; LP, mature form (probably fully lipid-modified) of Braun's lipoprotein. B, activity of the L. lactis SPase II in B. subtilis 8G5lsp. The steady-state levels of precursor, mature, and alternatively processed mature-like forms of PrsA in cells of B. subtilis 8G5lsp harboring plasmids pGDL48 (no SPase II; negative control), or pGDL64 (SPase II (Lla)) were analyzed by Western blotting. To this purpose, cells of overnight cultures grown in TY medium at 37 °C were collected by centrifugation, and samples for SDS-PAGE were prepared as described under "Experimental Procedures." The positions of pre-PrsA, mature, and mature-like forms of PrsA (PrsA*) are indicated.

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 MG1363Delta lsp 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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Table III
LspA-independent growth and secretion of PrtP
Growth rates of L. lactis MG1363 and its lspA deletion derivative MG1363Delta /sp were determined in M17 broth with glucose, whey-based permeate (WP) with glucose, or milk. The growth rates are expressed as the natural logarithm of the increase in A600/h of growth during the exponential growth phase. The activity of PrtP secreted by cells grown in whey-based permeate is expressed in arbitrary units (AU) measured as the increase of absorption at 405 nm/h per A600. Cell samples used for the PrtP measurement were collected from cultures at mid-exponential phase (NA600 of 0.7).

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 MG1363Delta lsp 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 MG1363Delta 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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Fig. 4.   Accumulation of lipoprotein precursors in L. lactis lacking SPase II. To monitor the processing of pre-PrtM, cells of L. lactis MG1363 (wild-type) and its lspA mutant derivative MG1363Delta lsp (no SPase II) were transformed with the plasmid pLP712 carrying the prtM and prtP genes. Cells from cultures in the mid-exponential growth phase were collected by centrifugation, and cell extracts were prepared as described by van de Guchte et al. (43). SDS-PAGE and Western blotting were carried out as described under "Experimental Procedures." Samples from cells with (+) or without (-) pLP712 are marked. These samples were used to visualize the processing of pre-PrtM (upper panel) and the chromosomally encoded lipoprotein pre-OppA (lower panel) with specific antibodies. The positions of pre-PrtM (~33 kDa), mature PrtM (~30.5 kDa), pre-OppA (~66 kDa), and mature OppA (~64 kDa) are indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

    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.

    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.

dagger This paper is dedicated to the memory of Roelke Venema. We recall sunny and cheerful days with Roelke in our midst.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: SPase, signal peptidase; IPTG, isopropyl-beta -D-thiogalactopyranoside; TY, tryptone/yeast extract; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; X-gal, 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside.

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DISCUSSION
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