INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Gram-positive lactic acid bacteria (LAB) are widely used in food industries for the production and preservation of fermented products. They are considered safe and even beneficial organisms. The potential of using LAB for new applications such as in production of heterologous proteins for biotechnology, in fermented food products, or in the digestive tract of humans or animals is currently under active study (3, 11, 13, 17, 19, 22, 30, 49, 54).

We are focused on optimizing heterologous protein secretion and export in Lactococcus lactis (30, 32), a well-characterized LAB for which genetic tools and the genome sequence are available (5, 11). To date, heterologous proteins such as bovine plasmin (3), bovine beta-lactoglobulin (BLG [6a], bovine rotavirus nonstructural protein 4 (NSP4 [13a]), murine interleukin-2 (IL-2) and IL-6 (54), or Listeria monocytogenes bacteriophage lysin (17) have been fused to lactococcal signal peptides (SPs) to direct their secretion in the medium. However, secretion efficiency (SE) has been rarely evaluated, and comparison of SE using native or heterologous SP has not been performed. The extent to which these and other features can be refined or improved to optimize protein secretion in L. lactis is the subject of this study.

In bacteria, most proteins that are secreted via the Sec pathway are synthesized as precursors containing the mature protein and an N-terminal SP (61) that is essential for precursor secretion. Although the primary sequences are poorly conserved, all SPs display a common tripartite structure including a positively charged N terminus, a hydrophobic core, and a neutral or negatively charged C terminus containing the SP cleavage site (61). Nevertheless, SPs of gram-positive bacteria are longer than those of gram-negative bacteria (61). Therefore, a gram-negative SP may be unable to direct secretion of a protein in a gram-positive host (8). Moreover, in a given species, the SE of a protein can vary with the SP chosen to direct its secretion (40, 47).

Even with the appropriate SP, secretion may be inefficient, and some heterologous proteins remain poorly or not at all secreted, even when fused to a homologous SP (6a, 13a, 46, 47). Notably, the N terminus of the mature moiety may greatly affect the translocation efficiency across the cytoplasmic membrane (2, 32). In Escherichia coli, the charge balance between the N termini of the SP and of the mature moiety may be critical for SE (26, 60). Although this charge balance rule was clearly demonstrated for gram-negative bacterial precursors, it may not apply to all gram-positive bacterial and eukaryotic precursors (25, 26). Until now, no detailed investigation was performed on charge balance in protein secretion in LAB.

Some precursors are synthesized as preproproteins, in which the SP is followed by a propeptide that is cleaved after translocation, giving rise to the mature protein (for a review, see reference 50). The propeptides can reportedly influence protein activities as well as SE. The antifolding activity and the role of the long class I propeptides (e.g., propeptides of proteases) in SE have been clearly demonstrated, whereas that of the short class II propeptides, e.g., the Staphylococcus aureus nuclease (Nuc), the Bacillus amyloliquefaciens barnase, or the Bacillus subtilis amylase, is less studied (39, 57). In S. aureus, the Nuc protein containing the propeptide (NucB) is localized in the cell wall, whereas the cleaved mature protein (NucA) is in the medium (9). Nevertheless, this localization is not observed in other hosts such as L. lactis or Corynebacterium glutamicum (32, 34). Results for E. coli demonstrated that the Nuc propeptide slows precursor folding, plays a positive role in SE of a fusion using an E. coli SP, and alleviates SecA dependency of precursor secretion in E. coli (55).

The secretion capacity of L. lactis was previously investigated using Nuc as a secretion reporter (32). Nuc, a small and stable secreted protein, is genetically and biochemically well characterized (51). Its enzymatic activity is readily detectable on petri plates as well as in zymograms (31, 34). Translational fusions to the N terminus and/or the C terminus of the mature protein are enzymatically active (30, 32, 41). SP_Nuc is atypical, as it is unusually long (60 residues) and contains two hydrophobic stretches that may form a hairpin in the cytoplasmic membrane during translocation (27). In L. lactis, as in E. coli, the native Nuc propeptide greatly affects SE. Furthermore, replacement of the native Nuc propeptide by a synthetic one can restore or even enhance SE (32).

Here, we examine the effects of changing the SP and/or propeptide on the secretion and enzymatic activity of the Nuc reporter. We found that the use of the homologous Usp45 SP (SP_Usp [58,59]) significantly improves SE. Furthermore, the Nuc propeptide is required for an optimal Nuc SE but can be replaced by synthetic propeptides that are acidic or neutral. The activities and role of charge balance in the enhancement capacity of these propeptides are discussed. The combination of SP_Usp and a synthetic propeptide resulted in significant enhancement in SE and also in overall production yields of Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains, plasmids, media, and growth conditions. E. coli strain TG1 (20) and L. lactis strains MG1363 (18) and NZ9000 (28) were used as hosts. Plasmids used are described in Fig. 1 and listed in Table 1. E. coli was grown on Luria-Bertani medium (48) and incubated at 37°C. L. lactis was grown on M17 medium (56) in which lactose was replaced by 0.5% glucose (M17-glu; Difco) and on brain heart infusion (Difco) and incubated at 30°C. SA medium was used to grow L. lactis for pulse-chase experiments (24). Antibiotics were added at the given concentrations: erythromycin, 5 µg/ml for L. lactis or 150 µg/ml for E. coli; chloramphenicol, 5 µg/ml for L. lactis and E. coli; and ampicillin, 100 µg/ml for E. coli. Induction of the nisin promoter was carried out as follows: an overnight culture was diluted 1:250 into fresh medium and incubated at 30°C until the optical density at 600 nm reached ~0.5. The culture was then divided into two equal volumes, and 1 ng of nisin/ml was added in one tube. The other tube was kept as the noninduced culture control. Cultures were further incubated, and protein samples were prepared after 1 h of induction.

DNA manipulation. Whole-cell lysates were prepared as described previously (44), except that proteinase K was added after lysozyme treatment. This additional proteolytic step eliminates mature Nuc forms associated with protoplasts prior to cell lysis.

Design of synthetic propeptides. PCRs were performed with a Perkin-Elmer Cetus (Norwalk, Conn.) apparatus using Thermophilus aquaticus DNA polymerase (Promega) as recommended by the manufacturer. Oligonucleotides were synthesized by MWG Biotech (see Table 2).

Derivatives of SP_usp:nuc fusions. To generate a Nuc derivative devoid of its propeptide, a DNA fragment containing nucT (32) was isolated from NsiI-XbaI-cut pBS:Nuc5 and cloned into an NsiI-XbaI-cut pBS:UNuc3 backbone, resulting in pBS:UNuc5. The NucT mature form contains three positive charges in its first 10 residues (32). A synthetic oligonucleotide encoding LEISSTCDA (32) was inserted into NsiI-cut pBS:UNuc3, resulting in pBS:UNuc5. To generate an SP_Usp:LEISSTCDA:NucT fusion, an NsiI-XbaI-cut nucT fragment was cloned into an NsiI-XbaI-cut pBS:UNuc5 backbone, resulting in pBS:UNuc6. These SP_usp:nuc derivative cassettes were introduced in L. lactis MG1363 as SacI-XhoI or SacI-EcoRI fragments cloned into a SacI-XhoI- or SacI-EcoRI-digested pVE3556 backbone vector resulting in plasmids pUNuc1 and pUNuc3 (SacI-XhoI cloning) or pUNuc4 and pUNuc5 (SacI-EcoRI cloning), respectively (Table 1).

Inducible expression. Nisin-controlled expression is a tightly controlled expression system with high levels of induction (10). Abundant precursor is accumulated using this system, which allowed us to examine the enzymatic activities of the two SP_Usp:NucB and SP_Usp:NucT precursor forms. The corresponding encoding cassettes were placed under the transcriptional control of the nisin-inducible promoter (P_nisA), resulting in plasmids pSEC1 (6a) and pSEC11. For each pSEC plasmid, a XhoI-BamHI SPuspnucB cassette was cloned into a XhoI-BamHI-cut pVE3655 backbone vector, which contains P_nisA, followed by a multicloning site. The latter plasmid is a derivative of pNZ8010 (10) (kindly provided by Oscar Kuipers), from which the gus gene, expressed from P_nisA, was deleted by XbaI digestion. Constructions were obtained in E. coli TG1 and then established in L. lactis NZ9000 (kindly provided by O. Kuipers [28]), a derivative of L. lactis MG1363 that carries the nisRK regulatory genes.

Expression of novel propeptide fusions to Nuc in L. lactis. To test the effect of the different synthetic propeptides on secretion efficiency (the proportion of total protein present in mature secreted form, SE) in L. lactis, plasmids pNuc13 to pNuc17 were established in strain MG1363 as cointegrates between XbaI-cut pVE3556 and XbaI-cut pBS:Nuc1, pBS:Nuc2, pBS:Nuc7, pBS:Nuc8, and pBS:Nuc9. The orientation of the resulting cointegrates was determined by restriction analysis, and plasmids harboring the same backbone structure were selected for further experiments.

Preparation of protein extracts and detection of Nuc fusions by immunoblotting. Protein samples from L. lactis cultures were prepared as described previously (32). Briefly, for cell fractionation, 2 ml of L. lactis exponential-phase cultures was harvested after a 5-min centrifugation at 6,000 × g at 4°C. Cell and supernatant fractions were treated separately. Supernatants were filtered on 0.2-µm-pore-size filters (Millipore, Bedford, Mass.) and precipitated with trichloroacetic acid (15% final concentration). Cell pellets were washed and resuspended in TES, prior to trichloroacetic acid precipitation (10% final concentration). Cell pellets were then washed once with 1 ml of cold acetone, dried, and resuspended in TES containing lysozyme (1 mg/ml; 30 min at 37°C). Cells were lysed with 20% sodium dodecyl sulfate (SDS). Equal volumes of 2× loading buffer were added to all samples. Both supernatant and cell fractions were denatured (5 min at 95°C) prior to SDS-polyacrylamide gel electrophoresis (PAGE).

Pulse-chase conditions. Pulse-chase experiments were performed essentially as described previously (32). An overnight culture of the appropriate L. lactis strain grown on SA medium (24) was used to inoculate, at 2%, 20 ml of SA medium with 33.5 µM methionine (Met). Cells were grown at 30°C to an optical density at 600 nm of 0.5; 10 ml of culture was then harvested and washed in SA medium without Met. Cells were resuspended in 2 ml of SA medium without Met and incubated at 30°C for 2 min. Cultures were pulse-labeled for 1 min by the addition of 10 µl of [³⁵S]Met (10 mCi/ml). Seven hundred microliters of Met (5%; 2,500,000-fold excess) was added (chase), and 250-µl samples were taken at given time intervals. Samples were prepared and immunoprecipitated as previously described (32, 37).

Nuc plate activity assays and zymogram. Nuc plate assays were performed as described previously (31). Nuc enzyme activity was evaluated on zymograms. After SDS-PAGE, protein samples were renatured as described elsewhere (34). A 2-mm toluidine blue-DNA agar (TBD-agar) layer was poured in the vertical support used for the polyacrylamide gel (PAG) (Protean II; Bio-Rad). After polymerization, the TBD-agar layer is kept on a single glass plate and dried at 55°C for 30 min. The renatured PAG is then placed on the TBD-agar layer covered with plastic film and incubated at 37°C for 1 to 5 h, depending on the amount of Nuc protein that was loaded in the PAG. After the appearance of bands corresponding to Nuc activity, the zymogram is photographed. Note that the SDS-PAG can be stained with Coomassie blue prior to renaturation treatment. The staining remains after renaturation and does not prevent visualization of Nuc activity (data not shown).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Replacement of SP_Nuc by the lactococcal SP_Usp enhances Nuc secretion in L. lactis. Nuc secretion in L. lactis driven by the SP_Nuc signal is inefficient (32). We therefore tested the effect of replacing SP_Nuc by SP_Usp, the SP of the major L. lactis secreted protein Usp45 (58). SP_Usp comprises 27 residues and is typical of gram-positive bacterial SPs (59). The SP_Usp:NucB fusion was constructed and expressed from the P_usp promoter (plasmid pUNuc1) (Table 1 and Fig. 1). Western blotting was performed to compare secretion of SP_Usp:NucB to that of SP_Nuc:NucB (the native Nuc protein is encoded by pNuc6) (data not shown). SE was around 95% with SP_Usp, compared to 60% with the native SP_Nuc. However, in these experiments, expression was driven by different-strength promoters (P_usp on pUNuc1 is weaker than P₅₉ on pNuc6); Northern blotting confirmed that Nuc expression from pUNuc1 was about eightfold lower than that observed from pNuc6 (data not shown). To test whether improved SE of SP_Usp:NucB was due to lower-level Nuc expression on pUNuc1, expression was ensured by the P₅₉ promoter (on plasmid pUNuc2) (Table 1 and Fig. 1). Nuc secretion was also very efficient with this construction compared to that of SP_Nuc:NucB (Fig. 2). Note that, in Western experiments, some mature NucA is found associated with the cell fraction (in the cell wall), as already shown and discussed by Dieye et al. (12) and Liebl et al. (34), in L. lactis and C. glutamicum, respectively. Immunodetection of L. lactis Usp45 protein on these samples showed no accumulation of intracellular precursor, indicating that Usp45 secretion was not altered by high-level secretion of Nuc driven by a common SP (data not shown). These results show that replacement of SP_Nuc by SP_Usp leads to efficient secretion of Nuc even at high expression levels.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Expression cassettes for Nuc production and export using the host SPUsp. Schematic structures of fusion proteins (right panel) expressed under the indicated promoters and carried by the indicated plasmids (left panel) are shown. For details of plasmid construction, see the text and Table 1. Black arrowheads indicate L. lactis promoter sequences of either the usp45 gene (Pusp), the strong promoter (P59), or the nisin-inducible promoter (Pnis). RBS, ribosome binding site of the usp45 gene; gray bar (SP), Usp45 SP coding region; gray checkered bar, native propeptide coding region; black bar, LEISSTCDA synthetic propeptide coding region; open bar, NucB or NucT coding sequence (not to scale).

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Replacement of SPNuc by SPUsp improves Nuc SE. Nuc SE was estimated by Western blot analysis on exponential-phase cultures of lactococcal strains containing pNuc6 (encoding SPNuc:NucB) and pUNuc2 (encoding SPUsp:NucB). Migration positions of precursor forms (prec) or mature forms of both NucA (A) and NucB (B) are indicated by arrows. C, cell lysates; S, supernatant fraction; SE, the proportion of total protein present in mature secreted form.

SP_Usp:NucB is more efficiently processed than SP_Nuc:NucB. Processing of the precursors SP_Usp:NucB and SP_Nuc:NucB was analyzed by pulse-chase labeling experiments using [³⁵S]Met (Fig. 3). The effect of SP replacement on SE in SA, the medium used for pulse-chase labeling, was found to be comparable with that observed in rich medium (data not shown). The proportions of precursor and mature NucB in pulse-labeled SP_Nuc:NucB and SP_Usp:NucB expressed from pNuc6 and pUNuc2, respectively, were comparable at time zero and 1 min after the chase. SP_Nuc:NucB was still present but at decreasing concentrations at 5, 10, and 20 min. In contrast, SP_Usp:NucB was detected in only trace amounts at 5 min and was absent at 10 and 20 min. These results are consistent with the conclusion that SP_Usp:NucB is more efficiently processed in L. lactis than is SP_Nuc:NucB.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Comparison of kinetics of SPNuc:NucB and SPUsp:NucB precursor processing by pulse-chase experiments. MG1363 containing pNuc6 (encoding SPNuc:NucB precursor) (upper panel) or pUNuc 2 (encoding SPUsp:NucB precursor) (lower panel) was grown in SA medium (24) and pulse-labeled with [35S]Met for 1 min. Samples were taken at different times after the pulse as indicated (in minutes). Time zero corresponds to a sample taken just at the end of the pulse. Migration positions of the different Nuc species are indicated by arrows, prec, precursor; B, NucB.

The antifolding activity of SP_Usp is not better than that of SP_Nuc. The more efficient processing of SP_Usp:NucB could be due to an antifolding activity of SP_Usp higher than that of SP_Nuc (intramolecular feature). The antifolding activities of the SPs were compared in vitro by means of a Nuc activity assay (zymogram). Constructs in which expression is driven by a nisin-inducible promoter were used to achieve high-level accumulation of the SP_Usp:Nuc(B or T) precursor forms (Table 1). Using this system, SP_Nuc:Nuc(B or T) and SP_Usp:Nuc(B or T) forms were detected in cell extracts by Western blotting (Fig. 4A). Enzymatically active forms were examined by zymograms on samples run on an SDS-PAG. No activity was detected for the precursors SP_Nuc:NucB and SP_Nuc:NucT (Fig. 4B) even after long exposure of zymograms, thus suggesting that precursor SP_Nuc:Nuc(B or T) is inactive in vitro. Similar results were already obtained with SP_Nuc:NucB produced from pNuc6 (32). In contrast, Nuc activity bands were detected for both SP_Usp:NucB and SP_Usp:NucT (Fig. 4B). Although amounts of precursor SP_Usp:Nuc(B or T) and SP_Nuc:Nuc(B or T) in cell fractions are comparable as revealed by the Western blot, only precursors comprising SP_Usp are active in zymograms. Nevertheless, the precursor forms show very weak activity compared to that of mature Nuc forms, suggesting that SP_Usp impairs enzyme activity in vitro.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Detection of enzymatic Nuc activity in precursor depends on the nature of the SP used to drive Nuc secretion. Protein samples were prepared from overnight cultures of lactococcal strains containing pNuc6 or pNuc9 (carrying cassette P59SPNuc:NucB or P59SPNuc:NucT, respectively) or nisin-induced cultures of lactococcal strains containing pSEC1 or pSEC11 (carrying cassette PnisASPUsp:NucB or PnisASPUsp:NucT, respectively). Strains containing pNuc6 and pNuc9 accumulate precursor forms in cell fraction. To achieve such accumulation with SPUsp, strains containing pSEC1 and pSEC11 were strongly induced with 10 ng of nisin/ml for 1 h. (A) Western blot analysis of cell fractions of lactococcal strains producing Nuc. A faint band is visible upon the precursor band for pSEC1 or pSEC11. This band corresponds probably to precursor aggregation due to overproduction of SPUsp:NucB and SPUsp:NucT. (B) Zymogram for detection of enzyme activity performed with the same protein samples after SDS-PAGE and gel renaturation. The positions of SPUsp:NucB (preUNucB), SPUsp:NucT (preUNucT), SPNuc:NucB (preNucB), and SPNuc:NucT (preNucT) precursor forms and of NucB (B), NucT (T), and NucA (A) mature forms are indicated by arrows.

Deletion of the natural propeptide severely reduces Nuc SE in L. lactis. The native Nuc propeptide is necessary for efficient secretion of Nuc driven by its native SP in L. lactis (32). The putative positive effects of the Nuc propeptide were also evaluated with the SP_Usp in place of the native SP_Nuc. A transcriptional and translational fusion between usp45 expression and secretion signals and a fragment encoding NucT (devoid of its natural propeptide [32]) was constructed to produce SP_Usp:NucT (encoded by pUNuc4) (Table 1 and Fig. 1). SP_Usp:NucB (pUNuc1) and SP_Usp:NucT (pUNuc4) secretion levels were compared by Western blotting on protein samples prepared on exponential cultures of the corresponding L. lactis strains (Fig. 5). The total amounts of detected Nuc forms are comparable for the two strains. However, SE of SP_Usp:NucT is only 30%, compared to around 95% for SP_Usp:NucB. Some mature NucT is also found associated with the cell fraction; this was previously observed for native NucA and/or NucT forms in L. lactis and C. glutamicum (12, 32, 34). It could be due to electrostatic interactions between negatively charged cell wall and charged residues in the N terminus of NucA and NucT (12). These results confirm that the Nuc propeptide is needed for efficient Nuc secretion in L. lactis and that this effect is independent of the SP used.

View larger version (42K): [in this window] [in a new window]   FIG. 5.   Deletion of the natural propeptide strongly decreases Nuc SE. Nuc SE was estimated by Western blot analysis on exponential-phase cultures of lactococcal strains containing pUNuc1 (encoding SPUsp:NucB) and pUNuc4 (encoding SPUsp:NucT). Migration positions of precursor forms (prec) or mature forms of NucA (A), NucB (B), and NucT (T) are indicated by arrows. C, cell lysates; S, supernatant fraction; SE, the proportion of total protein which is present in mature secreted form.

The synthetic propeptide LEISSTCDA improves both the SE and yields of NucB and NucT secreted via SP_Usp. The synthetic propeptide LEISSTCDA exerts a positive effect when acting in combination with the native SP_Nuc (32). To test its effects when combined with a nonnative SP, we constructed fusions SP_Usp:NucT (encoded by pUNuc4) and SP_Usp:LEISSTCDA-NucT (encoded by pUNuc5) (Table 1 and Fig. 1) and compared secretion profiles by Western blotting (Fig. 6). SP_Usp:LEISSTCDA-NucT processing was significantly more efficient (above 95%) than that of SP_Usp:NucT (30%). In addition, SP_Usp:LEISSTCDA-NucT also displayed a three- to fourfold-greater overall Nuc yield than did SP_Usp:NucT (Fig. 6).

View larger version (29K): [in this window] [in a new window]   FIG. 6.   The synthetic propeptide enhances Nuc SE when used in combination with SPUsp. Nuc SE was estimated by Western blot analysis on exponential-phase cultures of four lactococcal Nuc-producing strains. Right panel, MG1363 containing pUNuc4 (encoding SPUsp:NucT) and pUNuc5 (encoding SPUsp:LEISSTCDA:NucT). Left panel, MG1363 containing pUNuc1 (encoding SPUsp:NucB) and pUNuc3 (encoding SPUsp:LEISSTCDA:NucB). Migration positions of precursor forms (prec) or mature forms of NucA (A), NucB (B), NucT (T), LEISSTCDA:NucT (LEISS-T), and LEISSTCDA:NucB (LEISS-B) are indicated by arrows. C, cell lysates; S, supernatant fraction.

The SE of Nuc in L. lactis depends on the net global charge of the N terminus of the mature moiety. LEISSTCDA is characterized by its net global negative charge (2), conferred by acidic amino acid residues at positions +2 and +8. To test whether these two acidic residues are necessary for secretion enhancement by LEISSTCDA, two new propeptides were designed such that acidic amino acids were replaced by neutral or basic residues: LGISSTCNA (neutral global net charge) and LKISSTCHA (positive global net charge of +2). A third propeptide, LQVDDIPSA, was also obtained; it has a different primary structure but contains two acidic residues at positions +4 and +5 (see Materials and Methods) (Table 2 and Fig. 7). The effects of the different propeptides were evaluated on SP_Nuc:NucB. L. lactis MG1363 strains secreting native NucB and LEISSTCDA-NucB were used as controls. Exponential-phase L. lactis cultures containing the different Nuc fusions were processed and examined by Western blotting. In each case, the three expected Nuc forms (precursor and mature NucB and NucA) were detected (Fig. 7). The presence of basic residues (on LKISSTCHA) drastically reduced Nuc SE, to around 40% (Fig. 7, lanes 11 and 12), compared to 70% obtained with native Nuc (Fig. 7, lanes 3 and 4) and 90% obtained with SP_Nuc:LEISSTCDA-Nuc (Fig. 7, lanes 5 and 6). The LKISSTCHA-Nuc fusion was found mainly in precursor form. This effect is consistent with our previous finding that NucT (containing three positive charges in the first 10 residues of the mature moiety) has an SE of 30% (32). In contrast, introduction of two different negatively charged propeptides (Fig. 7, lanes 5, 6, 7, and 8) leads to a higher SE and an increased yield compared to that of native Nuc (Fig. 7, lanes 3 and 4). Insertion of a neutral propeptide also increases Nuc SE (Fig. 7, lanes 7 and 8) as well as the quantity of secreted Nuc. A maximum increase in secreted Nuc yield was approximately fourfold compared to the control (Fig. 7, lanes 3 and 4). These results show that (i) a mature protein with a positively charged N-terminal end is poorly secreted in L. lactis and (ii) both negatively charged and neutral propeptides enhance Nuc secretion in L. lactis and exhibit a dual effect of improving SE and increasing protein yield. Taken together, these findings suggest that a synthetic propeptide (devoid of basic residues) may act as a spacer that separates the globular Nuc protein from the region involved in Nuc maturation by the signal peptidase and thus facilitates precursor processing in L. lactis.

View larger version (35K): [in this window] [in a new window]   FIG. 7.   Secretion profiles of Nuc with or without synthetic propeptide derivatives. Nuc SE was estimated by Western blot analysis on cell and supernatant fractions extracted from exponential-phase L. lactis cultures. Supernatant and cell fractions were prepared separately, and immunodetection of the different Nuc forms was performed after SDS-PAGE. SE and the net charge of the first 10 residues of the mature moiety (net charge) are given below each lane. Strains contain the cloning vector alone (lanes 1 and 2); pNuc13 encoding SPNuc:Nuc (lanes 3 and 4); pNuc14 encoding the fusion protein containing the original synthetic propeptide SPNuc:LEISSTCDA-Nuc (lanes 5 and 6); and the fusion proteins containing the mutated propeptides SPNuc:LGISSTCNA-Nuc (lanes 7 and 8), SPNuc:LQVDDIPSA-Nuc (lanes 9 and 10), and SPNuc:LKISSTCHA-Nuc (lanes 11 and 12). We noted heterogeneity in the apparent sizes of precursors and secreted proproteins on SDS-PAGE, possibly due to charge modifications introduced on the synthetic propeptides. preNuc, native Nuc precursor form; B, NucB; A, NucA; pro-B, mature forms of propeptides fused to NucB.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

SP effects on Nuc secretion. SPs, although poorly conserved in their primary structure, are characterized by a conserved tripartite secondary structure (61). In comparison, SP_Nuc has an atypical structure (37). It is 60 residues long (the mean size of gram-positive bacterial SP is 28 residues [57]) and contains two highly hydrophobic stretches of approximately the same length separated by a hydrophilic region containing three basic residues. Miller et al. (37) proposed a model for insertion of native SP_Nuc:Nuc precursor in the bacterial membrane where SP_Nuc forms a hairpin. This atypical structure may be poorly recognized by the lactococcal secretion machinery, thereby explaining why Nuc precursor accumulates in L. lactis.

How does the homologous SP_Usp enhance Nuc SE? The SP reportedly acts as an intramolecular chaperone to retard protein folding. The SP thus facilitates interactions with chaperones dedicated to secretion and participates in maintaining the precursor in a conformation compatible with translocation (14, 45, 57). In vitro studies demonstrate that interaction between SP and the mature protein moiety can greatly retard the kinetics of protein folding (35). Nevertheless, in the absence of an external chaperone, although folding is retarded, it often occurs, resulting in precursor activity (as shown elsewhere for several enzymes [6, 15, 23, 53]). However, in vivo, cytoplasmic activity of a secreted protein could be lethal for the cell; precursor activity may be prevented through interactions with the secretion machinery or a dedicated inhibitor (1, 7, 21). Here, we confirmed that SP_Nuc:Nuc precursors are inactive in vitro (32), suggesting a strong intramolecular chaperone activity for SP_Nuc. In contrast, SP_Usp:Nuc precursors have some enzymatic activity in zymogram tests. This result shows that the two SPs have different antifolding capacities. The lower antifolding capacity of SP_Usp suggests that its intramolecular chaperone activity is not better than that of SP_Nuc. SP_Usp may then improve Nuc SE by allowing a better recognition of SP_Usp:Nuc precursor by the lactococcal secretion machinery (intermolecular interaction).

Effects of native and synthetic propeptides on SE of Nuc. Long propeptides that are present, for example, in proproteases have intramolecular chaperone activities and are involved in protein folding, protein secretion, and inhibition of enzyme activity (52). However, little is known about the role of short propeptides (e.g., those present in barnase, some amylases, and Nuc). Our studies rule out a role of Nuc propeptide in Nuc enzymatic activity, in keeping with previous reports (9, 32). In L. lactis, we observed a positive effect of the natural Nuc propeptide that is independent of the SP that precedes it. A synthetic propeptide, LEISSTCDA, was previously described as a secretion enhancer that can mimic the positive role of native Nuc propeptide in the SE and yield of both NucB and NucT (32).

Effect of synthetic propeptide on protein yield. The combination of the host SP_Usp with synthetic propeptide LEISSTCDA led to protein yields slightly higher (around 25 mg/liter) than those observed with pNuc7 (described in reference 32, combining SP_Nuc and LEISSTCDA). Possibly, in the absence of any synthetic propeptide, the precursor SP_Usp:Nuc is subject to a partial intracellular degradation; in this case, the real SE would actually be lower than the apparent SE. In the case of NucB, this may result in an apparent optimal SE. The insertion of a synthetic propeptide could affect the charge balance in the area of the SP cleavage site and/or the conformation of the precursor. The resulting effect could render the precursor less sensitive to intracellular degradation and/or could help it to escape the intracellular degradation thanks to a better SE. Degradation of hybrid precursor has already been observed in B. subtilis due to a poor SE of this precursor (38). Hybrid precursor could be a target for degradation by intracellular or membrane proteases such as ClpP (16) or HtrA (43). We are currently addressing this question by comparing yields and SEs of protein fusions in the different mutant backgrounds.

Combination of homologous SP and synthetic propeptide for the design of new secretion tools. The SP_Usp:LEISSTCDA combination can be used to direct secretion of other heterologous proteins in L. lactis (Ribeiro et al., submitted). In some cases, protein production is increased when LEISSTCDA propeptide is inserted between SP_Usp and the mature moiety of the hybrid precursor (Ribeiro et al., submitted). In those studies, the synthetic propeptide insertion did not interfere with antigenic properties or with activity of the heterologous protein. These results indicate that the synthetic propeptide can improve secretion of heterologous proteins other than Nuc. Recently, we successfully used, in L. lactis, the combination of SP_Usp and LEISSTCDA to improve the secretion of L7/L12, the Brucella abortus immunodominant antigen (Ribeiro et al., submitted). The host range of this combination is being currently evaluated. We propose that the combination of SP_Usp and a properly designed synthetic propeptide such as the nonapeptides reported here could be a valuable tool for enhancement of secretion of heterologous proteins in gram-positive bacteria, including various LAB species such as Streptococcus thermophilus, Lactobacillus casei, and Lactobacillus sakei.