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1 Unité d'Ecologie et de Physiologie du Système Digestif, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France
2 Unité des Bactéries Lactiques et Pathogènes Opportunistes, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France
3 Institute of Biological Sciences, Federal University of Minas Gerais (UFMG-ICB), Belo Horizonte, Minas Gerais, Brazil
4 Bioneer A/S, Hørsholm, Denmark
Correspondence
Philippe Langella
philippe.langella{at}jouy.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). As only a single secreted protein, Usp45 (van Asseldonk et al., 1990), can be detected by Coomassie blue staining, purification of heterologous secreted proteins could be simplified compared to other Gram-positive bacteria such as Bacillus subtilis. Nevertheless, degradation by host proteases remains a recurrent problem.
Up to now, only two major proteases, ClpP and HtrA, have been identified in plasmid-free strains of L. lactis. ClpP is an ATP-dependent enzyme reported as the major intracellular housekeeping protease in L. lactis (Frees & Ingmer, 1999) whereas HtrA is a trypsin-like serine protease essential for growth at high temperatures (39 °C for L. lactis) and which degrades misfolded proteins at the cell surface (Poquet et al., 2000; Foucaud-Scheunemann & Poquet, 2003). In this study, we constructed a L. lactis NZ9000 strain deficient in its both major proteases, ClpP and HtrA (hereafter called clpP-htrA), and evaluated its physiology and heterologous protein production potential under the control of the NICE (nisin-controlled expression) system (Kuipers et al., 1998). Comparison between the clpP-htrA double mutant and the wild-type control strain revealed, as expected, reduced proteolytic activities and increased stability of two heterologous proteins. However, growth thermosensitivity and lethality were found to be partly alleviated compared to either clpP or htrA single mutants, suggesting the occurrence of a secondary suppressive mutation event.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. As controls for growth studies, we used three strains where the erythromycin resistance gene (erm) is inserted by single crossover at various loci (resulting in an EmR phenotype): the previously described clpP (Bermúdez-Humarán et al., 2002) and htrA (Miyoshi et al., 2002) single mutant strains, hereafter called clpP- and htrA-NZ9000, respectively, as well as a his strain which was constructed by insertion of the erm gene into the non-essential histidine (his) locus (Delorme et al., 1993). The his strain has the same growth and viability behaviour as the wild-type (wt) (native EmS NZ9000) strain (data not shown) and it is hereafter called in this study L. lactis wt.
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). Antibiotics were added when required as follows: erythromycin (2.5 µg ml–1) and chloramphenicol (5 µg ml–1).
Construction of L. lactis clpP-htrA.
The L. lactis strain deficient in both its major proteases, ClpP and HtrA, was obtained by conjugation between the erythromycin-resistant (Emr) clpP-NZ9000 (clpP gene inactivated by insertion of the erm gene; Fig. 1(a); Bermúdez-Humarán et al., 2002) as donor strain and a chloramphenicol-resistant (CmR) derivative of the marker-free L. lactis NZ9000 htrA as recipient strain (Fig. 1c; Rigoulay et al., 2004). This latter strain was obtained by the introduction of pGhost3 plasmid (kindly provided by E. Maguin, INRA, Jouy en Josas) in the marker-free NZ9000 htrA strain: pGhost3 is a thermosensitive (Ts) plasmid (isolated after mutagenesis of pGK12; Maguin et al., 1992) that replicates in L. lactis at 28 °C but is lost above 37 °C. For conjugation, solid surface mating was carried out as described previously (Langella & Chopin, 1989) with the following modifications: 1 ml of saturated culture of the donor strain was mixed with 1 ml saturated culture of the recipient strain and cells were harvested, suspended in 200 µl and spread on non-selective GM17 medium. After 5 h of mating at 30 °C, cells were collected and plated at appropriate dilutions on GM17 medium containing antibiotics. Transconjugants were selected as double EmR/CmR clones and pGhost3 plasmid was then cured by a temperature shift, as follows. One transconjugant was grown in GM17 medium containing antibiotics at 30 °C until saturation. The culture was then diluted and plated without CmR at 35 °C: this temperature was chosen to allow loss of the plasmid and to limit thermal stress, as both ClpP and HtrA are heat-shock proteins (Frees & Ingmer, 1999; Poquet et al., 2000; Foucaud-Scheunemann & Poquet, 2003). After 48 h incubation, some colonies were screened on selective and non-selective GM17 media incubated for 24 h. Colonies of the clpP-htrA double mutant strain which had lost pGhost3 were Cm sensitive (CmS), but they were EmR, and Em addition was needed to maintain the clpP mutation (Fig. 1a). These candidates were also sensitive to both streptomycin (Str) and rifampicin (Rif), confirming that they were not transconjugants derived from the StrR/RifR donor strain. One of these plasmid-cured CmS transconjugants was chosen as the clpP-htrA strain.
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). Protein extracts were prepared from either exponential-phase (OD600 0.4-0.6) or stationary-phase (OD600 2–3) cultures.
Production of heterologous proteins in the clpP-htrA strain.
Production and secretion of two heterologous proteins, the staphylococcal nuclease (Nuc) and the human papillomavirus E7 protein fused to Nuc (Nuc-E7), were analysed in the clpP-htrA strain. For this, pSEC:Nuc or pSEC:Nuc-E7 plasmids, encoding Nuc and Nuc-E7, respectively (Bermúdez-Humarán et al., 2002, 2003a), were established in the four following L. lactis strains: wt, clpP-NZ9000, htrA-NZ9000 and clpP-htrA. Nuc and Nuc-E7 production was then examined in these strains by Western blotting essentially as described previously (Le Loir et al., 1998), using anti-Nuc or anti-E7 antibodies. Briefly, for cell fractionation, 2 ml of induced L. lactis cultures at a given OD600 was harvested by centrifugation at 4 °C and 10 000 r.p.m. The supernatant and cells were processed separately. To compare the amounts of secreted and cell-associated proteins, both cell and supernatant fractions were concentrated. Sample concentration was calculated as follows. The equivalent of 1 ml of 1 OD600 unit of culture (cell or supernatant) was concentrated in a 100 µl final volume as described below, and 10 µl was loaded for SDS-PAGE. Supernatants were precipitated by the addition of 10 % trichloroacetic acid, harvesting by centrifugation at 4 °C and 13 000 r.p.m. The precipitate was dissolved in 1 : 20 volume of 50 mM sodium hydroxide. Cell pellets were resuspended in 70 µl TES containing lysozyme (10 mg ml–1). After 30 min of incubation at 37 °C, cells were lysed with 30 µl 20 % SDS. Equal volumes of 2x loading buffer were added to all samples.
Determination of nuclease activity yields.
Nuclease (Nuc) activity in the supernatants of induced cultures was determined spectrophotometrically, essentially as described previously (Heins et al., 1967). Briefly, DNA added as substrate is degraded by Nuc to mono- and oligonucleotides, which are non-precipitable by perchloric acid. The products of the degradation are then quantified by measuring DNA as A260. Nuc concentration is estimated by comparison to a Nuc standard of known concentration.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The presence of a 618 bp PCR product (containing the intact clpP gene: 600 bp) from genomic DNA of the wt (his) and htrA-NZ9000 strains but not in strains clpP-NZ9000 (used as positive control) and clpP-htrA indicates the disruption of the clpP gene in our mutant strain (Fig. 1b). This genotype was confirmed by a second PCR analysis with primers 5E (CATGCCATGGCATGCAAAAGAAAAACGAAATGATACACC) and 3B (GAGCGATTAGAATTATCAGCAAGG). These primers were designed to hybridize with the integrated plasmid (erm gene, primer 5E) and with the target clpP gene (primer 3B) (Fig. 1a). As shown in Fig. 1(c), the presence of a 
1500 bp amplified band from the clpP-NZ9000 (used as positive control) and clpP-htrA strains again confirms the disruption of the clpP gene of our mutant strain.
htrA inactivation was then confirmed in the clpP-htrA strain by PCR analysis with primers 5H and 3H, designed to hybridize with upstream and downstream regions, respectively, of the htrA gene (Fig. 2a). The presence of a 902 bp PCR product (containing a fragment of the htrA gene with a 322 bp internal deletion) from genomic DNA of strains htrA-NZ9000 (used as positive control) and clpP-htrA indicates the disruption of the htrA gene in our mutant strain (Fig. 2b). PCR amplification from genomic DNA of wt and clpP-NZ9000 strains results in a 1228 bp product (containing an intact htrA gene: 1224 bp) (Fig. 2b). In addition, we confirmed the absence of HtrA protein in strain clpP-NZ9000 by Western blot analysis using antibodies raised against S. aureus HtrA1 protease (its highly conserved catalytic site) and found to specifically recognize L. lactis HtrA (Rigoulay et al., 2004). As shown in Fig. 2(c), protein extracts prepared from exponential-phase wt cultures revealed a band corresponding to the lactococcal HtrA protein (42 kDa; Rigoulay et al., 2004). Interestingly, no HtrA signal was detected in extracts prepared from stationary-phase cultures, suggesting that HtrA is mainly produced in the exponential phase (data not shown). htrA inactivation either by single crossover in htrA-NZ9000 (Miyoshi et al., 2002) or by double crossover in clpP-htrA (Fig. 2a) leads to the absence of HtrA (Fig. 2c) in exponential-phase protein extracts.
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). Surprisingly, the growth of the clpP-htrA double mutant strain was improved compared to the clpP-NZ9000 single mutant (Fig. 3a). All strains reached the same growth levels at 10 h (Fig. 3a). Viability was then analysed by monitoring strains at 30 °C for 3 days (72 h). Bacterial populations were estimated every 24 h using a spiral plater (Spiral System DS, Interscience) on GM17 agar (Em 2.5 µg ml–1) and viability was measured as c.f.u. ml–1. At 30 °C, viability of all the strains was comparable (Fig. 3b).
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), whereas the htrA mutant was severely affected at 37 °C in liquid culture medium but completely stopped growing at 39 °C (Poquet et al., 2000). We thus checked the growth phenotype of clpP-htrA at 37 °C.
At 37 °C, clpP-NZ9000 was unexpectedly found to grow almost normally in liquid culture medium. A significant improvement in growth of the clpP-htrA strain was observed when compared to both single clpP- and htrA-NZ9000 mutant strains (Fig. 4a). Furthermore, the clpP-htrA strain was able to form colonies at 37 °C (20-fold lower frequency than at 30 °C, data not shown). Thus, unexpectedly high growth rates at both 30 °C and 37 °C were observed for the clpP-htrA strain. Survival analyses showed a marked defect at 37 °C for all mutant strains: 107 c.f.u. ml–1 were measured for the wt strain after 72 h versus 106 c.f.u. ml–1 for the clpP-htrA and htrA-NZ9000 mutants and 105 c.f.u. ml–1 for the clpP-NZ9000 mutant (Fig. 4b).
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We then evaluated the response of the clpP-htrA mutant to higher temperature, ethanol and oxidative stress conditions as described previously (Foucaud-Scheunemann & Poquet, 2003). We first tested temperature stress: briefly, overnight cultures were diluted 100-fold and after 2 h, once OD600 0.10 was reached, a temperature shift from 30 °C to 39 °C was performed and growth was measured by OD600 for 10 h (Fig. 5a). After the temperature upshift, the wt strain grew to OD600 2 whereas the growth of the htrA-NZ9000 strain was rapidly arrested as previously described (Poquet et al., 2000; Foucaud-Scheunemann & Poquet, 2003). Unexpectedly, but in agreement with the growth characteristics at 37 °C, the clpP-NZ9000 strain grew better than htrA-NZ9000 (OD600 1.2), and the clpP-htrA strain even better (OD600 1.5) (Fig. 5a). Again, our data are in agreement with the presence of a secondary suppressor mutation.
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). Finally, we tested the effect of different concentrations (5, 10 and 15 mM) of hydrogen peroxide (data not shown): growth of all strains was affected to similar extent, as previously observed (Foucaud-Scheunemann & Poquet, 2003).
To explain our data, we propose that a secondary suppressor mutation occurred allowing improved growth of the double mutant strain; such an event might have been favoured during the temperature shift necessary to eliminate pGhost from the potential transconjugant candidates. It has previously been shown that in both wt and clpP mutant strains of L. lactis, trmA inactivation increased thermal stress tolerance and proteolysis of puromycyl peptides (the protease involved remained unknown, although different from FtsH or HtrA), without having any effect of oxidative stress tolerance, and TrmA was proposed to be a negative regulator of stress tolerance and proteolysis (Frees et al., 2001). Comparing the thermosensitive phenotype of our double mutant strain with a trmA-clpP mutant strain (kindly provided by Karin Hammer, Technical University of Denmark, DK-2800 Lyngby, Denmark) which is known to grow at normal rates in stress conditions (e.g. at 37 °C or in the presence of puromycin), we observed that our double mutant has a lower growth rate than the trmA-clpP mutant (data not shown). Alternatively, htrA suppressors might have arisen, as previously shown in Escherichia coli, where sohA and sohB are multi-copy suppressors of htrA (Baird & Georgopoulos, 1990; Baird et al., 1991).
Stable production of heterologous proteins in L. lactis clpP-htrA
To determine the capacities of the clpP-htrA strain to produce and secrete heterologous proteins, production of Nuc and Nuc-E7 was compared in wt, clpP-NZ9000, htrA-NZ9000 and clpP-htrA strains. For Nuc protein, Western blot analysis with anti-Nuc antibodies revealed two bands in the cell fractions of wt(pSEC:Nuc) and clpP-NZ9000(pSEC:Nuc) strains that correspond to Nuc precursor (preNuc, synthesized as a preproprotein: 22 kDa) and to NucB protein (product of preNuc signal peptide maturation: 19 kDa). Note that separation of preNuc and NucB bands could not be well achieved, perhaps due to the high concentration of SDS-PAGE (15 %) used to separate NucA from NucB. In the supernatant of these strains, a third form of Nuc was detected and it corresponds to NucA protein [product of a secondary cleavage of a 21 aa NucB propeptide: 19 kDa (Le Loir et al., 1998) performed by HtrA in L. lactis (Poquet et al., 2000)] plus NucB protein (Fig. 6). In contrast, in htrA-NZ9000(pSEC:Nuc) and clpP-htrA(pSEC:Nuc) extracts, no NucA in supernatant fractions was observed, in agreement with the absence of HtrA protease (responsible for NucB-to-NucA processing) in these strains (Poquet et al., 2000). Interestingly, supernatant samples of strains htrA-NZ9000(pSEC:Nuc) and clpP-htrA(pSEC:Nuc) showed a slightly higher concentration of NucB as determined by comparison with the signals of known amounts of Nuc by quantitative scanning of blots after immunodetection (Image-Quant; Bermúdez-Humarán et al., 2002; data not shown). To confirm these observations, Nuc yields were determined by a Nuc activity test (see Methods). The results revealed that Nuc activity was significantly higher in htrA-NZ9000(pSEC:Nuc) and clpP-htrA(pSEC:Nuc) (94.4±9.9 µg ml–1 and 85.5±5.1 µg ml–1 respectively; means±SD, n=3), than in wt(pSEC:Nuc) and clpP-NZ9000(pSEC:Nuc) supernatants fractions (73±4.4 µg ml–1 and 62.9±7.3 µg ml–1, respectively), confirming higher Nuc secretion in our double mutant strain than in wt and clpP strains.
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, 2003b). In the supernatant, one major band corresponding to the secreted Nuc-E7 fusion was observed (Fig. 7). In the cell fractions of the clpP-NZ9000(pSEC:Nuc-E7) strain, preNuc-E7 and Nuc-E7 forms were detected. Surprisingly, no Nuc-E7 signal was observed in the supernatant, suggesting that secretion of this protein in a single clpP mutant cannot be achieved. This observation could be explained by the lower metabolism of the clpP mutant once induced with nisin. Indeed, in the induced L. lactis cultures for the preparation of proteins, the OD600 reached by the clpP mutant was 15-fold lower than other strains, suggesting significant decreases both of growth rate and of protein synthesis. We can conclude that the clpP strain is not a good candidate for heterologous protein production with the NICE system. In the cell fractions of htrA-NZ9000(pSEC:Nuc-E7) and clpP-htrA(pSEC:Nuc-E7) strains, Western blotting revealed the same three bands observed in the wt strain (Fig. 7). Strikingly, the amounts of Nuc-E7 in the supernatant fractions of these two strains were about threefold higher in htrA-NZ9000(pSEC:Nuc-E7) and clpP-htrA(pSEC:Nuc-E7) strains compared to the wt(pSEC:Nuc-E7) strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Baird, L., Lipinska, B., Raina, S. & Georgopoulos, C. (1991). Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype. J Bacteriol 173, 5763–5770.
Bermúdez-Humarán, L. G., Langella, P., Miyoshi, A., Gruss, A., Guerra, R. T., Montes de Oca-Luna, R. & Le Loir, Y. (2002). Production of human papillomavirus type 16 E7 protein in Lactococcus lactis. Appl Environ Microbiol 68, 917–922.
Bermúdez-Humarán, L. G., Langella, P., Commissaire, J., Gilbert, S., Le Loir, Y., L'Haridon, R. & Corthier, G. (2003a). Controlled intra or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis. FEMS Microbiol Lett 224, 307–313.[CrossRef][Medline]
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Received 18 November 2005;
revised 10 April 2006;
accepted 28 April 2006.
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) was monitored by OD600 measurements for 10 h at 30 °C and compared to that of the wt (
), clpP-NZ9000 (
) and htrA-NZ9000 (
) strains. Viability was measured for all strains by estimating bacterial populations every 24 h for 3 days.
