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A xylose-inducible expression system for Lactococcus lactis
FEMS Microbiology Letters 239 (2004) 205–212 www.fems-microbiology.org
A xylose-inducible expression system for Lactococcus lactis Anderson Miyoshi a, Emmanuel Jamet b, Jacqueline Commissaire c, Pierre Renault b, Philippe Langella c,*, Vasco Azevedo a,** a
b
Laborato´rio de Gene´tica Celular e Molecular, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil Unite´ de Ge´ne´tique Microbienne, Inst. National de la Recherche Agronomique, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France c Unite´ de Recherches Laitie`res et de Ge´ne´tique Applique´e, Inst. National de la Recherche Agronomique, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France Received 3 July 2004; received in revised form 6 August 2004; accepted 18 August 2004 First published online 8 September 2004 Edited by A. Klier
Abstract A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lactococcus lactis NCDO2118. It is based on the use of a xylose-inducible lactococcal promoter, PxylT. The capacities of this system to produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene (nuc) fused or not to the lactococcal Usp45 signal peptide. Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein production, and correct targeting either to the cytoplasm or to the extracellular medium. Furthermore, this expression system is versatile and can be switched on or off easily by adding either xylose or glucose, respectively. These results confirm the potential of this expression system as an alternative and useful tool for the production of proteins of interest in L. lactis. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lactococcus lactis; Xylose; Staphylococcal nuclease; Inducible promoter
1. Introduction Lactococcus lactis is a food-grade Gram-positive lactic acid bacterium (LAB) that is widely used in the dairy industry for production and preservation of fermented foods. Since 1990s, many studies concern the potential use of L. lactis as a cellular factory for production and secretion of recombinant proteins for the following reasons: (i) it does not produce endotoxins [1]; (ii) a plas*
Corresponding authors. Tel.: +33 01 3465 2070; fax: +33 01 3465 2065. ** Tel./fax: +55 31 3499 2610. E-mail addresses: [email protected] (P. Langella), vasco@ mono.icb.ufmg.br (V. Azevedo).
mid-free strain does not produce the extracytoplasmic protease PrtP [2]; and (iii) relatively few proteins are known to be secreted by L. lactis, and only one, Usp45 (unknown secreted protein of 45 kDa) is secreted in detectable quantities by Coomassie blue staining [3]; a feature that facilitates the purification and analysis of a protein of interest. Thereafter, L. lactis has been extensively engineered for production of biotechnological proteins with high added value, such as enzymes and antigens (see review [4]). To date, several gene expression systems for L. lactis have been developed (for reviews, see [5,6]). The design of these systems has been achieved through studies focusing on the regulatory elements of gene expression, such as promoters, inducers and repressors. Among
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.08.018
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them, the most commonly used expression system is the nisin-controlled expression (NICE) system [7,8], which is based on a combination of the PnisA promoter and the nisRK regulatory genes. This system has proven to be highly versatile [9] and has already been used to overproduce several heterologous proteins [10]. Sugar-inducible expression systems have also been developed and some of them are alternative laboratorial tool for heterologous proteins production in L. lactis [11–16]. These sugar-dependent systems offer certain advantages: (i) sugar utilization has been extensively studied in LAB showing most systems are subject to a dual control by a dedicated regulator and by CcpA-dependent catabolite repression (for reviews, see [17,18]); (ii) most genes involved in sugar transport and catabolism are organized into strongly expressed and controlled operons; (iii) their use is reliable in a number of environmental conditions and do not require expensive inputs. However, in L. lactis, all sugar inducible systems are based on the use of the promoter controlling the plasmid lactose PTS system which retains a strong basal activity in most conditions, or is used to control the expression of the heterologous T7 polymerase making this system not suitable to produce food or food ingredients. In this context, the development of a more tightly regulated system can be an alternative and promising tool for protein production in L. lactis. In a previous study, the promoter of xylT, the xylose permease gene, (PxylT) from L. lactis NCDO2118 was described and functionally characterized [19]: PxylT presents a conserved cre site [20] and it is strongly induced (10,000fold) during mid-exponential-phase (OD600 = 0.4) in the presence of xylose [19]. Otherwise, in the presence of PTS transported sugars (as glucose, fructose and/or mannose), PxylT was shown to be tightly repressed; and finally, L. lactis PxylT is transcriptionally activated by the protein XylR [12,19,21,22]. Lastly, this promoter could thus be successively switched on by adding xylose
and off by washing the cells and grow them on glucose [19]. All these results were obtained using the Vibrio fischeri luciferase as the reporter protein [11]. Thus, based on these data, we developed a new lactococcal xylose-inducible expression system that also incorporates the ability to target heterologous proteins to cytoplasm or extracellular medium. The system, which combines the PxylT [19], the ribosome-binding site (RBS) and the signal peptide (SP) of the lactococcal secreted protein, Usp45 [23] and the Staphylococcus aureus nuclease gene (nuc) as the reporter [24,25], were successfully applied to high-level Nuc production and correct protein targeting in the vegetable L. lactis subsp. lactis strain NCDO2118.
2. Materials and methods 2.1. Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli TG1 [26] was aerobically grown in Luria–Bertani medium at 37 C. L. lactis strains (NCDO2118, IL1403 [27], MG1363 [2] and NZ9000 [8]) were anaerobically grown in M17 medium supplemented with glucose (GM17) or 0.5% xylose (XM17) at 30 C. Plasmids were selected by addition of antibiotics as follows (concentrations in micrograms per milliliter): for E. coli, ampicillin (100) and chloramphenicol (10); for L. lactis strains, chloramphenicol (10). 2.2. DNA manipulations Chromosomal DNA from L. lactis and plasmid DNA from E. coli were isolated as described previously [28,29]. General DNA manipulation techniques were carried out according to standard procedures [30]. Unless otherwise indicated, DNA restriction and modification enzymes were used as recommended by the suppliers. When re-
Table 1 Bacterial strains and plasmids used in this work Strain/plasmid
Relevant characteristics
Source/reference
Bacterial strains E. coli TG1 L. lactis NCDO2118 L. lactis IL1403 L. lactis MG1363 L. lactis NZ9000
supE, hsd, D5, thi, DlacproAB), F 0 (traD36 proABlacZD M15) L. lactis subsp. lactis (vegetable strain, plasmid free) L. lactis subsp. lactis (wild type strain, plasmid free) L. lactis subsp. cremoris (wild type strain, plasmid free) L. lactis subsp. cremoris (derivative strain of MG1363, carrying nisRK genes on the chromosome)
[26] Collection straina [27] [2] [8]
Plasmids pGEM-T Easy pGEM:PxylT pCYT:Nuc pSEC:Nuc pXYCYT:Nuc pXYSEC:Nuc
ColE1/Apr pGEM-T Easy vector carrying 548-bp PCR fragment of PxylT pWV01/Cmr; expression vector containing the fusion rbsUsp45::nucB, under the control of PnisA pWV01/Cmr; expression vector containing the fusion rbsUsp45::spUsp45::nucB, under the control of P nisA pWV01/Cmr; expression vector containing the fusion rbsUsp45::nucB, under the control of PxylT pWV01/Cmr; expression vector containing the fusion rbsUsp45::spUsp45::nucB, under the control of PxylT
Promega This work [32] [32] This work This work
a
Unite´ de Ge´ne´tique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy en Josas, cedex, France.
A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212
quired, DNA fragments were isolated from agarose gels by using the ConcertTM Rapid Gel Extraction System (Gibco BRL). PCR amplifications, using Taq DNA polymerase (Invitrogen), were performed with a DNA thermocycler (Perkin–Elmer). DNA sequencing was carried out on double-stranded plasmid DNA by the dideoxy chain termination method [31] with the MegaBACE Sequencing Systems (Amersham Biosciences). 2.3. Isolation of the xylT gene promoter and nucleotide sequence analysis The entire DNA sequence of PxylT, was isolated as follows. A 548-bp DNA fragment was PCR amplified using the following oligonucleotides, designated on the basis of the genomic DNA sequence from L. lactis IL1403 (GenBank Accession No. NC002662): A51 (5 0 -GGTAATGATTGTTGGCTTGGC-3 0 ) and A52 (5 0 -GACCAAAACGGTCACTCATTGG-3 0 ). The amplified PCR product was cloned into pGEM-T Easy Vector (Promega), resulting in pGEM:PxylT (Table 1), and was established by transformation in E. coli TG1 [30]. The integrity of the isolated sequence was confirmed by sequencing. This plasmid was then used, as template, for further plasmid constructions. The sequence data manipulations were performed with the Genetic Computer Group (GCG). Nucleic acid homology searches were performed by the Basic Local Alignment Search Tool (BLAST) service at the National Center for Biotechnology Information (NCBI). 2.4. Construction of xylose-inducible expression plasmids Plasmids designed for xylose-inducible expression were constructed as follows. A 305-bp DNA fragment was PCR amplified using the following oligonucleotides, containing one artificial restriction site at each end: XylT1 (5 0 -GGAGATCTGGTAATGATTGTTGGCTTG-3 0 – BglII site is underlined) and XylT3 (5 0 -GCGGATCCTTATTTGCAAGTCTTCTTGC-3 0 – BamHI site is underlined). The amplified PCR product was digested with BglII and BamHI restriction endonucleases and then cloned into purified backbones isolated from BglII–BamHI-cut pCYT:Nuc and pSEC:Nuc expression vectors where the expression cassettes encoding cytoplasmic or secreted Nuc under the PnisA promoter, respectively, were deleted (Table 1; [32]). The resulting plasmids, pXYCYT:Nuc and pXYSEC:Nuc (Table 1), were first obtained in E. coli TG1 and then transferred to L. lactis strains by electroporation [33].
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in fresh GM17 medium supplemented with nisin A (Sigma) at a final concentration of 1 ng/mL. Xylose-induced nuc expression: L. lactis strains harboring pXYCYT:Nuc and pXYSEC:Nuc (Table 1) were grown overnight in GM17. Cells were then harvested by centrifugation and washed twice in M17. After the second wash, the cell pellet was suspended in fresh M17 (at the same volume used for the overnight growth) and inoculated (1:50) in XM17. Kinetic of Nuc production, mediated by nisin or xylose induction, was monitored in both exponential and stationary growth phases. After induction, L. lactis cultures were grown until optical density at 600 nm (OD600) 0.4 (exponential-phase) or 1.5 (stationaryphase), before performing cell fractionation and protein extractions. 2.6. Protein extractions and Western blotting Proteins sample preparation from L. lactis cultures was performed as previously described [34] except the introduction of protease inhibitors and mild precipitation procedures. Briefly, protein samples were prepared from 2 ml of cultures. Cell pellet and supernatant were treated separately, essentially as described previously [34]. To inhibit proteolysis in supernatant samples, 1 mM phenylmethylsulfonyl fluoride and 10 mM dithiothreitol were added. Proteins were then precipitated by addition of 100 ll of 100% trichloroacetic acid, incubated 10 min on ice, and centrifuged 10 min at 17,500 · g at 4 C. For the cell fraction, TES-Lys buffer (25% sucrose, 1 mM EDTA, 50 mM Tris–HCl [pH 8.0], lysozyme [10 mg/ml]) was complemented with 1 mM phenylmethylsulfonyl fluoride and 10 mM dithiothreitol. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting, using anti-Nuc antibodies, was performed as described previously [30]. Immunodetections were carried out with protein G horseradish peroxidase conjugate (BioRad) and ECL Kit (Dupont-NEN) as recommended by the suppliers. Quantification of Nuc was performed by scanning blots after immunodetection and comparing signals to those of known amounts of a purified commercial NucA (Sigma) (ImageQuant) [25]. 2.7. Determination of nuclease activity Nuclease (Nuc) plate activity assay [35] was used to determine nuclease activity of induced or non-induced colonies of lactococci harbouring pXYCYT:Nuc or pXYSEC:Nuc plasmids.
2.5. Conditions of nisin and xylose induction 2.8. Nucleotide sequence accession number Nisin-induced nuc expression: L. lactis NZ9000 harboring pCYT:Nuc and pSEC:Nuc (Table 1; [32]) were grown overnight in GM17 and then inoculated (1:50)
The 548-bp DNA fragment, harboring the L. lactis NCDO2118 xylT gene promoter sequence, used in this
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study has been deposited in the GenBank database under Accession No. AY702978.
3. Results and discussion 3.1. Molecular characterization of the xylT gene promoter Nucleotide sequence analysis of a DNA fragment harboring the entire sequence of PxylT, the xylT gene promoter from L. lactis NCDO2118, revealed that the sequence has 96% identity with the one from L. lactis IL1403 (Fig. 1). The xylT gene promoter presents (i) the RBS; (ii) the potential –35 and –10 sequences, and (iii) a consensus cre site based on Bacillus subtilis genome sequence data [20] (Fig. 1). Further, it also comprises the 3 0 part of the xylX gene (coding for a putative acetyltransferase in xylose utilization operon; [1,19]) and its transcriptional terminator sequence, characterized by an inverted repeated sequence; and the 5 0 part of xylT gene (coding for the xylose permease gene [1,19]) (Fig. 1; GenBank Accession No. AY702978). 3.2. Xylose-inducible expression vectors for intra- and extracellular production of the staphylococcal nuclease (Nuc) We first examined whether the PxylT could drive the expression of the nuc gene, encoding for either cytoplasmic or secreted Nuc forms. For this purpose, the PxylT was transcriptionally fused to either (i) the RBS of the lactococcal usp45 gene [23] plus the DNA fragment encoding mature Nuc [24,25] (PxylT::RBSUsp45::nucB; Fig. 2(a)), or (ii) the RBS and the signal peptide (SP) of the lactococcal usp45 gene plus the DNA fragment encoding mature Nuc (PxylT::RBSUsp45::SPUsp45::nucB; Fig. 3(a)). These expression cassettes were inserted on the backbone of the pCYT:Nuc and pSEC:Nuc vectors (Table 1, [32]), devoid of the PnisA promoter, resulting in
Fig. 2. Intracellular production of Nuc using the pXYCYT:Nuc expression vector. (a) Schematic representation of the xylose-inducible expression vector for intracellular production of Nuc. For details of plasmid construction, see the text and Table 1. PxylT: xylose-inducible promoter; RBSUsp45: ribosome binding site of usp45; nucB: S. aureus nucB coding sequence; Cmr: chloramphenicol resistance; T: transcriptional terminator of the xylX gene (not to scale). (b) Cytoplasmic Nuc production on exponential and stationary growth phase cultures. Protein extracts of xylose induced (lanes Xyl) and non-induced (lanes Glu) culture samples of L. lactis NCDO2118(pXYCYT:Nuc) strain were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antibodies, in exponential- (OD600 0.4; lanes Exp) or stationary-phase (OD600 1.5; lanes Stat). The migration positions of mature NucA/ B forms are indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the standard (lane Std).
pXYCYT:Nuc and pXYSEC:Nuc vectors. In both cases, nucB expression is placed under the control of PxylT, however, in the first case, nucB expression product (Nuc) is targeted to the cytoplasm, and in the second case, Nuc is targeted to the extracellular medium (Table 1). These two vectors were then introduced into L. lactis NCDO2118 strain, resulting in NCDO2118(pXYCYT:Nuc) and NCDO2118(pXYSEC:Nuc) strains. 3.3. How does the xylose inducible expression system function? To test the potentiality of the xylose inducible expression system (XIES), these two NCDO2118(pXYCYT:-
Fig. 1. Nucleotide sequence of the xylT gene promoter from L. lactis NCDO2118. The transcriptional terminator sequence of the xylX gene is indicated in bold by arrows. The cre site, the potential –35 and –10 sequences, and the RBS of the xylT gene promoter are underlined in red bold. The conserved nucleotide positions of the cre site are in bold capital letter. The entire 548-bp DNA fragment containing the xylT promoter, the 3 0 part of the xylX gene, and the 5 0 part of xylT gene can be Accessed in GenBank through the Number AY702978 .
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Fig. 3. Extracellular production of Nuc using the pXYSEC:Nuc expression vector. (a) Schematic representation of the xylose-inducible expression vector for extracellular production of Nuc. For details of plasmid construction, see the text and Table 1. PxylT: xylose-inducible promoter; RBSUsp45: ribosome binding site of usp45; SPUsp45: signal peptide of usp45; nucB: S. aureus nucB coding sequence; Cmr: chloramphenicol resistance; T: transcriptional terminator of the xylX gene (not to scale). (b) Secreted Nuc production on exponential and stationary growth phase cultures. Protein extracts of xylose induced (lanes Xyl) and non-induced (lanes Glu) culture samples of L. lactis NCDO2118(pXYSEC:Nuc) strain were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antibodies, in exponential- (OD600 0.4; lanes Exp) or stationary-phase (OD600 1.5; lanes Stat). The migration positions of Nuc forms (preNuc [SP-NucB] and mature NucA/B) are indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the standard (lane Std). Note that the upper band in the C fraction of the xylose-grown exponential culture could be due either to an aggregation product or to an alternative start of translation.
Nuc) and NCDO2118(pXYSEC:Nuc) strains were grown in absence or in presence of xylose, counted on plates and Nuc activity was analyzed using the Nuc plate assay [35]. No Nuc activity was observed with the non-induced cultures suggesting a tight regulation of this expression system. Nuc + clones (colonies surrounded by a pink halo corresponding to Nuc activity; [25]) were only detected with the xylose-induced colonies of the NCDO2118(pXYSEC:Nuc) strain (not shown). In contrast, no Nuc activity was observed for xylose-induced colonies of the NCDO2118(pXYCYT:Nuc) strain, which is in agreement with the intracellular location of Nuc, since this Nuc plate assay is suitable to detect only secreted Nuc form (not shown). These first observations indicate that Nuc production and secretion were properly induced in presence of xylose and its product was correctly secreted to the external medium. Moreover, the system is tightly regulated, considering that no Nuc activity was detected in non-induced cultures of both strains. To check the suitability of the system in other lactococcal strains, the pXYCYT:Nuc and pXYSEC:Nuc vectors were then introduced into L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1363 strains. Note that both are derived from dairy strains. These four L. lactis strains, IL1403([pXYCYT:Nuc] or [pXYSEC:Nuc]) and MG1363([pXYCYT:Nuc] or [pXYSEC:Nuc]), grew normally on GM17 but poorly on XM17, reaching, after an overnight culture, a maximum OD600 nm 0.6 compared to OD600 nm 1.5 for the corresponding NCDO2118 derivative strains. This suggests that L. lactis IL1403 and MG1363 strains (isolated from dairy media) are not well equipped to use xylose as the carbon source in contrast to the L. lactis NCDO2118 strain, isolated from vegetal media. The Nuc phenotypes
of the resulting strains were further analyzed as described above and no Nuc activity was detected with xylose-induced cultures of these four strains. This absence of Nuc production in the dairy strains was then confirmed by Western blot experiments using anti-Nuc antibodies (not shown) confirming that they are not suitable for XIES. As previously reported [36,37], and confirmed here, xylose metabolism, in lactococcal strains, is a variable property, probably due to artificial selection which can lead to mutations in genes essential for xylose uptake and degradation (xylR, xylA and xylB). Otherwise, plant environmental isolates, such as L. lactis NCDO2118, retain this capacity. 3.4. Xylose-induced intra- or extracellular Nuc production in L. lactis NCDO2118 The production and targeting capacities of the system were analyzed by Western blotting using anti-Nuc antibodies in both exponential-(OD600 0.4) and stationary-phase (OD600 1.5) xylose induced and non-induced culture cellular (C) and supernatant (S) fractions of NCDO2118(pXYCYT:Nuc) and NCDO2118(pXYSEC:Nuc). Such analysis of the protein contents of C fractions of both exponential- and stationary phase xylose-induced NCDO2118(pXYCYT:Nuc) cultures revealed the presence of two bands, corresponding to NucB and its degradation product, NucA. In the case of the secreted form of Nuc, NucA results from the cleavage of NucB by the unique L. lactis housekeeping extracellular protease, HtrA [38]. Its presence in the C fraction could be due either to a deleterious effect during protein precipitation with trichloroacetic acid (TCA) or a residual activity of HtrA during protein preparation. These
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mature forms were detected in the C fraction at the expected size (20 kDa), whereas no signal was detected in the S fraction (Fig. 2(b)). Note that in stationary phase induced NCDO2118(pXYCYT:Nuc) culture samples, Nuc yield is around 5-fold higher than in exponential-phase culture samples. Analyses on exponential-phase cultures of induced NCDO2118(pXYSEC:Nuc) strain revealed (i) two slight bands corresponding to the intracellular precursor SPUsp45-NucB and NucB in the C fraction; and (ii) only a faint band corresponding to mature NucB in the S fraction (Fig. 3(b)). Otherwise, in stationary-phase (Fig. 3(b)), yields of Nuc in C and S fractions of induced NCDO2118(pXYSEC:Nuc) culture samples, showed to be 4- to 5-fold higher than on exponential-phase, as previously observed (Fig. 2(b)), and provided the visualization of NucB and NucA (Fig. 3(b)). In both situations, the secretion efficiency (SE; the ratio of mature protein secreted in the supernatant) was evaluated around 60% which corresponds to 15 lg of secreted active Nuc/mL. 3.5. Comparison of the rate of Nuc production using either the xylose-induced or the nisin-induced expression system To further examine the production capacity of the XIES system, comparative analyses between xyloseand nisin-induced Nuc production were performed. For this purpose, nisin-induced cultures of L. lactis NZ9000, harboring pCYT:Nuc or pSEC:Nuc expression vectors [8,32], were submitted to the same conditions described above. In exponential-phase, cytoplasmic and secreted Nuc nisin-induced productions were 10-fold more efficient than the ones observed using the XIES system (Fig. 4(a)). However, in stationary-phase, the nisin- or xylose-induced cytoplasmic Nuc productions were comparable (Fig. 4(b)). In both situations, Nuc
was correctly addressed to the desired location: cytoplasm or extracellular medium. 3.6. The xylose-induced expression system is tightly controlled by carbon source We previously observed that the transcription induced by nisin continues even after the elimination of the nisin and 10 h after the nisin-pulse [32]. Here, we tried to evaluate the versatility of the XIES system. To do this, the L. lactis NCDO2118(pXYSEC:Nuc) strain was grown on three types of sugar: (i) one non-PTS transported sugar considered as neutral versus the XIES, galactose and (ii) two PTS-transported sugars, xylose and glucose considered as inducer and repressor of the XIES, respectively. L. lactis NCDO2118(pXYSEC:Nuc) was first grown overnight in 5 mL of M17 Galactose 0.5% (GalM17) to be sure that no induction could be observed during this pre-culture. This absence of induction by galactose was confirmed by Western blots experiments on protein samples of this overnight pre-culture where no trace of Nuc was detected (data not shown). This strain was then inoculated (1:50) in 20 mL of fresh GalM17 and grown until OD600 = 0.2 where 0.5% of xylose was added. Once OD600 = 0.5 was reached, protein extracts were performed from 2 mL of this culture and analyzed by Western blot experiments which confirm the induction by xylose of the production of Nuc (Fig. 5(a); lanes Xyl/Exp). This culture was then divided in three 5 mL-aliquots: one was maintained in presence of xylose (Fig. 5(a); lanes Xyl/Stat), a second was properly washed twice with fresh culture medium M17 and the cell pellet was suspended in 5 mL of GM17 to eliminate all traces of xylose (Fig. 5(b); lanes Glu/Stat) whereas glucose 0.5% was added in the third culture (Fig. 5(b); lanes Xyl + Glu/Stat).
Fig. 4. Comparative analyses between xylose- and nisin-induced Nuc production. Protein extracts of xylose- or nisin-induced culture samples of L. lactis (i) NCDO2118([pxylT:CYT:Nuc] or [pxylT:SEC:Nuc]) and (ii) NZ9000([pCYT:Nuc] or [pSEC:Nuc]) strains were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antiserum, in exponential- (a; OD600 0.4) or stationaryphase (b; OD600 1.5). The migration positions of Nuc forms (preNuc [SP-NucB] and mature NucA/B) are indicated by arrows.
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Fig. 5. The xylose-inducible-expression-system is tighly controlled by the sugar present in the growth medium. Protein extracts of xylose-induced (panel a; lanes Xyl/Exp and Xyl/Stat) and glucose-repressed (panel b; lanes Xyl + Glu/Stat and Glu/Stat) culture samples of L. lactis NCDO2118(pXYSEC:Nuc) strain were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antibodies, in exponential-phase using xylose (OD600 0.5; lanes Exp) or in stationary-phase (OD6001.5; lanes Stat) using either xylose (lanes Xyl) or xylose plus glucose (lanes Xyl + Glu) or glucose (lanes Glu). In this last case, the cell pellet of exponential-phase culture on xylose was recovered, washed and resuspended in fresh M17 containing glucose. Growth was then pursued for 10 h and protein extracts were performed. The migration positions of Nuc forms (preNucB and mature NucA/B) are indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the standard (lane Std). These data are representative of three different experiments showing similar results.
These three cultures were grown for several hours until OD600 = 1.5. Protein extractions were performed on C and S fractions of 2 mL of each culture and the production of Nuc was followed by Western blot experiments (Fig. 5(a) and (b); lanes Xyl/Stat, Glu/Stat and Xyl + Glu/Stat). In the presence of xylose only (Fig. 5(a); lanes Xyl/Exp and Xyl/Stat), in both exponential- and stationary-phase, we observed the expected profile of Nuc distribution as previously observed in the preceding experiments with the precursor SPUsp45-NucB in the C fraction and two mature Nuc B and A forms (Fig. 5). In contrast, the presence of glucose (Fig. 5(b); lanes Xyl + Glu/Stat and Glu/Stat) led to a significant decrease of the intensity of Nuc detected bands (corresponding to the precursor) in the C fraction. No mature Nuc was detected in the S fraction of the washed sample (Fig. 5(b); lanes Glu/Stat) whereas mature Nuc B and a minor band of NucA were detected in the non-washed sample. These observations suggest that the induction by xylose and the repression by glucose of the XIES system are quite effective and rapidly established (Fig. 5). Elimination of xylose by washing and resuspension in fresh culture medium is more efficient to repress the expression system than simple addition of glucose. This aspect is an interesting property of the XIES system allowing transitory gene expression if needed.
4. Concluding remarks In this work, we described the design of a new lactococcal xylose-inducible expression system. Here, the combination of the strong PxylT from L. lactis NCDO2118 and the well-recognized genetic elements (ribosome binding site and the signal peptide) of lactococcal protein Usp45, were applied to produce and target a model reporter protein, the S. aureus nuclease (Nuc) to either cytoplasm or extracellular medium. Our results demonstrate that our xylose-inducible expression system allowed comparable high-level induced Nuc production rate on stationary-phase as
Fig. 4 the one measured with Nisin-inducible expression system. Despite founds concerning the inability of L. lactis strains which are IL1403 and MG1363 to metabolize xylose, we cannot exclude the potential application of the system to other lactococcal strains able to metabolize this carbohydrate by using xyl gene products. L. lactis NCDO2118 is a robust strain isolated from vegetal that can grow in less complex media that most dairy strains allowing its use in lower input production systems. Lastly, the xylose system could be sequentially switched on and off without washing the cells, offering thus a higher control versatility that most inducible systems known to date. In summary, the above results show that xylose-inducible expression system becomes as an alternative and useful tool for over-expression of desired proteins in L. lactis.
Acknowledgements We are grateful to Luis Bermu´dez-Humara´n for providing pCYT:Nuc and pSEC:Nuc expression vectors. We also thank Yves Le Loir and Alexandra Gruss for valuable discussions during the course of this work. Vasco Azevedo and Philippe Langella share credit in this work for senior authorship. This work was supported by COFECUB (Comite´ Franc¸ais dÕEtudes et de Coope´ration Universitaire avec le Bre´sil) and CAPES (Coordenac¸a˜o de Aperfeic¸oamen´ vel Superior, Brasil). to de Pessoal de Nı
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[20] Miwa, Y., Nakata, A., Ogiwara, A., Yamamoto, M. and Fujita, Y. (2000) Evaluation and characterization of catabolite-responsive elements (cre) of Bacillus subtilis. Nucl. Acids Res. 28, 1206– 1210. [21] Lokman, B.C., Leer, R.J., van Sorge, R. and Pouwels, P. (1994) Promoter analysis and transcriptional regulation of the Lactobacillus pentosus xylose regulon. Mol. Gen. Genet. 245, 117–125. [22] Chaillou, S., Bor, Y.C., Batt, C.A., Postma, P.W. and Pouwels, P.H. (1998) Molecular cloning and functional expression in Lactobacillus plantarum 80 of xylT, encoding the D -xyloseH + symporter of Lactobacillus brevis. Appl. Environ. Microbiol. 64, 4720–4728. [23] van Asseldonk, M., de Vos, W.M. and Simons, G. (1993) Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous a-amylase. Mol. Gen. Genet. 240, 428–434. [24] Shortle, D. (1983) A genetic system for analysis of staphylococcal nuclease. Gene 22, 181–189. [25] Le Loir, Y., Gruss, A., Ehrlich, S.D. and Langella, P. (1994) Direct screening of recombinants in Gram-positive bacteria using the secreted staphylococcal nuclease as a reporter. J. Bacteriol. 176, 5135–5139. [26] Gibson, T.J. (1984) Studies on the Epstein-Bart virus genome, Ph.D. thesis. University of Cambridge, Cambridge, England. [27] Chopin, A., Chopin, M.C., Moillo-Batt, A. and Langella, P. (1984) Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid 11, 260–263. [28] Poquet, I., Ehrlich, S.D. and Gruss, A. (1998) An export-specific reporter designed for gram-positive bacteria: application to Lactococcus lactis. J. Bacteriol. 180, 1904–1912. [29] Birnboim, H. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513–1523. [30] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [31] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. [32] Bermu´dez-Humara´n, L.G., Langella, P., Commissaire, J., Gilbert, S., Le Loir, Y., LÕHaridon, R. and Corthier, G. (2003) Controlled intra- or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis. FEMS Microbiol. Lett. 224, 307–313. [33] Langella, P., Le Loir, Y., Ehrlich, S.D. and Gruss, A. (1993) Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis. J. Bacteriol. 175, 5806–5813. [34] Le Loir, Y., Gruss, A., Ehrlich, S.D. and Langella, P. (1998) A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J. Bacteriol. 180, 1895–1903. [35] Dieye, Y., Usai, S., Clier, F., Gruss, A. and Piard, J.C. (2001) Design of a protein-targeting system for lactic acid bacteria. J. Bacteriol. 183, 4157–4166. [36] Erlandson, K.A., Park, J.H., El Khal, W., Kao, H.H, Basaran, P., Brydges, S. and Batt, C.A. (2000) Dissolution of xylose metabolism in Lactococcus lactis. Appl. Environ. Microbiol. 66, 3974– 3980. [37] Erlandson, K.A., Delamarre, S.C. and Batt, C.A. (2001) Genetic evidence for a defective xylan degradation pathway in Lactococcus lactis. Appl. Environ. Microbiol. 67, 1445–1452. [38] Poquet, I., Saint, V., Seznec, E., Simoes, N., Bolotin, A. and Gruss, A. (2000) HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing. Mol. Microbiol. 35, 1042–1051.
A xylose-inducible expression system for Lactococcus lactis Anderson Miyoshi a, Emmanuel Jamet b, Jacqueline Commissaire c, Pierre Renault b, Philippe Langella c,*, Vasco Azevedo a,** a
b
Laborato´rio de Gene´tica Celular e Molecular, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil Unite´ de Ge´ne´tique Microbienne, Inst. National de la Recherche Agronomique, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France c Unite´ de Recherches Laitie`res et de Ge´ne´tique Applique´e, Inst. National de la Recherche Agronomique, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France Received 3 July 2004; received in revised form 6 August 2004; accepted 18 August 2004 First published online 8 September 2004 Edited by A. Klier
Abstract A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lactococcus lactis NCDO2118. It is based on the use of a xylose-inducible lactococcal promoter, PxylT. The capacities of this system to produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene (nuc) fused or not to the lactococcal Usp45 signal peptide. Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein production, and correct targeting either to the cytoplasm or to the extracellular medium. Furthermore, this expression system is versatile and can be switched on or off easily by adding either xylose or glucose, respectively. These results confirm the potential of this expression system as an alternative and useful tool for the production of proteins of interest in L. lactis. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lactococcus lactis; Xylose; Staphylococcal nuclease; Inducible promoter
1. Introduction Lactococcus lactis is a food-grade Gram-positive lactic acid bacterium (LAB) that is widely used in the dairy industry for production and preservation of fermented foods. Since 1990s, many studies concern the potential use of L. lactis as a cellular factory for production and secretion of recombinant proteins for the following reasons: (i) it does not produce endotoxins [1]; (ii) a plas*
Corresponding authors. Tel.: +33 01 3465 2070; fax: +33 01 3465 2065. ** Tel./fax: +55 31 3499 2610. E-mail addresses: [email protected] (P. Langella), vasco@ mono.icb.ufmg.br (V. Azevedo).
mid-free strain does not produce the extracytoplasmic protease PrtP [2]; and (iii) relatively few proteins are known to be secreted by L. lactis, and only one, Usp45 (unknown secreted protein of 45 kDa) is secreted in detectable quantities by Coomassie blue staining [3]; a feature that facilitates the purification and analysis of a protein of interest. Thereafter, L. lactis has been extensively engineered for production of biotechnological proteins with high added value, such as enzymes and antigens (see review [4]). To date, several gene expression systems for L. lactis have been developed (for reviews, see [5,6]). The design of these systems has been achieved through studies focusing on the regulatory elements of gene expression, such as promoters, inducers and repressors. Among
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.08.018
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them, the most commonly used expression system is the nisin-controlled expression (NICE) system [7,8], which is based on a combination of the PnisA promoter and the nisRK regulatory genes. This system has proven to be highly versatile [9] and has already been used to overproduce several heterologous proteins [10]. Sugar-inducible expression systems have also been developed and some of them are alternative laboratorial tool for heterologous proteins production in L. lactis [11–16]. These sugar-dependent systems offer certain advantages: (i) sugar utilization has been extensively studied in LAB showing most systems are subject to a dual control by a dedicated regulator and by CcpA-dependent catabolite repression (for reviews, see [17,18]); (ii) most genes involved in sugar transport and catabolism are organized into strongly expressed and controlled operons; (iii) their use is reliable in a number of environmental conditions and do not require expensive inputs. However, in L. lactis, all sugar inducible systems are based on the use of the promoter controlling the plasmid lactose PTS system which retains a strong basal activity in most conditions, or is used to control the expression of the heterologous T7 polymerase making this system not suitable to produce food or food ingredients. In this context, the development of a more tightly regulated system can be an alternative and promising tool for protein production in L. lactis. In a previous study, the promoter of xylT, the xylose permease gene, (PxylT) from L. lactis NCDO2118 was described and functionally characterized [19]: PxylT presents a conserved cre site [20] and it is strongly induced (10,000fold) during mid-exponential-phase (OD600 = 0.4) in the presence of xylose [19]. Otherwise, in the presence of PTS transported sugars (as glucose, fructose and/or mannose), PxylT was shown to be tightly repressed; and finally, L. lactis PxylT is transcriptionally activated by the protein XylR [12,19,21,22]. Lastly, this promoter could thus be successively switched on by adding xylose
and off by washing the cells and grow them on glucose [19]. All these results were obtained using the Vibrio fischeri luciferase as the reporter protein [11]. Thus, based on these data, we developed a new lactococcal xylose-inducible expression system that also incorporates the ability to target heterologous proteins to cytoplasm or extracellular medium. The system, which combines the PxylT [19], the ribosome-binding site (RBS) and the signal peptide (SP) of the lactococcal secreted protein, Usp45 [23] and the Staphylococcus aureus nuclease gene (nuc) as the reporter [24,25], were successfully applied to high-level Nuc production and correct protein targeting in the vegetable L. lactis subsp. lactis strain NCDO2118.
2. Materials and methods 2.1. Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli TG1 [26] was aerobically grown in Luria–Bertani medium at 37 C. L. lactis strains (NCDO2118, IL1403 [27], MG1363 [2] and NZ9000 [8]) were anaerobically grown in M17 medium supplemented with glucose (GM17) or 0.5% xylose (XM17) at 30 C. Plasmids were selected by addition of antibiotics as follows (concentrations in micrograms per milliliter): for E. coli, ampicillin (100) and chloramphenicol (10); for L. lactis strains, chloramphenicol (10). 2.2. DNA manipulations Chromosomal DNA from L. lactis and plasmid DNA from E. coli were isolated as described previously [28,29]. General DNA manipulation techniques were carried out according to standard procedures [30]. Unless otherwise indicated, DNA restriction and modification enzymes were used as recommended by the suppliers. When re-
Table 1 Bacterial strains and plasmids used in this work Strain/plasmid
Relevant characteristics
Source/reference
Bacterial strains E. coli TG1 L. lactis NCDO2118 L. lactis IL1403 L. lactis MG1363 L. lactis NZ9000
supE, hsd, D5, thi, DlacproAB), F 0 (traD36 proABlacZD M15) L. lactis subsp. lactis (vegetable strain, plasmid free) L. lactis subsp. lactis (wild type strain, plasmid free) L. lactis subsp. cremoris (wild type strain, plasmid free) L. lactis subsp. cremoris (derivative strain of MG1363, carrying nisRK genes on the chromosome)
[26] Collection straina [27] [2] [8]
Plasmids pGEM-T Easy pGEM:PxylT pCYT:Nuc pSEC:Nuc pXYCYT:Nuc pXYSEC:Nuc
ColE1/Apr pGEM-T Easy vector carrying 548-bp PCR fragment of PxylT pWV01/Cmr; expression vector containing the fusion rbsUsp45::nucB, under the control of PnisA pWV01/Cmr; expression vector containing the fusion rbsUsp45::spUsp45::nucB, under the control of P nisA pWV01/Cmr; expression vector containing the fusion rbsUsp45::nucB, under the control of PxylT pWV01/Cmr; expression vector containing the fusion rbsUsp45::spUsp45::nucB, under the control of PxylT
Promega This work [32] [32] This work This work
a
Unite´ de Ge´ne´tique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy en Josas, cedex, France.
A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212
quired, DNA fragments were isolated from agarose gels by using the ConcertTM Rapid Gel Extraction System (Gibco BRL). PCR amplifications, using Taq DNA polymerase (Invitrogen), were performed with a DNA thermocycler (Perkin–Elmer). DNA sequencing was carried out on double-stranded plasmid DNA by the dideoxy chain termination method [31] with the MegaBACE Sequencing Systems (Amersham Biosciences). 2.3. Isolation of the xylT gene promoter and nucleotide sequence analysis The entire DNA sequence of PxylT, was isolated as follows. A 548-bp DNA fragment was PCR amplified using the following oligonucleotides, designated on the basis of the genomic DNA sequence from L. lactis IL1403 (GenBank Accession No. NC002662): A51 (5 0 -GGTAATGATTGTTGGCTTGGC-3 0 ) and A52 (5 0 -GACCAAAACGGTCACTCATTGG-3 0 ). The amplified PCR product was cloned into pGEM-T Easy Vector (Promega), resulting in pGEM:PxylT (Table 1), and was established by transformation in E. coli TG1 [30]. The integrity of the isolated sequence was confirmed by sequencing. This plasmid was then used, as template, for further plasmid constructions. The sequence data manipulations were performed with the Genetic Computer Group (GCG). Nucleic acid homology searches were performed by the Basic Local Alignment Search Tool (BLAST) service at the National Center for Biotechnology Information (NCBI). 2.4. Construction of xylose-inducible expression plasmids Plasmids designed for xylose-inducible expression were constructed as follows. A 305-bp DNA fragment was PCR amplified using the following oligonucleotides, containing one artificial restriction site at each end: XylT1 (5 0 -GGAGATCTGGTAATGATTGTTGGCTTG-3 0 – BglII site is underlined) and XylT3 (5 0 -GCGGATCCTTATTTGCAAGTCTTCTTGC-3 0 – BamHI site is underlined). The amplified PCR product was digested with BglII and BamHI restriction endonucleases and then cloned into purified backbones isolated from BglII–BamHI-cut pCYT:Nuc and pSEC:Nuc expression vectors where the expression cassettes encoding cytoplasmic or secreted Nuc under the PnisA promoter, respectively, were deleted (Table 1; [32]). The resulting plasmids, pXYCYT:Nuc and pXYSEC:Nuc (Table 1), were first obtained in E. coli TG1 and then transferred to L. lactis strains by electroporation [33].
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in fresh GM17 medium supplemented with nisin A (Sigma) at a final concentration of 1 ng/mL. Xylose-induced nuc expression: L. lactis strains harboring pXYCYT:Nuc and pXYSEC:Nuc (Table 1) were grown overnight in GM17. Cells were then harvested by centrifugation and washed twice in M17. After the second wash, the cell pellet was suspended in fresh M17 (at the same volume used for the overnight growth) and inoculated (1:50) in XM17. Kinetic of Nuc production, mediated by nisin or xylose induction, was monitored in both exponential and stationary growth phases. After induction, L. lactis cultures were grown until optical density at 600 nm (OD600) 0.4 (exponential-phase) or 1.5 (stationaryphase), before performing cell fractionation and protein extractions. 2.6. Protein extractions and Western blotting Proteins sample preparation from L. lactis cultures was performed as previously described [34] except the introduction of protease inhibitors and mild precipitation procedures. Briefly, protein samples were prepared from 2 ml of cultures. Cell pellet and supernatant were treated separately, essentially as described previously [34]. To inhibit proteolysis in supernatant samples, 1 mM phenylmethylsulfonyl fluoride and 10 mM dithiothreitol were added. Proteins were then precipitated by addition of 100 ll of 100% trichloroacetic acid, incubated 10 min on ice, and centrifuged 10 min at 17,500 · g at 4 C. For the cell fraction, TES-Lys buffer (25% sucrose, 1 mM EDTA, 50 mM Tris–HCl [pH 8.0], lysozyme [10 mg/ml]) was complemented with 1 mM phenylmethylsulfonyl fluoride and 10 mM dithiothreitol. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting, using anti-Nuc antibodies, was performed as described previously [30]. Immunodetections were carried out with protein G horseradish peroxidase conjugate (BioRad) and ECL Kit (Dupont-NEN) as recommended by the suppliers. Quantification of Nuc was performed by scanning blots after immunodetection and comparing signals to those of known amounts of a purified commercial NucA (Sigma) (ImageQuant) [25]. 2.7. Determination of nuclease activity Nuclease (Nuc) plate activity assay [35] was used to determine nuclease activity of induced or non-induced colonies of lactococci harbouring pXYCYT:Nuc or pXYSEC:Nuc plasmids.
2.5. Conditions of nisin and xylose induction 2.8. Nucleotide sequence accession number Nisin-induced nuc expression: L. lactis NZ9000 harboring pCYT:Nuc and pSEC:Nuc (Table 1; [32]) were grown overnight in GM17 and then inoculated (1:50)
The 548-bp DNA fragment, harboring the L. lactis NCDO2118 xylT gene promoter sequence, used in this
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study has been deposited in the GenBank database under Accession No. AY702978.
3. Results and discussion 3.1. Molecular characterization of the xylT gene promoter Nucleotide sequence analysis of a DNA fragment harboring the entire sequence of PxylT, the xylT gene promoter from L. lactis NCDO2118, revealed that the sequence has 96% identity with the one from L. lactis IL1403 (Fig. 1). The xylT gene promoter presents (i) the RBS; (ii) the potential –35 and –10 sequences, and (iii) a consensus cre site based on Bacillus subtilis genome sequence data [20] (Fig. 1). Further, it also comprises the 3 0 part of the xylX gene (coding for a putative acetyltransferase in xylose utilization operon; [1,19]) and its transcriptional terminator sequence, characterized by an inverted repeated sequence; and the 5 0 part of xylT gene (coding for the xylose permease gene [1,19]) (Fig. 1; GenBank Accession No. AY702978). 3.2. Xylose-inducible expression vectors for intra- and extracellular production of the staphylococcal nuclease (Nuc) We first examined whether the PxylT could drive the expression of the nuc gene, encoding for either cytoplasmic or secreted Nuc forms. For this purpose, the PxylT was transcriptionally fused to either (i) the RBS of the lactococcal usp45 gene [23] plus the DNA fragment encoding mature Nuc [24,25] (PxylT::RBSUsp45::nucB; Fig. 2(a)), or (ii) the RBS and the signal peptide (SP) of the lactococcal usp45 gene plus the DNA fragment encoding mature Nuc (PxylT::RBSUsp45::SPUsp45::nucB; Fig. 3(a)). These expression cassettes were inserted on the backbone of the pCYT:Nuc and pSEC:Nuc vectors (Table 1, [32]), devoid of the PnisA promoter, resulting in
Fig. 2. Intracellular production of Nuc using the pXYCYT:Nuc expression vector. (a) Schematic representation of the xylose-inducible expression vector for intracellular production of Nuc. For details of plasmid construction, see the text and Table 1. PxylT: xylose-inducible promoter; RBSUsp45: ribosome binding site of usp45; nucB: S. aureus nucB coding sequence; Cmr: chloramphenicol resistance; T: transcriptional terminator of the xylX gene (not to scale). (b) Cytoplasmic Nuc production on exponential and stationary growth phase cultures. Protein extracts of xylose induced (lanes Xyl) and non-induced (lanes Glu) culture samples of L. lactis NCDO2118(pXYCYT:Nuc) strain were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antibodies, in exponential- (OD600 0.4; lanes Exp) or stationary-phase (OD600 1.5; lanes Stat). The migration positions of mature NucA/ B forms are indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the standard (lane Std).
pXYCYT:Nuc and pXYSEC:Nuc vectors. In both cases, nucB expression is placed under the control of PxylT, however, in the first case, nucB expression product (Nuc) is targeted to the cytoplasm, and in the second case, Nuc is targeted to the extracellular medium (Table 1). These two vectors were then introduced into L. lactis NCDO2118 strain, resulting in NCDO2118(pXYCYT:Nuc) and NCDO2118(pXYSEC:Nuc) strains. 3.3. How does the xylose inducible expression system function? To test the potentiality of the xylose inducible expression system (XIES), these two NCDO2118(pXYCYT:-
Fig. 1. Nucleotide sequence of the xylT gene promoter from L. lactis NCDO2118. The transcriptional terminator sequence of the xylX gene is indicated in bold by arrows. The cre site, the potential –35 and –10 sequences, and the RBS of the xylT gene promoter are underlined in red bold. The conserved nucleotide positions of the cre site are in bold capital letter. The entire 548-bp DNA fragment containing the xylT promoter, the 3 0 part of the xylX gene, and the 5 0 part of xylT gene can be Accessed in GenBank through the Number AY702978 .
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Fig. 3. Extracellular production of Nuc using the pXYSEC:Nuc expression vector. (a) Schematic representation of the xylose-inducible expression vector for extracellular production of Nuc. For details of plasmid construction, see the text and Table 1. PxylT: xylose-inducible promoter; RBSUsp45: ribosome binding site of usp45; SPUsp45: signal peptide of usp45; nucB: S. aureus nucB coding sequence; Cmr: chloramphenicol resistance; T: transcriptional terminator of the xylX gene (not to scale). (b) Secreted Nuc production on exponential and stationary growth phase cultures. Protein extracts of xylose induced (lanes Xyl) and non-induced (lanes Glu) culture samples of L. lactis NCDO2118(pXYSEC:Nuc) strain were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antibodies, in exponential- (OD600 0.4; lanes Exp) or stationary-phase (OD600 1.5; lanes Stat). The migration positions of Nuc forms (preNuc [SP-NucB] and mature NucA/B) are indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the standard (lane Std). Note that the upper band in the C fraction of the xylose-grown exponential culture could be due either to an aggregation product or to an alternative start of translation.
Nuc) and NCDO2118(pXYSEC:Nuc) strains were grown in absence or in presence of xylose, counted on plates and Nuc activity was analyzed using the Nuc plate assay [35]. No Nuc activity was observed with the non-induced cultures suggesting a tight regulation of this expression system. Nuc + clones (colonies surrounded by a pink halo corresponding to Nuc activity; [25]) were only detected with the xylose-induced colonies of the NCDO2118(pXYSEC:Nuc) strain (not shown). In contrast, no Nuc activity was observed for xylose-induced colonies of the NCDO2118(pXYCYT:Nuc) strain, which is in agreement with the intracellular location of Nuc, since this Nuc plate assay is suitable to detect only secreted Nuc form (not shown). These first observations indicate that Nuc production and secretion were properly induced in presence of xylose and its product was correctly secreted to the external medium. Moreover, the system is tightly regulated, considering that no Nuc activity was detected in non-induced cultures of both strains. To check the suitability of the system in other lactococcal strains, the pXYCYT:Nuc and pXYSEC:Nuc vectors were then introduced into L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1363 strains. Note that both are derived from dairy strains. These four L. lactis strains, IL1403([pXYCYT:Nuc] or [pXYSEC:Nuc]) and MG1363([pXYCYT:Nuc] or [pXYSEC:Nuc]), grew normally on GM17 but poorly on XM17, reaching, after an overnight culture, a maximum OD600 nm 0.6 compared to OD600 nm 1.5 for the corresponding NCDO2118 derivative strains. This suggests that L. lactis IL1403 and MG1363 strains (isolated from dairy media) are not well equipped to use xylose as the carbon source in contrast to the L. lactis NCDO2118 strain, isolated from vegetal media. The Nuc phenotypes
of the resulting strains were further analyzed as described above and no Nuc activity was detected with xylose-induced cultures of these four strains. This absence of Nuc production in the dairy strains was then confirmed by Western blot experiments using anti-Nuc antibodies (not shown) confirming that they are not suitable for XIES. As previously reported [36,37], and confirmed here, xylose metabolism, in lactococcal strains, is a variable property, probably due to artificial selection which can lead to mutations in genes essential for xylose uptake and degradation (xylR, xylA and xylB). Otherwise, plant environmental isolates, such as L. lactis NCDO2118, retain this capacity. 3.4. Xylose-induced intra- or extracellular Nuc production in L. lactis NCDO2118 The production and targeting capacities of the system were analyzed by Western blotting using anti-Nuc antibodies in both exponential-(OD600 0.4) and stationary-phase (OD600 1.5) xylose induced and non-induced culture cellular (C) and supernatant (S) fractions of NCDO2118(pXYCYT:Nuc) and NCDO2118(pXYSEC:Nuc). Such analysis of the protein contents of C fractions of both exponential- and stationary phase xylose-induced NCDO2118(pXYCYT:Nuc) cultures revealed the presence of two bands, corresponding to NucB and its degradation product, NucA. In the case of the secreted form of Nuc, NucA results from the cleavage of NucB by the unique L. lactis housekeeping extracellular protease, HtrA [38]. Its presence in the C fraction could be due either to a deleterious effect during protein precipitation with trichloroacetic acid (TCA) or a residual activity of HtrA during protein preparation. These
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mature forms were detected in the C fraction at the expected size (20 kDa), whereas no signal was detected in the S fraction (Fig. 2(b)). Note that in stationary phase induced NCDO2118(pXYCYT:Nuc) culture samples, Nuc yield is around 5-fold higher than in exponential-phase culture samples. Analyses on exponential-phase cultures of induced NCDO2118(pXYSEC:Nuc) strain revealed (i) two slight bands corresponding to the intracellular precursor SPUsp45-NucB and NucB in the C fraction; and (ii) only a faint band corresponding to mature NucB in the S fraction (Fig. 3(b)). Otherwise, in stationary-phase (Fig. 3(b)), yields of Nuc in C and S fractions of induced NCDO2118(pXYSEC:Nuc) culture samples, showed to be 4- to 5-fold higher than on exponential-phase, as previously observed (Fig. 2(b)), and provided the visualization of NucB and NucA (Fig. 3(b)). In both situations, the secretion efficiency (SE; the ratio of mature protein secreted in the supernatant) was evaluated around 60% which corresponds to 15 lg of secreted active Nuc/mL. 3.5. Comparison of the rate of Nuc production using either the xylose-induced or the nisin-induced expression system To further examine the production capacity of the XIES system, comparative analyses between xyloseand nisin-induced Nuc production were performed. For this purpose, nisin-induced cultures of L. lactis NZ9000, harboring pCYT:Nuc or pSEC:Nuc expression vectors [8,32], were submitted to the same conditions described above. In exponential-phase, cytoplasmic and secreted Nuc nisin-induced productions were 10-fold more efficient than the ones observed using the XIES system (Fig. 4(a)). However, in stationary-phase, the nisin- or xylose-induced cytoplasmic Nuc productions were comparable (Fig. 4(b)). In both situations, Nuc
was correctly addressed to the desired location: cytoplasm or extracellular medium. 3.6. The xylose-induced expression system is tightly controlled by carbon source We previously observed that the transcription induced by nisin continues even after the elimination of the nisin and 10 h after the nisin-pulse [32]. Here, we tried to evaluate the versatility of the XIES system. To do this, the L. lactis NCDO2118(pXYSEC:Nuc) strain was grown on three types of sugar: (i) one non-PTS transported sugar considered as neutral versus the XIES, galactose and (ii) two PTS-transported sugars, xylose and glucose considered as inducer and repressor of the XIES, respectively. L. lactis NCDO2118(pXYSEC:Nuc) was first grown overnight in 5 mL of M17 Galactose 0.5% (GalM17) to be sure that no induction could be observed during this pre-culture. This absence of induction by galactose was confirmed by Western blots experiments on protein samples of this overnight pre-culture where no trace of Nuc was detected (data not shown). This strain was then inoculated (1:50) in 20 mL of fresh GalM17 and grown until OD600 = 0.2 where 0.5% of xylose was added. Once OD600 = 0.5 was reached, protein extracts were performed from 2 mL of this culture and analyzed by Western blot experiments which confirm the induction by xylose of the production of Nuc (Fig. 5(a); lanes Xyl/Exp). This culture was then divided in three 5 mL-aliquots: one was maintained in presence of xylose (Fig. 5(a); lanes Xyl/Stat), a second was properly washed twice with fresh culture medium M17 and the cell pellet was suspended in 5 mL of GM17 to eliminate all traces of xylose (Fig. 5(b); lanes Glu/Stat) whereas glucose 0.5% was added in the third culture (Fig. 5(b); lanes Xyl + Glu/Stat).
Fig. 4. Comparative analyses between xylose- and nisin-induced Nuc production. Protein extracts of xylose- or nisin-induced culture samples of L. lactis (i) NCDO2118([pxylT:CYT:Nuc] or [pxylT:SEC:Nuc]) and (ii) NZ9000([pCYT:Nuc] or [pSEC:Nuc]) strains were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antiserum, in exponential- (a; OD600 0.4) or stationaryphase (b; OD600 1.5). The migration positions of Nuc forms (preNuc [SP-NucB] and mature NucA/B) are indicated by arrows.
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Fig. 5. The xylose-inducible-expression-system is tighly controlled by the sugar present in the growth medium. Protein extracts of xylose-induced (panel a; lanes Xyl/Exp and Xyl/Stat) and glucose-repressed (panel b; lanes Xyl + Glu/Stat and Glu/Stat) culture samples of L. lactis NCDO2118(pXYSEC:Nuc) strain were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antibodies, in exponential-phase using xylose (OD600 0.5; lanes Exp) or in stationary-phase (OD6001.5; lanes Stat) using either xylose (lanes Xyl) or xylose plus glucose (lanes Xyl + Glu) or glucose (lanes Glu). In this last case, the cell pellet of exponential-phase culture on xylose was recovered, washed and resuspended in fresh M17 containing glucose. Growth was then pursued for 10 h and protein extracts were performed. The migration positions of Nuc forms (preNucB and mature NucA/B) are indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the standard (lane Std). These data are representative of three different experiments showing similar results.
These three cultures were grown for several hours until OD600 = 1.5. Protein extractions were performed on C and S fractions of 2 mL of each culture and the production of Nuc was followed by Western blot experiments (Fig. 5(a) and (b); lanes Xyl/Stat, Glu/Stat and Xyl + Glu/Stat). In the presence of xylose only (Fig. 5(a); lanes Xyl/Exp and Xyl/Stat), in both exponential- and stationary-phase, we observed the expected profile of Nuc distribution as previously observed in the preceding experiments with the precursor SPUsp45-NucB in the C fraction and two mature Nuc B and A forms (Fig. 5). In contrast, the presence of glucose (Fig. 5(b); lanes Xyl + Glu/Stat and Glu/Stat) led to a significant decrease of the intensity of Nuc detected bands (corresponding to the precursor) in the C fraction. No mature Nuc was detected in the S fraction of the washed sample (Fig. 5(b); lanes Glu/Stat) whereas mature Nuc B and a minor band of NucA were detected in the non-washed sample. These observations suggest that the induction by xylose and the repression by glucose of the XIES system are quite effective and rapidly established (Fig. 5). Elimination of xylose by washing and resuspension in fresh culture medium is more efficient to repress the expression system than simple addition of glucose. This aspect is an interesting property of the XIES system allowing transitory gene expression if needed.
4. Concluding remarks In this work, we described the design of a new lactococcal xylose-inducible expression system. Here, the combination of the strong PxylT from L. lactis NCDO2118 and the well-recognized genetic elements (ribosome binding site and the signal peptide) of lactococcal protein Usp45, were applied to produce and target a model reporter protein, the S. aureus nuclease (Nuc) to either cytoplasm or extracellular medium. Our results demonstrate that our xylose-inducible expression system allowed comparable high-level induced Nuc production rate on stationary-phase as
Fig. 4 the one measured with Nisin-inducible expression system. Despite founds concerning the inability of L. lactis strains which are IL1403 and MG1363 to metabolize xylose, we cannot exclude the potential application of the system to other lactococcal strains able to metabolize this carbohydrate by using xyl gene products. L. lactis NCDO2118 is a robust strain isolated from vegetal that can grow in less complex media that most dairy strains allowing its use in lower input production systems. Lastly, the xylose system could be sequentially switched on and off without washing the cells, offering thus a higher control versatility that most inducible systems known to date. In summary, the above results show that xylose-inducible expression system becomes as an alternative and useful tool for over-expression of desired proteins in L. lactis.
Acknowledgements We are grateful to Luis Bermu´dez-Humara´n for providing pCYT:Nuc and pSEC:Nuc expression vectors. We also thank Yves Le Loir and Alexandra Gruss for valuable discussions during the course of this work. Vasco Azevedo and Philippe Langella share credit in this work for senior authorship. This work was supported by COFECUB (Comite´ Franc¸ais dÕEtudes et de Coope´ration Universitaire avec le Bre´sil) and CAPES (Coordenac¸a˜o de Aperfeic¸oamen´ vel Superior, Brasil). to de Pessoal de Nı
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