1 MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway
2 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway
Correspondence
Lars Axelsson
lars.axelsson{at}matforsk.no
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ABSTRACT |
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INTRODUCTION |
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Many lactic acid bacteria (LAB) produce antimicrobial peptides called bacteriocins, and their production is often regulated via quorum-sensing mechanisms based on a secreted peptide pheromone (Eijsink et al., 2002; Quadri, 2002
). The pheromone activates a two-component regulatory system consisting of a histidine kinase receptor and a cognate response regulator. In strains producing class I bacteriocins (lantibiotics such as nisin), the bacteriocin itself acts as pheromone (Kuipers et al., 1995
). Strains producing class II bacteriocins, such as sakacin A and sakacin P, produce and secrete a separate, non-modified peptide pheromone whose gene is usually co-transcribed with the genes encoding the histidine kinase and the response regulator (Nes & Eijsink, 1999
). In both cases, the activated response regulator enhances transcription of all operons involved in bacteriocin production.
The promoters and regulatory genes from the Lactococcus lactis nisin gene cluster have been used to develop regulated gene expression systems for lactococci (de Ruyter et al., 1996), lactobacilli (Pavan et al., 2000
; Sørvig et al., 2003
) and other Gram-positive bacteria (Eichenbaum et al., 1998
; Bryan et al., 2000
). While being efficient and well regulated in lactococci, the plasmid-based nisin-controlled expression (NICE) systems often exhibit significant basal activity (i.e. activity without induction) in lactobacilli (Pavan et al., 2000
; Sørvig et al., 2003
). This problem can be circumvented by integrating the histidine kinase and responseregulator genes in the chromosome (Pavan et al., 2000
), thus limiting the expression systems to specially designed host strains.
Recently, we have developed one-plasmid inducible expression systems based on promoters and regulatory genes involved in the production of the class II bacteriocins sakacin A (sap gene cluster) and sakacin P (spp gene cluster) (Axelsson et al., 2003; Sørvig et al., 2003
). Sørvig et al. (2003)
constructed prototypes of the modular so-called pSIP expression vectors (Fig. 1
). These vectors contain a promoter element derived either from the sakacin A or from the sakacin P structural gene with an engineered NcoI site for translational fusion cloning, as well as the cognate two-component regulatory system. Using
-glucuronidase from Escherichia coli (gusA) as a reporter gene, sap-derived (pSIP300 series) and spp-derived (pSIP400 series) vectors were compared, and in addition the effect of the promoter driving expression of the two-component regulatory system was studied (Sørvig et al., 2003
), since this had earlier been shown to be important in the development of vectors in the NICE system (Kleerebezem et al., 1997
). The prototype pSIP vectors showed promising results in terms of expression levels and the ability to control these levels, but the maximum expression of the
-glucuronidase reporter protein was relatively low compared with results obtained with the NICE system, for example (Sørvig et al., 2003
). The potential for the use of regulatory elements from the bacteriocin regulons in protein production was further illlustrated by Mathiesen et al. (2004b)
, who constructed a plasmid containing the complete sakacin P regulon, in which the sakacin P structural gene and its cognate immunity gene had been replaced by the aminopeptidase N gene (pepN) from Lactococcus lactis. L. plantarum cells harbouring this construct produced PepN at levels amounting to 40 % of the total cellular protein. Unfortunately, the type of construct used by Mathiesen et al. (2004b)
was cumbersome to make, large (13·7 kb), difficult to change (e.g. reporter gene, replicon, marker) and unstable in one of the two Lactobacillus host strains used.
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METHODS |
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E. coli XL10 Gold (Stratagene) and Lc. lactis MG1363 were used as host strains for the construction of plasmids. E. coli XL10 Gold cells (Stratagene) were transformed according to the manufacturer's procedure; Lc. lactis MG1363 and Lactobacillus cells were electrotransformed according to Holo & Nes (1989) and Aukrust & Blom (1992)
, respectively.
Construction of plasmids.
Plasmids used and constructed in this study are listed in Table 1. Plasmids were constructed using standard molecular cloning and PCR fusion (overlap extension) techniques (Sambrook et al., 1989
; Horton & Pease, 1991
). Primers used in PCR reactions are listed in Table 2
. Restriction enzymes were purchased from Promega or New England Biolabs. Primers were purchased from MWG AG Biotech. PCR was performed with the Gene Amp PCR System 9700 (PE Biosystems) and Expand High Fidelity PCR System Polymerase (Roche Diagnostics) using standard procedures.
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For exchange of the 256rep replicon of pSIP300 and pSIP401 (Sørvig et al., 2003), SH71rep was PCR-amplified using primers Sip52c and Sip53 (Table 2
), with pVS2 (Von Wright et al., 1987
) as template. The PCR product was digested with NcoI (a site for which was inserted at the 5' end by the Sip52c primer) and Sau3AI (a site occurring naturally at the 3' end of SH71rep), and pSIP300 and pSIP401 were digested with BspHI and BamHI (restriction sites flanking the 256rep fragment), thus generating fragments with compatible ends.
The E. coli gusA gene fragment (Schlaman et al., 1994) was excised from pSIP302 (Sørvig et al., 2003
) using the 5' NcoI site that includes the start codon and the XhoI restriction site at the 3' end. The pepN gene was amplified with primers Sip32 and Sip41, which introduce NcoI and XhoI restriction sites at the 5' and 3' ends, respectively (Table 2
), using pSTO10 (Strøman, 1992
) as template. Thus, the gusA and pepN gene fragments generated could easily be introduced/exchanged using the NcoI and XhoI restriction sites present in the pSIP vectors (Fig. 1
).
The PorfX promoter fragment was obtained by PCR, using primers Sip56 and Sip57 (Table 2), and pMLS114 (Hühne et al., 1996
), as template. The primers introduce AgeI and NcoI restriction sites at the 5' and 3' ends of the promoter fragment, respectively. Since these two restriction enzymes also flank the PsapA and PsppA promoter fragments in other pSIP vectors (Fig. 1
), exchange of promoter fragments is straightforward.
New pSIP vectors were generated by ligation of the appropriate fragments and subsequent transformation of E. coli XL10 Gold or Lc. lactis MG1363. Vectors containing pUC(pGEM)ori/256rep were transformed into E. coli and vectors containing SH71rep were transformed into Lc. lactis. After plating on the appropriate agar containing erythromycin, recombinant plasmids were selected. The sequences of all constructs were verified using an ABI Prism 3100 Genetic Analyser, with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Biosystems), following the manufacturer's recommendations. To prevent changes in codon usage, silent mutations were not accepted; thus, the DNA sequences of the reporter genes (gusA or pepN) were identical in all constructs. The final plasmids were electrotransformed into L. sakei Lb790 and L. plantarum NC8. The integrity of the vectors in L. sakei Lb790 and L. plantarum NC8 was confirmed by plasmid isolation as described above. DNA rearrangements involving the vectors were not detected in any instance.
Assay for GUS activity.
A modified -galactosidase assay (Miller, 1972
) was used to determine
-glucuronidase activities in Lactobacillus strains harbouring vectors with gusA. Cultures were induced with 50 ng ml1 of the SapIP or SppIP inducing peptides (Molecular Biology Unit, University of Newcastle, UK) at OD600
0·3, and grown to OD600
1·8. Assays were performed as described by Axelsson et al. (2003)
. Activity was calculated as described by Miller (1972)
and expressed as Miller Unit equivalents (MU). Doseresponse studies were performed as described by Sørvig et al. (2003)
.
Assay for PepN activity.
Lactobacillus strains harbouring vectors with pepN were induced with 50 ng ml1 of SapIP or SppIP at OD600 0·3, and grown to OD600
1·8. Cells were harvested, resuspended in a buffer consisting of 0·01 M KCl, 0·05 M NaHPO4 and 0·001 M MgSO4, and disrupted by glass beads (106 µm and finer, Sigma), essentially as described by van de Guchte et al. (1991)
. The resulting cell-free extracts were used to assay aminopeptidase activity using L-lysine p-nitroanilide (Sigma) as substrate. PepN activity was determined according to the protocol described by Exterkate (1984)
, with the following modifications: reactions were conducted at 30 °C (instead of 37 °C), and in 0·1 M Tris/HCl, pH 8·5 (instead of a 0·1 M sodium phosphate buffer). Protein concentrations were determined using the Bio-Rad Protein Assay with BSA as standard.
Protein analysis.
For protein analysis of GusA, strains were induced and cultured as described above. Cells were harvested, resuspended in 0·05 M NaHPO4 and disrupted by glass beads (106 µm and finer, Sigma). The cell-free extracts were analysed on 12 % SDS-polyacrylamide gels. PepN-containing extracts prepared as described above were also analysed on 12 % SDS-polyacrylamide gels, and the amounts of PepN were quantified as a percentage of the total intracellular protein content by scanning the gels with a densitometer (Gel Doc 1000, Bio-Rad).
Determination of plasmid copy-number.
Plasmid copy-number was estimated in slot-blot experiments by hybridizing a plasmid-specific probe encompassing the ermB gene of the vectors to known amounts of total DNA isolated from L. sakei Lb790 or L. plantarum NC8, and to known amounts of purified plasmid DNA. All pSIP plasmids contain the ermB gene, allowing the use of the same probe in all hybridization reactions. The amount of plasmid DNA in a sample of total DNA was estimated by comparing the hybridization signal obtained with total DNA to the hybridization signal obtained with plasmid DNA. By taking into account the respective sizes of chromosomal and plasmid DNA, the number of plasmids per chromosome was calculated (Gardner et al., 2001).
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RESULTS |
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GusA expression
Previous work in our laboratories with similar expression systems has shown that maximum levels of GusA expression are obtained when the lactobacilli are harvested at OD600 1·52·0 (Axelsson et al., 2003; Sørvig et al., 2003
). Thus, in the present work, cells were harvested at OD600
1·8 to ensure maximum or near-maximum expression levels. Fig. 2
shows the results with the gusA-containing expression vectors in the host strains L. sakei Lb790 and L. plantarum NC8. The exchange of the 256rep replicon with the SH71rep replicon led to an approximately twofold increase in the levels of GUS activity for the sakacin P-based vector in both host strains (pSIP404 versus pSIP403). For the sakacin A-based vector the increase was approximately threefold in L. sakei Lb790, whereas no significant effect was observed in L. plantarum NC8 (pSIP304 versus pSIP302). Exchange of the PsppA promoter with the PorfX promoter from the same spp regulon (Brurberg et al., 1997
; Risøen et al., 2000
) clearly enhanced GusA production in both host strains (pSIP409 versus pSIP403). The highest production levels were obtained with pSIP411, which is based on the PorfX promoter and the SH71rep replicon. By changing from pSIP403 to pSIP411, GusA production in L. sakei Lb790 and L. plantarum NC8 was increased 12- and 10-fold, respectively. SDS-PAGE of cell-free extracts of GusA-producing strains showed a band representing the GusA protein, and confirmed that the highest levels of GusA were obtained in strains harbouring pSIP411 (Fig. 3a, b
).
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As observed previously (Sørvig et al., 2003), the levels of basal activity and, linked to them, the levels of induction factors, depend on both the vector and the host strain. Table 3
shows the basal activity and the induction factors for the different constructs, based on the expression of GusA activity, as depicted in Fig. 2
. For comparison, results from experiments with the two-plasmid NICE system were included (Kleerebezem et al., 1997
; Sørvig et al., 2003
). It can be seen that (i) in L. sakei, only pSIP411 of the pSIP vectors has a basal activity significantly higher than the background level for strains without the gusA gene, which is about 6 MU (Sørvig et al., 2003
); (ii) the L. plantarum variants generally show higher basal activity with all constructs and therefore generally lower induction factors than L. sakei; and (iii) the NICE system shows high levels of expression, but has higher basal activity.
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PepN expression
The experimental set-up assumed to yield near-maximum GusA expression was also applied to the expression of PepN. Fig. 4 shows the results obtained with constructs containing the pepN reporter gene. The lowest PepN levels were obtained with the pepN analogue of pSIP302, pSIP305, in both L. sakei Lb790 and L. plantarum NC8. Similarly to that which was observed with the gusA reporter gene, the exchange of 256rep with SH71rep (creating vector pSIP308) increased PepN production in L. sakei Lb790 (threefold), while PepN levels in L. plantarum NC8 were not affected (pSIP308 versus pSIP305, Fig. 4a, b
).
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In L. plantarum NC8, pSIP407 (PsppA promoter, 256rep) gave the highest level of PepN activity, while lower activities were observed with plasmids pSIP410 (PorfX promoter, 256rep) and pSIP412 (PorfX promoter, SH71rep).
In contrast to the GusA constructs, the increase in maximum PepN activity going from pSIP305 (corresponding to GusA construct pSIP302) to the pSIP412 vector (corresponding to pSIP411) in L. sakei was not accompanied by an increase in basal activity in non-induced strains. The induction factors with the pepN constructs generally followed the same trend as for GusA (Table 3), that is, the L. sakei variants had higher induction factors ranging from 60 to 300, compared to the L. plantarum variants with induction factors from 40 to 120 (results not shown). However, for the PepN variants, the basal activities were essentially the same in the two species, and the lower induction factors in L. plantarum were therefore due to generally lower maximum expression levels. Worthy of note is that for pSIP407, for which the highest PepN activity for L. plantarum was obtained, the basal activity was five- to tenfold higher than for the other constructs.
Copy number analysis
Copy number analysis was performed according to Gardner et al. (2001), as described in Methods. The chromosome sizes of L. sakei Lb790 and L. plantarum NC8 were taken as 1·9 and 3·3 Mb, respectively (Dudez et al., 2002
; Kleerebezem et al., 2003
). The copy numbers of pSIP302 and pSIP403 (plasmids containing 256rep) were estimated to be approximately three in L. sakei Lb790 and approximately six in L. plantarum NC8. This corresponds well with the estimated copy number of the p256 plasmid from L. plantarum NC7 (Sørvig et al., 2005
), from which 256rep originates. After exchanging the 256rep replicon with the SH71rep replicon (yielding pSIP304 and pSIP404), copy numbers in L. sakei Lb790 increased threefold, whereas the copy number in L. plantarum NC8 increased eightfold for pSIP304 and fourfold for pSIP404. Thus, the general picture is that vectors based on the SH71rep replicon have higher copy numbers than vectors based on the 256rep replicon.
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DISCUSSION |
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Mathiesen et al. (2004b) have recently described a sakacin P-based expression plasmid yielding PepN levels amounting to approximately 40 % of total intracellular protein in L. plantarum NC8. Mathiesen and co-workers kept the natural organization of the spp gene cluster more or less intact in the vector they created, because previous studies had shown that this could be important to obtain maximum expression levels. The best pSIP vectors developed in the present study show that equally high, or even slightly higher, expression levels may be obtained with a much more simple, smaller and versatile vector, in which all non-essential DNA has been removed, and in which the natural organization of the regulatory gene cluster has been rearranged. One example is the vector pSIP412, which gave PepN levels amounting to close to half of the total intracellular protein content in L. sakei Lb790. These PepN levels are similar to the amounts reported for overexpression of PepN in its natural host, Lc. lactis, using the NICE system (de Ruyter et al., 1996
). Another example is pSIP411, which yielded GUS levels that were five to ten times higher than the levels obtained with the best prototype vectors (Sørvig et al., 2003
) and that were comparable to the levels obtained with the NICE system (Table 3
). Previous studies have shown that nisin-based plasmid expression systems used in lactobacilli are poorly regulated (Pavan et al., 2000
; Sørvig et al., 2003
), and that integration of the regulatory genes into the chromosome may be necessary to sustain the inducibility of the vectors and to avoid basal activity (Pavan et al., 2000
). The work presented in the present study demonstrates that it is possible to obtain controlled, high-level expression with minor basal activities with a one-plasmid system and to sustain inducibility without integrating genes into the chromosome of the host.
The vectors described in this paper are easy to transform into the host strains and should function in any strain of L. sakei and L. plantarum. The present study and previous studies (Kleerebezem et al., 1997; Eichenbaum et al., 1998
; Kahala & Palva, 1999
; Mathiesen et al., 2004a
) show, however, that expression levels in both the induced and non-induced situations depend to a considerable extent on the host and reporter genes used. These differences may be due to factors such as mRNA stability, codon usage in the reporter gene, the level of transcription of the two-component regulatory system (Kleerebezem et al., 1997
; Pavan et al., 2000
; Sørvig et al., 2003
), differences in protein folding efficiency and turnover, host cell physiology, plasmid copy-number, and potential negative effects of the reporter gene product on the host cell. For example, in this work, the expression of gusA yielded less protein than the expression of pepN, although otherwise identical vectors were used (Fig. 3
). This could be due to the lactococcal pepN gene being better adapted to the Lactobacillus expression machinery than the gusA gene from E. coli. Indeed, the codon adaptation index (CAI), calculated using a web tool developed at UMBC, Maryland (http://www.evolvingcode.net/codon/CalculateCAIs.php), was significantly higher for pepN than for gusA (
0·6 versus 0·4) using highly expressed genes from L. plantarum as the reference set (Kleerebezem et al., 2003
).
While effects of reporter genes on expression levels are well known and to some extent explainable, other variables are more difficult to rationalize. For example, in the present study, results obtained with the gusA reporter gene indicated in almost all instances that exchanging the PsppA promoter with the PorfX promoter as well as increasing the plasmid copy-number was beneficial for maximizing protein production. However, in the case of the pepN reporter gene, the results were inconsistent and more strain-dependent. Remarkably, exchange of the promoter had no significant effect on PepN production, whereas it clearly affected GusA production. Thus, the effect of the promoter exchange depended on the reporter gene in both host strains. Another remarkable finding concerns the fact that increasing the plasmid copy-number had clear positive effects in the L. sakei Lb790 host, but not in the L. plantarum NC8 host. Given the fact that the integrity of the vectors was maintained in the lactobacilli hosts, recombination events with indigenous plasmids (only relevant for L. sakei Lb790) or the chromosome seem unlikely, and do not appear to offer an explanation for these results.
Generally, the L. plantarum host showed less-consistent results than the L. sakei host. A complicating factor in the case of L. plantarum NC8 comes from the presence of bacteriocin regulatory genes on the chromosome, which was discovered very recently (Maldonado et al., 2004). The induction factor and histidine protein kinase genes are novel, but the response regulator gene is identical to the response regulator involved in the production of plantaricins by L. plantarum C11 (Diep et al., 1995
). It has been shown previously that the products of the regulatory C11 genes can act on the regulated promoters in the sakacin P regulon (Brurberg et al., 1997
; Risøen et al., 2000
). Although the chromosomal system is not induced under the conditions used in this study (Maldonado et al., 2003
, 2004
) (G. Mathiesen & V. G. H. Eijsink, unpublished observations), it is possible that low background expression of one or more of the regulatory genes affects the performance of the pSIP vectors in the L. plantarum NC8 host. Although L. sakei Lb790 performed somewhat more predictably than L. plantarum NC8 in this study, it should be noted also that several L. sakei strains, including Lb790, harbour genes related to bacteriocin gene clusters in their chromosomes, although some of these are mutated and non-functional (Møretrø et al., 2005
).
In several of the settings used in this study, the PorfX promoter outperforms the PsppA promoter in terms of protein production upon induction. In previous studies, based on constructs in which the promoters were translationally coupled (but not fused) to a cat reporter gene, the PsppA and PorfX were found to be equally strong and strictly regulated (Risøen et al., 2000). Like PsppA, PorfX drives expression of a bacteriocin structural gene (Mathiesen et al., 2005
). The present study based on translational fusions reveals differences that may be due to one or more subtle differences in the promoter sequences. These include a one-nucleotide exchange in the characteristic direct repeat sequences upstream of the 10 region (Brurberg et al., 1997
), a one-nucleotide difference in the spacing between the putative ribosome-binding site (AGGAG in both cases) and the start codon, and sequence differences in the spacer regions. It is well known that changes in the length and/or sequence of the window between the ribosome-binding site and the start codon may affect expression (van de Guchte et al., 1992
; Vellanoweth & Rabinowitz, 1992
), and this has also been observed in an earlier variant of a PorfX-based expression system (Mathiesen et al., 2004a
).
In conclusion, we present a set of easy-to-use vectors which permit dose-dependent high-level gene expression in lactobacilli and which can easily be adapted for a particular purpose by exchanging one or more cassettes. The current set of pSIP vectors is based on two types of replicons and two types of pheromones, and similar vectors based on other replicons and other pheromones may be developed (Eijsink et al., 1995). For the practical purpose of expressing a gene, it may sometimes be useful to try several of the developed vectors, because it seems that the performance of the expression system depends on the combination of the gene of interest, the promoter, the replicon, and the host strain. Work is now in progress in our laboratories to exploit the new expression systems for the expression of proteins of industrial and medical importance.
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ACKNOWLEDGEMENTS |
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Received 1 April 2005;
accepted 18 April 2005.
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