1 Research Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, PO Box 56, Viikinkaari 9, FIN-00014 University of Helsinki, Finland
2 Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
Correspondence
Harri Savilahti
harri.savilahti{at}helsinki.fi
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ABSTRACT |
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INTRODUCTION |
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Transposons have been exploited as genetic tools in a variety of organisms (Craig et al., 2002). Although most transposons operate only in vivo, several of the more advanced transposon-based insertion mutagenesis systems are functional also in vitro (Boeke, 2002
). Furthermore, a combined in vitro and in vivo approach is currently feasible with two of the systems: Tn5 and Mu. With both of these transposons, the strategy involves an initial assembly of the transposition machinery in vitro, subsequent introduction of such machineries into recipient cells, and genomic integration of the delivered transposon DNA within these cells (Goryshin et al., 2000
; Lamberg et al., 2002
).
Bacteriophage Mu transposition is one of the best-characterized transposition systems (Chaconas & Harshey, 2002). In the simplest in vitro set-up, Mu transposition into an intermolecular target requires MuA transposase, transposon DNA and target DNA as the only macromolecular components (Haapa et al., 1999a
). This minimal-component reaction proceeds via initial in vitro assembly of a stable nucleoprotein complex, the Mu transpososome, which is catalytically inactive in the absence of divalent cations, but becomes activated for transposition chemistry in the presence of divalent cations such as Mg2+ (Savilahti et al., 1995
). Similarly, this activation can occur within bacterial cells following electroporation of assembled complexes, thus facilitating transposon integration into host chromosomal DNA (Fig. 1a
), as demonstrated with Gram-negative bacteria (Lamberg et al., 2002
).
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Most of the transposition mutagenesis strategies currently in use for Gram-positive bacteria are based on transposon mobilization in vivo. Typically, these strategies may involve conjugative transposons (Scott, 1993), or they may utilize transposon delivery systems that employ temperature-sensitive replication-deficient vector plasmids (Youngman, 1993
). Transposons Tn551, Tn917 and Tn916 have traditionally been used for insertional mutagenesis in staphylococci and streptococci, and these elements have proven valuable in gene identification in these organisms (Caparon & Scott, 1991
; Mei et al., 1997
; Novick, 1991
; Slater et al., 2003
). Similarly, Himar1-based transposons, as well as a derivative of Tn4001, have recently been utilized for efficient mutagenesis of staphylococci and streptococci (Bae et al., 2004
; Barnett & Scott, 2002
; Lyon et al., 1998
; May et al., 2004
).
Genomic-level approaches are needed for advanced studies on important bacterial processes such as pathogenicity, and many of these studies require the generation of large mutant collections. In this communication, we describe a general gene delivery strategy for introducing selectable markers into Gram-positive bacteria for the benefit of genomic studies, and discuss the merits and limitations of this DNA transposition-based strategy with respect to the most important technical variables. The methodology should be applicable to any bacterial species given that reasonable electroporation efficiencies can be achieved.
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METHODS |
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Standard DNA techniques.
Plasmid DNA from E. coli and Staph. aureus was isolated using purification kits from Qiagen, as recommended by the supplier, with the exception that collected Staph. aureus cells were resuspended in 300 µl PBS (Sambrook & Russell, 2001), and treated with lysostaphin (200 µg ml1) at 37 °C for 15 min prior to plasmid extraction. Standard DNA manipulation and cloning techniques, including PCR for plasmid engineering, were performed as described by Sambrook & Russell (2001)
, and DNA-modifying enzymes were used as recommended by the suppliers. DNA sequence determination was performed at the DNA sequencing facility of the Institute of Biotechnology (University of Helsinki) by using the BigDye terminator cycle sequencing kit and the ABI 377 XL sequencer, both from Applied Biosystems.
Chromosomal DNA isolation.
Chromosomal DNA from Gram-positive bacteria was isolated as follows. Cells from an overnight culture (5 ml) were collected by centrifugation and resuspended in 200 µl lysis buffer (6·7 % sucrose; 50 mM Tris/HCl, pH 7·0; 1 mM EDTA). RNase A and lysozyme (4 µl each from 10 mg ml1 and 100 mg ml1 stock solutions, respectively) were added to the suspension, which was then incubated at 37 °C for 30 min. Subsequently, 8 µl 20 mg proteinase K ml1 and 5 µl 20 % SDS were added, and the suspension was incubated at 56 °C for 30 min. The suspension was then extracted once with phenol, once with phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.), and once with chloroform/isoamyl alcohol (24 : 1, v/v). DNA was ethanol-precipitated from the water phase, and resuspended in 50 µl TE buffer (10 mM Tris/HCl, pH 7·5; 0.5 mM EDTA). DNA yields from standard 5 ml cultures ranged between 10 and 50 µg.
Plasmids.
Plasmid pUC19 was from New England Biolabs. Plasmid pLZ12-Km (Hanski et al., 1992) is an E. coliStreptococcus sp. shuttle vector that includes a gene (aphA3) encoding Km resistance, and it was obtained from Michael Caparon, Washington University, St Louis, MO, USA. Staphylococcus sp. plasmid pSK265 (Jones & Khan, 1986
) includes a gene (cat) encoding Cm resistance, and it was obtained from William Kelley. Plasmid pLEB21 (Qiao et al., 1996
) was obtained from Per Saris, University of Helsinki; this plasmid is a modified pUC6S (Vieira & Messing, 1991
) in which a cloned Lactobacillus reuteri gene (ermB) encoding Em resistance (Axelsson et al., 1988
) is inserted at a SmaI site. Plasmid pVTF1 is a derivative of plasmid pEntranceposon-KanR (F-766; Finnzymes), and it was obtained from Ville Tieaho, Finnzymes. Instead of the entire vector backbone between two BglII sites, pVTF1 contains a pUC19 origin of replication (nucleotides 8481571 of pUC19, numbered as indicated by New England Biolabs). Furthermore, an additional BglII site has been engineered into this origin at position 1197.
Mini-Mu transposons.
The pUC19-derived carrier plasmids pLEB620 and pHTH2 for transposons Em-Mu and Km-Mu, respectively (Fig. 1b), were constructed as follows. The 1·27 kb BamHI/BglII fragment of pLEB21 that includes the ermB gene was ligated to the 0·92 kb BamHI fragment of pVTF1 to yield pLEB620. Similarly, the 1·5 kb SalI fragment of pLZ12-Km that includes the aphA3 gene was end-filled with Klenow enzyme and ligated to the end-filled 0·92 kb BamHI fragment of pVTF1 to yield pHTH2. To confirm authenticity, these two plasmids were sequenced by using the Mu in vitro transposition-based DNA sequencing kit (Template Generation System, Finnzymes). From their respective carrier plasmids, mini-Mu transposons were released by BglII digestion that leaves four-nucleotide 5' overhangs flanking the exposed transposon ends. Such an end configuration in mini-Mu transposons ensures efficient assembly of stable transpososomes (Haapa et al., 1999b
; Savilahti et al., 1995
). DNA fragments were purified using anion-exchange chromatography, as described by Haapa et al. (1999b)
.
Electrocompetent cells
Electrocompetent cells for E. coli were prepared, stored and used as described previously (Lamberg et al., 2002). Electrocompetent cells for Gram-positive bacterial strains were prepared as described below. These preparations were used either directly (fresh) for electroporation, or they were frozen as aliquots in liquid nitrogen and stored at 80 °C until thawed for electroporation. The viable counts of these preparations were routinely
5x1010 c.f.u. ml1. Approximately 70 % of the cells survived the freezing step.
Staph. aureus.
An overnight culture in THY medium was diluted (1 : 500) in a total volume of 200 ml THY and grown at 37 °C with gentle agitation to an OD600 of 0·4. Cells were collected by centrifugation at 3000 r.p.m. (1248 g) in a Heraeus Biofuge Primo fixed-angle rotor at 4 °C for 15 min. Cells were then washed by resuspension in 20 ml ice-cold 0·5 M sucrose and collected by subsequent centrifugation as above. This washing step was repeated. The cells were then resuspended in 10 ml ice-cold solution containing 0·5 M sucrose and 10 % (v/v) glycerol, and sedimented again by centrifugation, as described above. Subsequently, the cell pellet was resuspended in 0·5 M sucrose and 10 % glycerol, in a total volume of 0·5 ml.
Strep. suis.
Cells were grown in THY medium supplemented with 30 mM glycine to an OD600 of 0·2. Subsequently, the cells were prepared as described previously (Pulliainen et al., 2003
).
Strep. pyogenes.
Cells were grown in THY medium to an OD600 of 0·3. Subsequently, the cells were prepared as described previously for Strep. suis (Pulliainen et al., 2003
).
Transpososome assembly.
The in vitro transpososome assembly was performed as described previously (Lamberg et al., 2002). The standard assembly reaction (40 µl) contained 2·2 pmol (
2·0 µg) transposon DNA, 9·8 pmol (0·8 µg) MuA, 150 mM Tris/HCl (pH 6·0), 50 % (v/v) glycerol, 0·025 % (w/v) Triton X-100, 150 mM NaCl and 0·1 mM EDTA. The reaction was carried out at 30 °C for 2 h, and the assembly of transpososomes was monitored by agarose (NuSieve 3 : 1) gels containing BSA (87 µg ml1) and heparin (87 µg ml1), as described previously (Lamberg et al., 2002
).
Concentration of transpososomes and electroporation.
Transpososomes were concentrated, and the preparation was desalted for electroporation as follows. Initially, a total of 16 standard assembly reactions were pooled, and the pool volume was brought to 4 ml with water. The mixture was then filtered using a Centricon YM-100 centrifugal cartridge (100 kDa cut-off; Millipore), according to the manufacturer's instructions. The retentate was then desalted by passage with 2 ml water, yielding a final transpososome stock for electroporation. An approximate tenfold increase in transpososome concentration was achieved by this method, as estimated from the final volume of the stock (60 µl).
For electroporation, thawed electrocompetent cells (50 µl) were initially mixed on ice with 1 or 2 µl transpososome preparation; transposon preparations without the addition of MuA and replicative plasmid preparations were used in control experiments. The mixture was next transferred to a pre-chilled 0·1 cm electrode spacing cuvette (Bio-Rad). Electroporation was then performed using a Gene Pulser II electroporator (Bio-Rad) with the following settings: Staph. aureus (100 , 2·3 kV, 25 µF), Strep. pyogenes and Strep. suis (200
, 1·8 kV, 15 µF). Subsequently, 1 ml pre-warmed THY medium supplemented with 0·3 M sucrose was added, and the suspension was incubated at 37 °C for 90 min with gentle agitation (Staph. aureus) or 2 h without agitation (Strep. pyogenes and Strep. suis). For the selection of genomic integrations, the cells were then spread on agar plates containing BHI (Staph. aureus) or THY (Strep. pyogenes and Strep. suis), and appropriate antibiotics. Alternatively, glycerol (100 %) was added to the cell suspension to the final concentration of 15 % (v/v), the suspension was frozen as aliquots in liquid nitrogen, and the aliquots were stored at 80 °C until thawed for selection as above.
Southern blotting.
For blotting, 2·5 µg chromosomal DNA was digested with PvuII and separated on a 0·8 % Seakem GTG agarose gel. The DNA was transferred with 0·4 M NaOH to a nylon filter (Hybond-N+, Amersham) and fixed with UV light (Stratalinker UV cross-linker; Stratagene). Southern hybridization was performed essentially as described by Sambrook & Russell (2001), with
-32P-labelled transposon (Em-Mu or Km-Mu) probes (Random Primed, Roche). Visualization was done by autoradiography using the Fujifilm Image Reader BAS-1500.
Determination of chromosomal target sites.
The chromosomal DNA of each antibiotic-resistant isolate was digested as follows: with BamHI for Km-Mu isolates of Strep. suis and Strep. pyogenes, with PvuII for Em-Mu isolates of Strep. pyogenes and Staph. aureus ATCC 29213, and with NheI+SpeI for Em-Mu isolates of Staph. aureus S30. In each case, the digestion generated a DNA fragment with an intact transposon attached to its flanking chromosomal DNA. These fragments were then cloned into the BamHI, SmaI or XbaI site of pUC19, respectively, using appropriate selection schemes with two antibiotics. DNA sequences of transposon borders were determined from these plasmids using the following primers that read the sequence outwards from within the transposons as specified in Fig. 1(b). SeqA, 5'-ATCAGCGGCCGCGATCC-3'; HSP-393, 5'-GAGGTCCCTAGCGCCTACG-3'; HSP-379, 5'-GGAATTTGTATCGTCGATCC-3'. Genomic transposon insertion site loci (Table 2
) were identified by comparing the insertion site data to publicly available genomic sequences using BLAST database searches on the National Center for Biotechnology Information and the Wellcome Trust Sanger Institute servers. ORFs were analysed by using web-based programs on the European Bioinformatics Institute server.
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RESULTS |
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Assembly and concentration of transpososomes
Mu transpososomes were assembled by incubating Em-Mu and Km-Mu transposons with MuA transposase in the absence of divalent metal ions (see Methods), and transpososome assembly was monitored using native agarose gel electrophoresis as described (Lamberg et al., 2002). In this gel retardation assay, transpososomes are visualized as proteinDNA complexes that can withstand a challenge by heparin embedded in the gel. Both Em-Mu and Km-Mu produced transpososomes in similar amounts (data not shown), and the yields were comparable to those observed in previous studies with analogous mini-Mu transposons (Lamberg et al., 2002
). The two transpososome preparations were then concentrated approximately tenfold by centrifugal cartridges, and the preparations were desalted prior to electroporation (see Methods). Analytical gel retardation assay verified successful concentration of transpososomes and indicated that the pre-assembled complexes tolerated the treatments without disassembly (data not shown).
Electroporation
Results from the studies with Gram-negative bacteria (Lamberg et al., 2002) indicated that the major factors affecting the colony-formation capacity of each recipient strain include the method of preparing electrocompetent cells and the electroporation parameters. Therefore, we first optimized these factors for each strain studied by using replicative plasmids for electroporation and by varying cell culture conditions and pulse parameters (data not shown). In parallel, viable counts were determined following electroporation to evaluate corresponding cell survival rates. The optimized conditions varied among species and strains (see Methods), and electroporation efficiencies ranged from 5x104 to 4x106 c.f.u. (µg introduced plasmid DNA)1 (Table 1
). With all strains, approximately 50 % of the cells survived electroporation under the optimized conditions. For genomic integration, an aliquot of the concentrated and desalted assembly reaction was electroporated into electrocompetent Staph. aureus, Strep. pyogenes or Strep. suis cells, and bacterial clones were selected for resistance of appropriate antibiotic. The colony-forming capacity among species ranged from 1x101 to 2x104 c.f.u. (µg introduced transposon DNA)1. Notably, these values correlated with the electroporation efficiency values obtained with replicative plasmids, being consistently two to three orders of magnitude lower (Table 1
); thus, the results underscored the importance of optimizing the method of preparing competent cells. Only those samples that contained detectable proteinDNA complexes yielded antibiotic-resistant colonies.
Genomic integration
Southern blot analysis with transposon-specific probes can be used to evaluate the presence and copy number of integrated transposons within genomic DNA. Digestion of chromosomal DNA with an enzyme that does not cleave the transposon DNA generates one fragment that hybridizes to the probe for each integrated transposon copy. Chromosomal DNA from seven Staph. aureus EmR, ten Strep. pyogenes EmR, and ten Strep. suis KmR clones was isolated, digested with PvuII (which does not cut the transposon sequence), and analysed by Southern hybridization with appropriate transposon probes. All isolates generated a single, prominent band with a discrete and, for each clone, different gel mobility (Fig. 2). Control DNA from the recipient strains did not produce detectable bands in the analyses. These and additional data obtained by using several other restriction enzymes for chromosomal DNA digestion (data not shown) indicated that a single copy of the transposon DNA was present in the bacterial chromosome of each of the antibiotic-resistant clones studied.
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DISCUSSION |
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The methodology is based on electroporation of in vitro-assembled Mu DNA transposition complexes that become activated for transposition chemistry within bacterial cells and subsequently integrate the delivered exogenous DNA into the genome of the recipient cell, as previously shown with Gram-negative bacteria (Lamberg et al., 2002). However, to be useful with Gram-positive bacteria, the original strategy needed to be modified in two critical ways. First, we utilized mini-Mu transposons containing gene expression cassettes that are known to be functional in Gram-positive bacteria, and which confer resistance to the common antibiotics Em and Km, even when present as a single genomic copy (Autret et al., 2001
; Qiao et al., 1996
). Second, an important transpososome concentration step was introduced in the protocol, which effectively increased the number of complexes available for penetration into cells in a given electroporation experiment.
Our data demonstrate the usefulness of the Mu transpososome delivery-based genomic DNA integration strategy with three pathogenic Gram-positive species: Staph. aureus, Strep. suis and Strep. pyogenes. The strategy was particularly efficient with Staph. aureus, and more than 104 antibiotic-resistant colonies were obtained per microgram of input transposon DNA (Table 1). Such a high number guarantees an easy generation of exhaustive mutant libraries for this species. However, as the efficiency was lower in the other two species studied, construction of very large libraries for Strep. suis and Strep. pyogenes would require scaling up the procedure, e.g. by electroporating more aliquots of competent cells and/or further optimizing the procedures of preparing competent cells for these species. We were not able to use Em-Mu for mutagenesis of Strep. suis, as this strain was naturally resistant to Em. In addition, the KmR cassette did not confer resistance to Km in Staph. aureus, even when present within in a multicopy plasmid, perhaps reflecting differences in the mechanisms of how the studied bacterial strains recognize critical translation signals. These results underscore the importance of preliminary tests prior to any planned mutagenesis projects, i.e. the strain aimed for mutagenesis should be properly tested with regard to its resistance properties and the functionality of the planned resistance-determining cassette.
Electroporation efficiencies are usually higher in Gram-negative bacteria than in Gram-positive bacteria, due most likely to structural differences between the Gram-positive cell wall and the Gram-negative cell envelope. Electroporation efficiencies with Strep. pyogenes and Strep. suis were higher when fresh cells were used (data not shown). In contrast, the electroporation efficiency with Staph. aureus was highest when frozen cells were used, emphasizing the importance of optimizing for each given strain the conditions and treatments ultimately leading to electrocompetence of the cells. In general, substances that inhibit the synthesis of the peptidoglycan cell wall of Gram-positive bacteria are beneficial when included in the growth medium (Eynard & Teissié, 2000). These reagents most probably directly affect the rigidity of the cell wall and make it more permeable to the incoming DNA. For this purpose, we used glycine in our experiments with Strep. suis. However, alternative reagents, such as penicillin G or DL-threonine, may also be used (Mercenier & Chassy, 1988
). Optimal electroporation pulse may also vary among different strains. Accordingly, prior to experimenting with transpososomes, the optimal pulse parameters should be determined for each strain by using a replicative control plasmid. Notably, the results of such control electroporation experiments are indicative of the usefulness of the Mu transpososome strategy; so far the yield of transposon-containing clones has correlated with the observed competence status of a given bacterial strain (Lamberg et al., 2002
; this study). In summary, for the generation of sizeable mutant banks, reasonable, but not necessarily very high, electroporation efficiencies are required. Because multiple consecutive electroporations can be performed without too much extra effort, electroporation efficiencies ranging from 104 to 105 c.f.u. (µg replicative plasmid DNA)1 should be high enough for the generation of sizeable mutant libraries, which are adequate, for example, for microbial pathogenesis studies to identify novel virulence genes.
For the generation of mutant banks and experiments thereafter, it is essential that each individual clone contains only one newly introduced mutation. This was the case in the present study, as each analysed clone included a single integrated transposon. In addition, all of these clones contained an accurate 5 bp target site duplication, indicating that the gapped Mu transposition DNA intermediate with single-stranded regions can be repaired correctly by the host machinery of Gram-positive bacteria, and that the possible presence of host-encoded restriction systems or proteases does not impede MuA-mediated transposition. Furthermore, only those samples that contained detectable proteinDNA complexes yielded antibiotic-resistant colonies, reflecting the inability of the studied cells to efficiently recombine the incoming naked transposon DNA. Our protocol yielded a large number of integrant clones, indicating that the methodology is extremely suitable for the construction of exhaustive mutant libraries. For an average 2·0 Mb microbial genome, when an average ORF is estimated to be 1 kb, a tenfold per-gene coverage would require 20 000 individual mutant clones within a given library. Such a comprehensive mutant library has now been generated for Staph. aureus, and its analysis has revealed novel genes involved in bacterial biofilm formation (P. H. Tu Quoc, P. Genevaux, M. I. Pajunen, H. Savilahti, J. Schrenzel and W. L. Kelley, unpublished data).
Similar to Mu transpososome mutagenesis of Gram-positive staphylococci and streptococci (this study), Tn5 transpososomes have been used to directly mutagenize Gram-positive corynebacteria, mycobacteria and rhodococci (Derbyshire et al., 2000; Fernandes et al., 2001
; Laurent et al., 2003
; Oram et al., 2002
; Takayama et al., 2003
; Tanaka et al., 2002
). In most cases, the reported genomic integration efficiencies were similar to those obtained in our study, and in some cases they were even higher. However, a direct comparison between the characteristics of these two related systems has not been documented.
Besides efficiency, the spectrum of selected target sites at the sequence level is a critical issue with regard to the usefulness of a given genomic mutagenesis strategy. At least in vitro, the Mu system selects its transposition target sites very randomly and without a strict sequence preference (Butterfield et al., 2002; Haapa-Paananen et al., 2002
). Essentially identical targeting preference with only a weak selectivity has also been observed in an in vivo study that employed the expression of MuA transposase within E. coli and integration site analysis from plasmid targets (Mizuuchi & Mizuuchi, 1993
). On the basis of the above studies, it is highly probable that a similar pattern will emerge with the MuA-catalysed genomic integration strategy as well, although we acknowledge that the conditions within cells may affect targeting to some extent. Determination of a statistically relevant number of transposon insertion sites within a given bacterial genome will ultimately reveal the targeting preference in each studied strain.
We have extended the Mu-transpososome-mediated genomic transposon integration methodology to Gram-positive bacteria, in particular to staphylococci and streptococci, allowing direct generation of sizeable insertion mutant libraries for genomics studies. In principle, it should be possible to use a similar strategy with other Gram-positive bacterial species, given that a reasonable protocol for the preparation of competent cells is available. Furthermore, the mini-Mu transposons can easily accommodate modifications that would enable their advanced usage, e.g. for the generation of reporter gene fusions (Hayes, 2003) and for signature-tagged mutagenesis (Hensel et al., 1995
).
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ACKNOWLEDGEMENTS |
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Received 3 December 2004;
revised 4 January 2005;
accepted 17 January 2005.
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