School of Biological Sciences, Macleay Building A12, University of Sydney, New South Wales 2006, Australia
Correspondence
Ronald A. Skurray
skurray{at}bio.usyd.edu.au
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ABSTRACT |
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The GenBank accession numbers for pSK5630, pSK5632 and pSK5645 are AY182780, AY182781 and AY182783, respectively.
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INTRODUCTION |
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Several studies have linked this phenomenon to the replication mechanism of RC plasmids involving ssDNA intermediates, which can result in deletions due to illegitimate recombination (Ballester et al., 1989; Michel & Ehrlich, 1986
) or the formation of linear high-molecular-mass plasmid multimers (Gruss & Ehrlich, 1988
). The latter has been proposed to provide a selection against recombinant plasmids, thereby also encouraging the enrichment of deletion derivatives (Leonhardt & Alonso, 1991
). This selective disadvantage also typically results in segregational instability of RC-based vectors containing cloned DNA inserts. The segregational stability of plasmids containing foreign DNA has also been found to be inversely proportional to the size of the insert, perhaps reflecting a reduced copy number (Bron & Luxen, 1985
; Bron et al., 1988
). An alternative approach, which has been successful in partially circumventing the problems associated with S. aureus cloning and expression vectors, has been the development of plasmids for the stable integration of genes into the S. aureus chromosome (Lee et al., 1991
). However, the inability of these vectors to be transferred from S. aureus back into E. coli for additional manipulations is an inherent drawback to this approach, making their use cumbersome.
In contrast to the small RC plasmids, which routinely possess only a single antimicrobial resistance gene, S. aureus has a propensity for accumulating multiple resistance determinants on large, low-copy-number, theta-mode plasmids, e.g. pSK1 (Firth et al., 2000) and pSK41 (Berg et al., 1998
). In our laboratory, the analysis of resistance and putative virulence-associated genes encoded by plasmids such as pSK1 has often been problematic, due to an inability to clone and/or express many of these genes. These difficulties are likely to reflect a further potential drawback of RC-based vectors for this kind of application: their substantially elevated copy number in comparison to the large theta-mode multiresistance plasmids. This difference can also complicate efforts to dissect the regulatory pathways influencing the expression of genes that are encoded by low-copy-number plasmids or the chromosome. It has been previously demonstrated that mini replicons based on pSK1, a staphylococcal incompatibility group Inc1 plasmid (Firth et al., 2000
), exhibit a segregational stability approaching that of the parental plasmid, although this required the presence of both the pSK1 rep gene, and also that of the divergently transcribed par (formerly known as orf245) determinant, which encodes a plasmid partitioning mechanism (Firth, et. al., 2000
; A. Simpson, R. A. Skurray & N. Firth, unpublished results). Considering the relatively large size of pSK1 (28·1 kb), it would not be unwarranted to expect that shuttle vectors developed from this plasmid would continue to be stable in S. aureus following the cloning of even large DNA inserts. Thus, a range of shuttle vectors based on the replication and maintenance functions of the pSK1-multiresistance plasmid were constructed to provide stable, low-copy-number cloning and expression vectors for use in this species. This paper also describes the production and utilization of shuttle vectors that permit the generation of either transcriptional or translational fusions to an S. aureus
-lactamase (blaZ) reporter gene.
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METHODS |
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DNA isolation and manipulations.
The Quantum Prep kit (Bio-Rad) and small-scale alkaline lysis procedure (Lyon et al., 1983) were employed to isolate plasmid DNA from E. coli and S. aureus, respectively. Restriction enzymes, T4 DNA ligase, T4 DNA polymerase (all from New England Biolabs), shrimp alkaline phosphatase (Promega) and Pfu DNA polymerase for PCR amplification (Stratagene) were each used according to the manufacturers' instructions. Oligonucleotides were purchased from Sigma, PCR products were purified with the Wizard PCR Prep kit (Promega) and DNA fragments were isolated from agarose gels using the Concert gel extraction kit (Gibco-BRL). Automated DNA sequencing, performed at the Australian Genome Research Facility, was employed to verify the relevant sequences in all instances where a cloning step involved either the generation of blunt ends or the insertion of a DNA fragment generated by PCR.
Construction of shuttle vectors.
An S. aureus chloramphenicol resistance (CmR) determinant was amplified from the plasmid pWN1819 (Wang et al., 1987) using the primers pC194 Cm3' and pC194 Cm5' (Table 1
), producing a DNA fragment equivalent to bp 10812017 of the published pC194 sequence (Horinouchi & Weisblum, 1982
), which included a potential downstream transcription terminator. To provide a ready source of this CmR gene, the PCR fragment obtained was cloned into the HindIII and SmaI sites of pBluescript II KS+ to produce pSK5299 (Fig. 1
). The initial step in the construction of the first shuttle vector involved the replacement of the AatIIHindIII portion of pSK5601 with the HindIIISmaI CmR fragment from pSK5299, to generate pSK5605 (Fig. 1
; step 1). The rrnBTI terminator and multiple cloning site (MCS) removed in step 1 were then reinserted to produce pSK5623 (Fig. 1
; step 2). Construction of the shuttle vector pSK5630 was completed by the addition of the S. aureus pSK1 plasmid replication and partitioning genes, rep and par, respectively; the relevant region (equivalent to bp 32287 of GenBank entry AF203376) was amplified by PCR from pSK1-template DNA employing the primers orf245 3'BamHI and orf306 3'BamHI (Table 1
). The flanking BamHI sites facilitated the insertion of this reppar fragment into pSK5623 at the BglII site that had been previously incorporated into the 3' end of the CmR gene for this purpose (Fig. 1
; step 3). A further shuttle vector, pSK5632, was constructed in a similar fashion, as outlined in Fig. 1
, steps 46.
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Cloning of the pSK1 dfrA, orf172 and orf288 genes.
Genes from the pSK1-multiresistance plasmid were cloned as a test of the versatility of the shuttle vectors. A 2·7 kb dfrA TpR fragment originating from pSK1 (bp 1362871 of GenBank entry X13290) was obtained in both orientations by digestion of the subclones pSK4707 and pSK4708 (Table 1) with SmaI and SalI. These two fragments were subsequently ligated into the equivalent sites in the MCSs of pSK5630 and pSK5632 to produce derivatives of each of these shuttle vectors which contained dfrA cloned in either orientation. The pSK1 orf288 gene (N. Firth, S. Apisiridej & R. A. Skurray, unpublished results) was PCR amplified as a 1·08 kb fragment from pSK1-template DNA using the primers orf288r BamHI and orf288f BamHI (Table 1
) and ligated into the pSK5632 MCS to produce pSK5767 and pSK5768, with the orf288 DNA fragment inserted in the opposite and same orientation as lacZ
, respectively. To clone the pSK1 orf172 gene (N. Firth, S. Apisiridej & R. A. Skurray, unpublished results), PCR amplification from an existing clone, pSK5902 (Table 1
), using the primers orf172r BamHI and orf172f BamHI (Table 1
), generated a 0·88 kb DNA fragment that was ligated into the BamHI site of pSK5632 to produce pSK5766, which contained orf172 in the opposite orientation to the lac promoter. The inability to obtain clones in which orf172 was transcribed from the lac promoter suggested that overexpression of this gene is toxic to E. coli cells. To acquire orf172 inserted in the same orientation as lacZ
, a directional cloning was performed in which orf172 was cleaved from pSK5902 and cloned as an SphIBamHI fragment into the pSK5632 MCS, resulting in the isolation of pSK5800.
Cloning the promoters for the pSK1 genes dfrA, qacA and qacR.
Transcriptional fusions to the blaZ reporter gene were produced by cloning promoter-containing sequences into the BamHI and HindIII sites of pSK5645. A fragment containing the pSK1 dfrA promoter (PdfrA), equivalent to bp 764868 of the published dfrA nucleotide sequence (Rouch et al., 1989), was obtained by PCR amplification from pSK4707 template DNA using the primers dfrAP BamHI and dfrAP HindIII (Table 1
), resulting in the PdfrAblaZ fusion construct pSK5780. The qacR and qacA promoters were excised from pSK5202 and pSK5203 (Grkovic et al., 1998
), respectively, and inserted into pSK5645 to produce the blaZ fusion plasmids pSK5802 (PqacR) and pSK5874 (PqacA). pSK5803, a PqacAblaZ fusion construct, which also contained the qacR regulatory gene in cis (equivalent to bp 42837 of the published qacAqacR DNA sequence; Rouch et al., 1990
), was provided by PCR amplification from pSK1 template DNA using the primers qacA837HindIII and qacR42BamHI (Table 1
).
Metabolic labelling of lipoproteins in S. aureus.
Overnight cultures of S. aureus cells harbouring pSK1, pSK5632 or pSK5768 were diluted 1 : 20 in LB supplemented with 10 mg glucose ml-1 and grown until an OD600 of 0·5 was reached, at which point metabolic labelling using [3H]palmitic acid (Amersham Pharmacia Biotech) and isolation of lipoproteins was carried out essentially as previously described (Navarre et al., 1996). Solubilized cell-wall proteins and pre-stained SDS-PAGE standards (Bio-Rad) were separated on SDS-15 % polyacrylamide gels, dried onto 3MM paper (Whatman), and visualized by autoradiography after exposure for 2 weeks at -70 °C.
Nitrocefin assays.
A stationary-phase culture of S. aureus strain RN4220 containing the plasmid of interest was diluted 1 : 1000 and grown for 16 h, after which 10 ml of cells were collected by centrifugation and washed with 10 ml cold 50 mM NaH2PO4, before being resuspended in a final volume of 0·7 ml cold 50 mM NaH2PO4. -Lactamase activities for 0·3 ml samples of the washed whole cells, or an appropriate dilution thereof, were determined using the chromogenic substrate nitrocefin (kindly provided by GlaxoWellcome, UK) (O'Callaghan et al., 1972
) as previously described (Yoon et al., 1991
) and are presented such that 1 unit corresponds to 1 µM nitrocefin hydrolysed min-1 (µg total cellular protein)-1 at 37 °C. Results are the mean of at least two experiments.
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RESULTS |
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Construction of the expression shuttle vector pSK5632
An additional plasmid was constructed that utilized pUC19 components to provide E. coli replication and selection functions, an improved MCS, and a lacZ gene to assist the identification of clones in E. coli strains carrying a lacZ
M15 mutation. The resultant shuttle vector, pSK5632 (Fig. 1
; steps 46), simplified the preparation of plasmid DNA from E. coli due to possession of the high-copy-number pUC19 origin of replication (Fig. 1
; ColE1HC). The vector pSK5632 could be readily electroporated into S. aureus strain RN4220, where it was maintained at 9·7±0·9 copies per cell. The strong lac promoter located upstream of the MCS in pSK5632 should also be advantageous for the overexpression of cloned genes (see below).
Variants of pSK5632, in which the pSK1 reppar encoding fragment was in the opposite orientation, were found to transform S. aureus strain RN4220 significantly less efficiently. Additionally, a reduced yield of plasmid DNA was obtained from E. coli and S. aureus cells harbouring such plasmids (data not shown). Taken together with the failure to isolate the alternative variant of pSK5630, and the small percentage of clones obtained in which the rep gene was in the opposite orientation to that in pSK5632, it would appear that the alternative arrangement of the rep and par genes produces vectors which are not maintained as readily in either S. aureus or E. coli.
Versatility of shuttle vectors for cloning and expression
To confirm the functionality of the shuttle vectors, a 2·7 kb DNA fragment containing the pSK1 dfrA dihydrofolate reductase (TpR) determinant was cloned. The high-level TpR conferred by this dfrA gene has been shown to be the result of the inclusion of this gene in an operon whose transcription is initiated from a strong hybrid promoter, the -35 region of which is contributed by an upstream insertion sequence element, IS257 (Leelaporn et al., 1994). The 2·7 kb dfrA- and PdfrA-containing DNA fragment was inserted into the MCSs of pSK5630 and pSK5632 to produce plasmids with dfrA cloned in both orientations. All of the dfrA clones conferred levels of TpR equal to that observed for pSK1 (1600 µg ml-1), irrespective of the orientation of their DNA insert or the nature of the parental shuttle vector, whereas cells containing either of the vectors failed to grow on the plate with the lowest Tp concentration (100 µg ml-1).
Previously, our attempts to experimentally confirm the cellular location of ORF172 and ORF288, two hypothetical proteins encoded by a conserved region of pSK1-family plasmids (N. Firth, S. Apisiridej & R. A. Skurray, unpublished results), had not been successful. In addition to the failure of metabolic-labelling experiments utilizing S. aureus cells harbouring pSK1, we were unable to clone the orf288 and orf172 genes into either E. coli or S. aureus using available E. coliS. aureus shuttle vectors, possibly due to inappropriate expression or post-translational processing; ORF172 contains a surface-anchoring motif for Gram-positive cocci, whereas ORF288 is predicted to be a lipoprotein (N. Firth, S. Apisiridej & R. A. Skurray, unpublished results). Therefore, to facilitate their analysis, these two genes were individually cloned into the expression shuttle vector pSK5632.
Initially, orf172 clones could be obtained only in the orientation opposite to the pSK5632 lac promoter (Table 1; pSK5766). This suggested that overexpression of orf172 was deleterious to E. coli cells, and that the putative promoter for the orf172orf288orf84 operon, which was included in the pSK5766 insert DNA, is either not active in E. coli or is sufficiently weak that it did not direct the production of harmful quantities of the orf172 product. Although a directional cloning to insert orf172 in the same orientation as lacZ
did not produce viable E. coli transformants, a number of clones were obtained by electroporation of S. aureus strain RN4220, one of which was designated pSK5800. This plasmid could be successfully introduced into E. coli strain DH5
only in the presence of pREP-4, a plasmid which overexpresses the LacI repressor, preventing transcription of orf172 from the pSK5800 lac promoter/operator. In comparison, the cloning of orf288 proved to be straightforward, generating the constructs pSK5767 and pSK5768, which contained the gene inserted in the opposite and same direction as the vector lac promoter, respectively (Table 1
).
Detection of the pSK1 orf288 gene product
To verify that orf288 encodes a lipoprotein, metabolic-labelling experiments were carried out by growing S. aureus cells in the presence of [3H]palmitic acid, a lipoprotein constituent (Navarre et al., 1996). Because the orf288 clones lacked an endogenous promoter, only pSK5768 was chosen for analysis in S. aureus, as expression of ORF288 in this clone should be facilitated by the strong lac vector promoter. The results of a metabolic-labelling experiment are depicted in Fig. 2
, where a 3H-labelled protein of approximately 30·5 kDa can be clearly seen in the lane containing extracts from cells harbouring pSK5768, whereas no equivalent band is detectable in either the vector control (pSK5632) or parental multiresistance plasmid (pSK1) lanes. ORF288 migrated at a position corresponding to the molecular size expected for this protein after the removal of the first 17 amino acids by cleavage at the predicted peptidase II site (30·2 kDa; N. Firth, S. Apisiridej & R. A. Skurray, unpublished results). Detection of the ORF288 product when expressed from pSK5768, but not pSK1, is presumably attributable to orf288 expression being under control of the strong lac promoter in the former; transcription of the putative operon to which orf288 belongs on pSK1 must be insufficient to allow detection under the conditions used.
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Construction of transcriptional- and translational-fusion shuttle vectors
The successful employment of the pSK1-based shuttle vectors for cloning and expression purposes suggested that the production of further derivatives to facilitate the generation of low-copy-number transcriptional and translational reporter-gene fusions would likewise be advantageous. The -lactamase protein encoded by the blaZ gene originating from the S. aureus plasmid pI258 forms a membrane-bound mature protein and a secreted enzyme that are both processed and secreted normally when the precursor contains N-terminal translational extensions (Nielsen & Lampen, 1982
; Wang et al., 1987
). This BlaZ determinant has been previously successfully utilized as a reporter enzyme in S. aureus (Ji et al., 1995
; Wang et al., 1987
; Yoon et al., 1991
), in large part due to the availability of a chromogenic substrate, nitrocefin (O'Callaghan et al., 1972
), which provides for an easy and reliable colorimetric assay for the determination of
-lactamase activities. Importantly, the E. coli blaM ApR gene already present in the pSK1-based shuttle vectors does not exhibit
-lactamase activity in S. aureus and also has no significant homology at the DNA level to the pI258 Gram-positive blaZ gene, which meant that the employment of blaZ would not provide sites for homologous recombination.
To obtain a blaZ gene with upstream sequences suitable for the creation of transcriptional fusions, the pI258 blaZ gene was PCR amplified from pWN1819 (Wang et al., 1987) using the primer pair pI258 blaZ3' and pI258 blaZ5' (Table 1
), which incorporated an upstream ribosome-binding site (RBS) and stop codons in all three frames (Fig. 3
). The resultant PCR fragment was inserted into the HindIII and PvuII sites of pSK5630 (Fig. 1
) to construct the shuttle vector pSK5645 (Fig. 3a
), which possesses an rrnBT1 transcription terminator upstream of the blaZ reporter gene to prevent readthrough from vector promoters. Determination of the copy number of this plasmid indicated that it was maintained at 5·0±0·9 copies per cell. A shuttle vector suitable for the generation of translational fusions was produced by replacing the HindIIIXbaI fragment of pSK5645 with the equivalent region from the blaZ gene of pWN1819, generating pSK5805 (Fig. 3b
). To verify that the E. coli ApR gene in pSK5645 does not result in
-lactam degradation when present in S. aureus, a portion of the blaM gene in this plasmid was deleted by removal of the 0·25 kb BglIPvuI fragment internal to the E. coli ApR determinant (Fig. 3a
), producing pSK5775. Nitrocefin assays performed in S. aureus produced almost identical low levels of
-lactamase activities for RN4220 cells harbouring the transcriptional fusion vector pSK5645 (1·32 ± 0·64 units), the translational fusion vector pSK5805 (0·93 ± 0·35 units), or the plasmid with a blaM deletion, pSK5775 (1·11 ± 0·63 units), which confirmed that blaM does not contribute to
-lactamase activity in S. aureus. The low
-lactamase levels observed for cells containing these vectors is likely to be attributable to the minor
-lactamase activity of a cell-wall-synthesizing enzyme, as cells harbouring a shuttle vector which completely lacked a blaZ gene produced a similar result (pSK5630; 0·61 ± 0·27 units) to that of the transcriptional and translational fusion vectors. Thus, the E. coli rrnBT1 terminator largely prevents readthrough from any upstream promoters in the pSK5645 and pSK5805 shuttle vectors, an important feature for the construction of reporter-gene fusions.
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DISCUSSION |
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The presence of Plac in the pSK5632 shuttle vector may also be of assistance in the analysis of cloned genes in E. coli, since some staphylococcal genes that possess sequences resembling the E. coli promoter consensus still require a cognate promoter for expression in this species (Lee & Iandolo, 1986; Ranelli et al., 1985
). PqacA provides a good example of such a promoter, as it possesses ideally spaced hexamers that are a close match to the E. coli canonical -10 and -35 sequences (Fig. 4a
), yet this promoter directs only relatively low-level transcription in E. coli, even in the absence of the QacR repressor (Grkovic et al., 1998
). However, by creating a fusion to the pSK5645 blaZ reporter gene, the unrepressed PqacA was found to be capable of promoting strong transcription in the natural S. aureus host species (Fig. 4b
), a finding that is more in line with the DNA sequence of this element. Additionally, although PqacR, a poor match to the E. coli consensus hexamers (Fig. 4a
), is a much stronger promoter in E. coli than PqacA (Grkovic et al., 1998
), the opposite results were obtained when the strengths of these two promoters were compared in S. aureus (Fig. 4b
). These conflicting findings could be related to PqacR containing a 5'-TGN-3' motif immediately upstream of its -10 hexamer (Fig. 4a
), which creates an extended -10 region that is known to enhance promoter recognition by the E. coli
70 subunit (Barne et al., 1997
). The canonical S. aureus
SA factor may not recognize the PqacR TGN motif, or alternatively, E. coli may lack some other transcription factor(s) that influences the transcription of one or both of these genes in S. aureus. Interestingly, despite these significant differences in promoter strengths when analysed in either E. coli or S. aureus, the qacR gene cloned in cis to PqacA resulted in an approximately sixfold decrease in transcription from this promoter (Fig. 4b
) (Grkovic et al., 1998
). This relatively weak level of repression confirmed the observation that QacR allows a significant basal level of qacA expression (Fig. 4b
), which has been suggested to permit the QacA multidrug transporter to confer a significant degree of resistance to substrates that do not cause induction of qacA transcription (Grkovic et al., 2001
). The above findings emphasize the importance of vectors which allow the analysis of genes and their regulatory sequences both at the appropriate copy number and in the species from which they were originally derived.
Overall, the stability of the pSK1-based shuttle vectors suggests that they should be useful for a wide range of future applications. Of particular relevance was their elevated segregational and structural stability in comparison to that reported for the higher-copy-number RC-based vectors, which are often unstable following the cloning of even relatively small DNA fragments, or in some instances even in the absence of any foreign DNA insert (Bruckner, 1992). The success of the pSK1-based vectors is likely to be due to the use of a theta-mode replicon and the inclusion of the pSK1 par gene; the low copy number of these vectors may also assist the stable cloning of deleterious genes. Alternative broad-host-range vectors which replicate in Gram-positive bacteria, and maintain their structural integrity even following the cloning of DNA segments up to 33 kb in size, have also been developed based on the low-copy-number, theta-replicating plasmid pAM
1 from Enterococcus faecalis (Jannière et al., 1990
; Renault et al., 1996
). However, many of these pAM
1-based vectors replicate at a high copy number in Gram-positive hosts due to the deletion of a copy-control region, and most of the more sophisticated derivatives lack regions of pAM
1 known or hypothesized to encode functions involved in plasmid segregational stability (Jannière et al., 1990
; Renault et al., 1996
; Simon & Chopin, 1988
; Swinfield et al., 1991
). In contrast, the vectors constructed in this study utilize the complete pSK1 replication initiation region with adjacent segregational stability system. Hence, for experiments in S. aureus that require stable, low-copy-number cloning, gene expression, or the construction and analysis of genetic fusions, the use of pSK1-based shuttle vectors is likely to prove advantageous. Equally, the availability of vectors based on different and compatible replication systems is crucial for experiments that require the maintenance of two or more plasmids in the one cell. In addition to their demonstrated utility for the analysis of antimicrobial resistance and potential virulence determinants from multiresistance plasmids, the low copy number of the pSK1-based shuttle vectors should also make them suitable for the analysis of chromosomally encoded genes in S. aureus.
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ACKNOWLEDGEMENTS |
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Received 19 August 2002;
revised 29 November 2002;
accepted 12 December 2002.
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