Stable low-copy-number Staphylococcus aureus shuttle vectors

Steve Grkovic, Melissa H. Brown, Kate M. Hardie, Neville Firth and Ronald A. Skurray

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A series of Staphylococcus aureus–Escherichia coli shuttle vectors were constructed which contained the replication and maintenance functions of the S. aureus theta-mode multiresistance plasmid pSK1. The utility of the newly constructed vectors was demonstrated by the successful cloning and expression of several genes that had previously proven difficult to express in S. aureus. Additional vectors which permit the generation of transcriptional and translational fusions to an S. aureus blaZ reporter gene were also produced and subsequently employed to determine the relative strengths in S. aureus of a number of promoters. By utilizing the theta-mode replication functions of pSK1, the shuttle vectors described largely avoided the segregational and structural stability problems frequently encountered with Gram-positive rolling-circle-based vectors. In addition, these plasmids represent vectors which are suitable for the analysis of genes in S. aureus at low copy number.


Abbreviations: MCS, multiple cloning site; RC, rolling circle

The GenBank accession numbers for pSK5630, pSK5632 and pSK5645 are AY182780, AY182781 and AY182783, respectively.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Staphylococcus aureus is an important human pathogen that exhibits resistance to a startling range of antimicrobial compounds (Paulsen et al., 1997). However, molecular analysis of this Gram-positive species is often hindered by a shortage of reliable cloning and expression vectors. The large number of stable vectors that have been successfully developed specifically for use in Escherichia coli are based on plasmids that replicate via a theta mode, which involves the simultaneous synthesis of both DNA strands, the predominant style of plasmid replication in this species (Helinski et al., 1996). In contrast to E. coli, the small plasmids isolated from Gram-positive organisms primarily replicate via a rolling circle (RC) mechanism, where the leading strand is first synthesized, and then used as a template for production of the lagging strand (del Solar et al., 1998; Helinski et al., 1996; Novick, 1989). In the past, these small RC plasmids have provided a ready source of Gram-positive replication functions and resistance determinants. Thus, to date, most of the vectors constructed for use in Gram-positive species are derivatives of small RC S. aureus plasmids, such as pT181, pC194, pE194 and pUB110 (Bruckner, 1992; Gruss & Ehrlich, 1988; Novick, 1989). However, a recurring problem with such vectors is that after the insertion of even relatively small foreign DNA fragments, many of the resulting plasmids have exhibited structural and/or segregational instability.

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 {beta}-lactamase (blaZ) reporter gene.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Media, strains and culture conditions.
The E. coli strain DH5{alpha} (lacZ{Delta}M15) (Sambrook et al., 1989) and the S. aureus strain RN4220 (Kreiswirth et al., 1983) were employed in all procedures carried out in the respective species. Unless stated otherwise, all strains were cultured at 37 °C on Luria–Bertani (LB) broth or agar (Sambrook et al., 1989) containing, where appropriate, 100 µg ampicillin (Ap) ml-1 or 50 µg kanamycin (Km) ml-1 for E. coli and 10 µg chloramphenicol (Cm) ml-1 or 100 µg trimethoprim (Tp) ml-1 for S. aureus. E. coli was transformed by standard procedures (Sambrook et al., 1989) and S. aureus by electroporation as previously described (Schenk & Laddaga, 1992), employing a pulse of 1·3 kV. Minimum inhibitory concentrations for Tp were determined as previously described (Leelaporn et al., 1994).

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 1081–2017 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 AatII–HindIII portion of pSK5601 with the HindIII–SmaI 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 3–2287 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 rep–par 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 4–6.


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Table 1. Plasmids and oligonucleotide primers

 


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Fig. 1. Construction of shuttle vectors utilizing the pSK1 rep and par plasmid replication and partitioning functions. The cloning steps 1–3 were performed to generate the general-purpose shuttle vector pSK5630, whereas the cloning and expression vector pSK5632 was produced as a result of steps 4–6. The restriction endonuclease site(s) at which each plasmid was cut in order to perform a subsequent step is indicated in bold, with the specific sites at which this occurred for each manipulation recorded adjacent to the relevant arrow. Where necessary, blunt ends were created by removal of 3' overhangs with T4 DNA polymerase. More detailed information on these manipulations can be found in the text. Genes: ApR, E. coli blaM gene for {beta}-lactamase (black arrow); ColEI ori or ColE1HC ori, moderate- or high-copy-number E. coli plasmid origins of replication, respectively (dark grey arrow); CmR, S. aureus gene originating from pC194 encoding Cm acetyltransferase (checkerboard arrow); f1 ori, E. coli single-stranded phage origin of replication (light grey box); lacZ{alpha}, gene encoding the LacZ{alpha} peptide for blue/white selection of clones in the E. coli host DH5{alpha}, which carries a lacZ{Delta}M15 mutation (light grey arrow); MCS (black box); par, gene from S. aureus multiresistance plasmid pSK1 encoding plasmid partitioning function (cross-hatched arrow); rep, pSK1 plasmid replication gene (striped arrow); rrnBT1, transcription terminator from the E. coli rrnB rRNA operon (dark grey box). Restriction endonuclease sites: A, AatII; B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NdeI; P, PstI; Pv, PvuII; PvI, PvuI; S, SalI; Sm, SmaI; Sp, SphI. Sites in parentheses indicate those that were eliminated in a preceding step, whereas those denoted by an asterisk occur only once in the relevant completed shuttle vector. The GenBank accession numbers for the complete sequences of pSK5630 and pSK5632 are AY182780 and AY182781, respectively.

 
Determination of plasmid copy number and segregational stability.
Plasmid copy numbers were determined by densitometric analysis of electrophoretically fractionated DNA isolated from an S. aureus strain harbouring both the plasmid of interest and pUB110, a plasmid with a copy number of 10 per cell (Novick, 1990), as previously described (Weaver et al., 1993). Plasmid segregational stability assays (Firth et al., 2000) were performed using LB plates supplemented with Cm to identify S. aureus cells that retained the plasmid under test. For both types of assays, results are the mean of at least two experiments.

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 136–2871 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{alpha}, 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{alpha}, a directional cloning was performed in which orf172 was cleaved from pSK5902 and cloned as an SphI–BamHI 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 764–868 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 PdfrA–blaZ 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 PqacA–blaZ fusion construct, which also contained the qacR regulatory gene in cis (equivalent to bp 42–837 of the published qacA–qacR 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. {beta}-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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of the general-purpose shuttle vector pSK5630
The plasmid pSK5601 provided determinants necessary for plasmid function in E. coli, viz. a ColE1 moderate-copy-number plasmid replication function and an ApR antibiotic resistance marker, in addition to an E. coli transcription terminator, rrnBT1 (Fig. 1). pSK5601 was derived from the plasmid pKK232-8 (Brosius, 1984) by deletion of two rrnBT1T2 terminators, which removed areas of homology that could potentially provide sites for plasmid instability in recombination-proficient S. aureus strains. A selectable antibiotic resistance marker for use in S. aureus was provided by the CmR gene originating from the small RC plasmid pC194 (Horinouchi & Weisblum, 1982), chosen for its small size and previous successful use in other vectors (Jannière et al., 1990; Wang et al., 1987). The completed shuttle vector pSK5630 (Fig. 1) contained all the above elements, in addition to the rep and par genes from the multiresistance plasmid pSK1, which encode S. aureus theta-mode replication and partitioning functions, respectively. All of the analysed clones proved to have the rep–par BamHI fragment inserted in the same orientation as illustrated for pSK5630, i.e. with the pSK1 rep gene transcribed in the same direction as that encoding CmR (Fig. 1). S. aureus RN4220 cells could be readily transformed with pSK5630 DNA, as confirmed by small-scale isolations of plasmid DNA. Determination of the copy number of this plasmid indicated that it was maintained at 5·1±0·7 copies per cell. The rrnBT1 transcription terminator adjacent to the pSK5630 MCS (Fig. 1) should enable correctly orientated cloned genes to be analysed when expressed from their endogenous promoters, as E. coli terminators have been previously demonstrated to function in Gram-positive bacteria (Peschke et al., 1985).

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{alpha} gene to assist the identification of clones in E. coli strains carrying a lacZ{Delta}M15 mutation. The resultant shuttle vector, pSK5632 (Fig. 1; steps 4–6), 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 rep–par 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. coli–S. 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 orf172–orf288–orf84 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{alpha} 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{alpha} 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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Fig. 2. Metabolic labelling of the orf288 product. S. aureus RN4220 cells harbouring either pSK1, the pSK5632 shuttle vector (SV), or a clone of the pSK1 orf288 gene inserted into pSK5632, such that it was transcribed from the lac promoter (pSK5768), were used in labelling experiments employing [3H]palmitic acid, a lipoprotein constituent. The position of migration of ORF288 (30·5 kDa) is indicated on the left; the positions of migration and sizes (in kDa) of pre-stained molecular size markers are shown on the right.

 
Segregational and structural stability of shuttle vectors
To provide an indication of the segregational and structural stability of the pSK1-based shuttle vectors, the percentages of S. aureus RN4220 cells that continued to retain various plasmids over the length of a 100-generation experiment were determined. Since pSK5632 had been used to clone a number of different genes (viz. dfrA, orf288 and orf172), this shuttle vector and its derivatives were chosen for the assessment of plasmid segregational and structural stability in S. aureus. After more than 100 generations of growth in the absence of plasmid selection, pSK5632 was found to be maintained by 74·0±1·4 % of a population of S. aureus RN4220 cells, which is comparable to the value of approximately 80 % reported previously for pSK1 in this strain (Firth et al., 2000). The derivatives pSK5758 and pSK5800, which contain 2·7 kb dfrA and 0·88 kb orf172 DNA inserts, were found to be retained by 69·6±0·4 % and 65·4±0·3 % of RN4220 populations, respectively. 51·9±5·5 % of a RN4220 population retained pSK5768, which suggests that the 1·08 kb orf288 fragment carried by this derivative impaired the plasmid's segregational stability. Analysis of plasmid DNA prepared from representative CmR 100-generation colonies revealed that all the shuttle vectors were of the expected size, which provided an indication that they had also retained their structural integrity during the course of this experiment.

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 {beta}-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 {beta}-lactamase activities. Importantly, the E. coli blaM ApR gene already present in the pSK1-based shuttle vectors does not exhibit {beta}-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 HindIII–XbaI 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 {beta}-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 BglI–PvuI fragment internal to the E. coli ApR determinant (Fig. 3a), producing pSK5775. Nitrocefin assays performed in S. aureus produced almost identical low levels of {beta}-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 {beta}-lactamase activity in S. aureus. The low {beta}-lactamase levels observed for cells containing these vectors is likely to be attributable to the minor {beta}-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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Fig. 3. Shuttle vectors suitable for the generation of transcriptional and translational fusions to the S. aureus blaZ reporter gene. (a) Circular map of the transcriptional fusion vector pSK5645. The abbreviations for restriction endonuclease sites and genes are as described in the legend for Fig. 1, with the addition of the promoterless S. aureus {beta}-lactamase (blaZ) gene (stippled arrow). The locations of the RBS and stop codons in all three reading frames upstream of the blaZ gene are also indicated. The GenBank accession number for the complete sequence of pSK5645 is AY182783. (b) The DNA sequence surrounding the start codon for BlaZ in the transcriptional fusion vector pSK5645 and the translational fusion vector pSK5805, which illustrates the only differences between these two shuttle vectors. In pSK5645, the partially deleted, inoperative RBS present in pWN1819 and pSK5805 has been replaced with the original blaZ RBS sequence (Wang & Novick, 1987), in addition to the introduction of upstream stop codons in each reading frame. The TTG methionine initiation codon was also altered to an ATG in pSK5645 to ensure efficient initiation of translation (Rocha et al., 1999).

 
Analysis of the dfrA, qacA and qacR promoters in S. aureus
To assess the ability of pSK5645 to act as a shuttle vector for the generation of transcriptional fusions, a number of S. aureus promoters that had previously been analysed in E. coli, but not in their natural staphylococcal background, were fused to the blaZ reporter gene in this vector. The dfrA promoter (PdfrA) provided a sequence that has been shown to direct high-level transcription in E. coli (Leelaporn et al., 1994), whereas the promoters for the qacA multidrug resistance determinant (PqacA) and qacR transcriptional repressor (PqacR) were chosen as elements that exhibit low- and moderate-level transcription in E. coli, respectively (Grkovic et al., 1998). Furthermore, the transcriptional activity of PqacA was compared in the presence and absence of qacR, the multidrug-sensing repressor of qacA transcription (Grkovic et al., 1998). The sequences of the various promoters fused to the pSK5645 blaZ reporter gene are shown in Fig. 4(a). After introduction of the plasmids into S. aureus strain RN4220, nitrocefin assays were performed to determine the level of transcription originating from the cloned promoter-containing fragments, serving to demonstrate that pSK5645 functioned correctly as a transcriptional-fusion vector. As expected, PdfrA proved to be an exceptionally strong promoter in S. aureus, producing a {beta}-lactamase activity in excess of 1000 units when fused to the pSK5645 blaZ reporter gene (pSK5780; Fig. 4b). PqacA was also found to be a relatively strong promoter, directing approximately 60-fold more transcription in S. aureus than did PqacR (pSK5874, 338·99 units versus pSK5802, 5·32 units; Fig. 4b). Not unexpectedly, the presence of the QacR regulatory protein resulted in an almost sixfold decrease in transcription from PqacA (pSK5803, 58·67 units versus pSK5874, 338·99 units; Fig. 4b), although the repressed PqacA still exhibited over 10 times the transcription level observed for PqacR.



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Fig. 4. Analysis of promoter elements fused to the blaZ reporter gene in the transcriptional fusion shuttle vector pSK5645. (a) Nucleotide sequences of the dfrA (PdfrA), qacR (PqacR) and qacA (PqacA) promoters, with the -10 and -35 hexamers delineated by black rectangles. The asterisks indicate the TGN sequence in PqacR predicted to form an extended -10 region recognized by the {sigma}70 subunit of E. coli RNA polymerase. (b) Level of {beta}-lactamase activity determined for each plasmid when harboured by the S. aureus strain RN4220. The {beta}-lactamase values have been corrected for the level of activity observed for pSK5645 in the absence of a cloned promoter element and represent the mean of three separate experiments. The results are expressed such that 1 unit is equivalent to 1 µM nitrocefin hydrolysed min-1 (µg total cellular protein)-1 at 37 °C.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The S. aureus–E. coli shuttle vectors constructed in this work were successfully employed to clone and express several genes that had previously proven difficult to work with. Additionally, the production of a low-copy-number transcriptional-fusion shuttle vector not only permitted the analysis of three important S. aureus antimicrobial resistance promoters for the first time in the natural host background, but also facilitated their analysis at the appropriate copy number. As an initial test, clones of the pSK1 dfrA gene reintroduced into S. aureus continued to confer high-level TpR. This high-level resistance is associated with PdfrA (Leelaporn et al., 1994), a promoter sequence that was shown to direct high-level transcription in S. aureus (Fig. 4b). Cloning of the pSK1 orf172 and orf288 genes into the pSK5632 shuttle vector has facilitated the initiation of more in-depth analyses of these determinants. Strong evidence was obtained to indicate that the orf288 gene product is a lipoprotein (Fig. 2), and as such it could be exposed to the external environment. This is suggestive of a possible role for ORF288 in pathogenicity, as the virulence of S. aureus is primarily attributed to the large number of cell-wall-associated and extracellular proteins that this bacterium expresses to aid the establishment of infections (Projan & Novick, 1997). The segregational and structural stability of pSK5632 derivatives containing either the cloned orf172 or orf288 gene suggests that these constructs are ideally suited for further experiments directed at elucidating the function of these genes, such as mammalian infection models (Projan & Novick, 1997). Additionally, the elevated expression of orf288 from the vector lac promoter (Plac) in the pSK5768 construct (Fig. 2) should provide a further significant advantage over employing the natural pSK1 plasmid for the analysis of this determinant.

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 {sigma}70 subunit (Barne et al., 1997). The canonical S. aureus {sigma}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{beta}1 from Enterococcus faecalis (Jannière et al., 1990; Renault et al., 1996). However, many of these pAM{beta}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{beta}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.


   ACKNOWLEDGEMENTS
 
The chromogenic substrate nitrocefin was kindly provided by GlaxoWellcome, UK. This work was supported by Project Grants 153816 and 153818 from the National Health and Medical Research Council (Australia) to R. A. S. and N. F., and to R. A. S. and M. H. B., respectively. The contributions of Brendon O'Rourke and Sumalee Apisiridej to the work described are gratefully acknowledged.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ballester, S., Lopez, P., Espinosa, M., Alonso, J. C. & Lacks, S. A. (1989). Plasmid structural instability associated with pC194 replication functions. J Bacteriol 171, 2271–2277.[Medline]

Barne, K. A., Bown, J. A., Busby, S. J. & Minchin, S. D. (1997). Region 2·5 of the Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the ‘extended-10’ motif at promoters. EMBO J 16, 4034–4040.[Abstract/Free Full Text]

Berg, T., Firth, N., Apisiridej, S., Hettiaratchi, A., Leelaporn, A. & Skurray, R. A. (1998). Complete nucleotide sequence of pSK41: evolution of staphylococcal conjugative plasmids. J Bacteriol 180, 4350–4359.[Abstract/Free Full Text]

Bron, S. & Luxen, E. (1985). Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis. Plasmid 14, 235–244.[Medline]

Bron, S., Luxen, E. & Swart, P. (1988). Instability of recombinant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts. Plasmid 19, 231–241.[Medline]

Brosius, J. (1984). Plasmid vectors for the selection of promoters. Gene 27, 151–160.[CrossRef][Medline]

Bruckner, R. (1992). A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 122, 187–192.[CrossRef][Medline]

del Solar, G., Giraldo, R., Ruiz-Echevarría, M. J., Espinosa, M. & Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62, 434–464.[Abstract/Free Full Text]

Firth, N., Apisiridej, S., Berg, T., O'Rourke, B. A., Curnock, S., Dyke, K. G. H. & Skurray, R. A. (2000). Replication of staphylococcal multiresistance plasmids. J Bacteriol 182, 2170–2178.[Abstract/Free Full Text]

Grkovic, S., Brown, M. H., Roberts, N. J., Paulsen, I. T. & Skurray, R. A. (1998). QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA. J Biol Chem 273, 18665–18673.[Abstract/Free Full Text]

Grkovic, S., Brown, M. H. & Skurray, R. A. (2001). Transcriptional regulation of multidrug efflux pumps in bacteria. Semin Cell Devel Biol 12, 225–237.[CrossRef][Medline]

Gruss, A. & Ehrlich, S. D. (1988). Insertion of foreign DNA into plasmids from gram-positive bacteria induces formation of high-molecular-weight plasmid multimers. J Bacteriol 170, 1183–1190.[Medline]

Helinski, D. R., Toukdarian, A. E. & Novick, R. P. (1996). Replication control and other stable maintenance mechanisms of plasmids. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 2295–2324. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.

Horinouchi, S. & Weisblum, B. (1982). Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150, 815–825.[Medline]

Jannière, L., Bruand, C. & Ehrlich, S. D. (1990). Structurally stable Bacillus subtilis cloning vectors. Gene 87, 53–61.[Medline]

Ji, G., Beavis, R. C. & Novick, R. P. (1995). Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc Natl Acad Sci U S A 92, 12055–12059.[Abstract]

Kreiswirth, B. N., Lofdahl, S., Betley, M. J., O'Reilly, M., Schlievert, P. M., Bergdoll, M. S. & Novick, R. P. (1983). The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305, 709–712.[Medline]

Lee, C. Y. & Iandolo, J. J. (1986). Lysogenic conversion of staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene. J Bacteriol 166, 385–391.[Medline]

Lee, C. Y., Buranen, S. L. & Ye, Z. (1991). Construction of single-copy integration vectors for Staphylococcus aureus. Gene 103, 101–105.[CrossRef][Medline]

Leelaporn, A., Firth, N., Byrne, M. E., Roper, E. & Skurray, R. A. (1994). Possible role of insertion sequence IS257 in dissemination and expression of high- and low-level trimethoprim resistance in staphylococci. Antimicrob Agents Chemother 38, 2238–2244.[Abstract]

Leonhardt, H. & Alonso, J. C. (1991). Parameters affecting plasmid stability in Bacillus subtilis. Gene 103, 107–111.[CrossRef][Medline]

Lyon, B. R., May, J. W. & Skurray, R. A. (1983). Analysis of plasmids in nosocomial strains of multiple-antibiotic-resistant Staphylococcus aureus. Antimicrob Agents Chemother 23, 817–826.[Medline]

Michel, B. & Ehrlich, S. D. (1986). Illegitimate recombination occurs between the replication origin of the plasmid pC194 and a progressing replication fork. EMBO J 5, 3691–3696.[Abstract]

Navarre, W. W., Daefler, S. & Schneewind, O. (1996). Cell wall sorting of lipoproteins in Staphylococcus aureus. J Bacteriol 178, 441–446.[Abstract]

Nielsen, J. B. & Lampen, J. O. (1982). Membrane-bound penicillinases in Gram-positive bacteria. J Biol Chem 257, 4490–4495.[Abstract/Free Full Text]

Novick, R. P. (1989). Staphylococcal plasmids and their replication. Annu Rev Microbiol 43, 537–565.[CrossRef][Medline]

Novick, R. P. (1990). The Staphylococcus as a molecular genetic system. In Molecular Biology of the Staphylococci, pp. 1–37. Edited by R. P. Novick. New York: VCH.

O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method for the detection of {beta}-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1, 283–288.[Medline]

Paulsen, I. T., Firth, N. & Skurray, R. A. (1997). Resistance to antimicrobial agents other than {beta}-lactams. In The Staphylococci in Human Disease, pp. 175–212. Edited by K. B. Crossley & G. L. Archer. New York: Churchill Livingstone.

Peschke, U., Beuck, V., Bujard, H., Gentz, R. & Le Grice, S. (1985). Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis. J Mol Biol 186, 547–555.[Medline]

Projan, S. J. & Novick, R. P. (1997). The molecular basis of pathogenicity. In The Staphylococci in Human Disease, pp. 55–81. Edited by K. B. Crossley & G. L. Archer. New York: Churchill Livingstone.

Ranelli, D. M., Jones, C. L., Johns, M. B., Mussey, G. J. & Khan, S. A. (1985). Molecular cloning of staphylococcal enterotoxin B gene in Escherichia coli and Staphylococcus aureus. Proc Natl Acad Sci U S A 82, 5850–5854.[Abstract]

Renault, P., Corthier, G., Goupil, N., Delorme, C. & Ehrlich, S. D. (1996). Plasmid vectors for Gram-positive bacteria switching from high to low copy number. Gene 183, 175–182.[CrossRef][Medline]

Rocha, E. P., Danchin, A. & Viari, A. (1999). Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis. Nucleic Acids Res 27, 3567–3576.[Abstract/Free Full Text]

Rouch, D. A., Messerotti, L. J., Loo, L. S., Jackson, C. A. & Skurray, R. A. (1989). Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257. Mol Microbiol 3, 161–175.[Medline]

Rouch, D. A., Cram, D. S., DiBerardino, D., Littlejohn, T. G. & Skurray, R. A. (1990). Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol Microbiol 4, 2051–2062.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schenk, S. & Laddaga, R. A. (1992). Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett 73, 133–138.[Medline]

Simon, D. & Chopin, A. (1988). Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70, 559–566.[CrossRef][Medline]

Swinfield, T.-J., Jannière, L., Ehrlich, S. D. & Minton, N. P. (1991). Characterization of a region of the Enterococcus faecalis plasmid pAM{beta}1 which enhances the segregational stability of pAM{beta}1-derived cloning vectors in Bacillus subtilis. Plasmid 26, 209–221.[Medline]

Wang, P. Z. & Novick, R. P. (1987). Nucleotide sequence and expression of the {beta}-lactamase gene from Staphylococcus aureus plasmid pI258 in Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. J Bacteriol 169, 1763–1766.[Medline]

Wang, P. Z., Projan, S. J., Leason, K. R. & Novick, R. P. (1987). Translational fusion with a secretory enzyme as an indicator. J Bacteriol 169, 3082–3087.[Medline]

Weaver, K. E., Clewell, D. B. & An, F. (1993). Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication. J Bacteriol 175, 1900–1909.[Abstract]

Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]

Yoon, K. P., Misra, T. K. & Silver, S. (1991). Regulation of the cadA cadmium resistance determinant of Staphylococcus aureus plasmid pI258. J Bacteriol 173, 7643–7649.[Medline]

Received 19 August 2002; revised 29 November 2002; accepted 12 December 2002.



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