1. Analysis of the prototypical Staphylococcus aureus multiresistance plasmid
pSK1
Slade O. Jensen a
, Sumalee Apisiridej a,1
, Stephen M. Kwong a
, Yee Hwa Yang b
,
Ronald A. Skurray a
, Neville Firth a,*
a
School of Biological Sciences, University of Sydney, New South Wales 2006, Australia
b
School of Mathematics and Statistics, University of Sydney, New South Wales 2006, Australia
a r t i c l e i n f o
Article history:
Received 20 April 2010
Accepted 6 June 2010
Available online 12 June 2010
Communicated by C. Jeffery Smith
Keywords:
Staphylococcus aureus
Multiresistance plasmid
Toxin–antitoxin system
a b s t r a c t
The Staphylococcus aureus multiresistance plasmid pSK1 is the prototype of a family of
structurally related plasmids that were first identified in epidemic S. aureus strains isolated
in Australia during the 1980s and subsequently in Europe. Here we present the complete
28.15 kb nucleotide sequence of pSK1 and discuss the genetic content and evolution of
the 14 kb region that is conserved throughout the pSK1 plasmid family. In addition to
the previously characterized plasmid maintenance functions, this backbone region encodes
12 putative gene products, including a lipoprotein, teichoic acid translocation permease,
cell wall anchored surface protein and an Fst-like toxin as part of a Type I toxin–antitoxin
system. Furthermore, transcriptional profiling has revealed that plasmid carriage most
likely has a minimal impact on the host, a factor that may contribute to the ability of
pSK1 family plasmids to carry multiple resistance determinants.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
Clinical isolates of Staphylococcus aureus and coagulase-
negative staphylococci commonly carry one or more resis-
tance plasmids, which are important vehicles of the genet-
ic transfer that facilitates the acquisition, maintenance and
dissemination of antimicrobial resistance determinants in
staphylococci (Firth and Skurray, 2006). Although the biol-
ogy of small rolling-circle (RC)-replicating staphylococcal
plasmids (1-5 kb) has been analyzed in detail, compara-
tively little attention has been paid to the larger theta-rep-
licating plasmids beyond the resistance genes that they
carry. Three groups of theta-replicating plasmids have
been recognized in staphylococci; viz., the pSK639 family,
the multiresistance plasmids (heavy metal/b-lactamase
plasmids and the pSK1 family) and the conjugative multi-
resistance plasmids (pSK41 family) (Firth and Skurray,
2006).
Prior to 1976 methicillin-resistant S. aureus (MRSA)
strains isolated in Australia were gentamicin-sensitive and
not associated with widespread epidemics; they were sub-
sequently characterized as ST250-MRSA-I (Robinson and
Enright, 2003). However, in the late 1970s epidemic out-
breaks of multiresistant (including gentamicin resistant)
MRSA associated with considerable morbidity and mortality
were reported in Eastern Australian (EA) hospitals (King
et al., 1981; Pavillard et al., 1982). These new EA-MRSA
strains were genetically different from those previously iso-
lated (Townsend et al., 1985) and resistance to gentamicin
and the related aminoglycosides, tobramycin and kanamy-
cin, was mediated by an aacA-aphD gene (Rouch et al.,
1987). This gene was carried by the transposon Tn4001 or
a related Tn4001-like element, which between 1976 and
1980 were located at various chromosomal sites (Gillespie
0147-619X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.plasmid.2010.06.001
* Corresponding author. Address: Neville Firth, School of Biological
Sciences A12, University of Sydney, Sydney, New South Wales 2006,
Australia. Fax: +61 2 9351 4771.
E-mail address: neville.firth@sydney.edu.au (N. Firth).
1
Present address: Division of Molecular Genetic and Molecular Biology
in Medicine, Department of Preclinic, Faculty of Medicine, Thammasat
University, Rangsit Campus, Prathum Thani, Thailand.
Plasmid 64 (2010) 135–142
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2. et al., 1987; Gillespie et al., 1984; Lyon et al., 1983). How-
ever, from 1980 onwards Tn4001 was predominantly lo-
cated on a group of structurally related multiresistance
plasmids, designated the pSK1 family, that were prevalent
in clinical S. aureus strains isolated in Australia and the UK
(Gillespie et al., 1987; Lyon et al., 1984, 1987; Wright et al.,
1998; Cookson and Phillips, 1988; Townsend et al., 1987).
pSK1 family plasmids commonly confer resistance to
antiseptics and disinfectants (via qacA), aminoglycosides
(via Tn4001) and trimethoprim (via dfrA) (Firth and Skur-
ray, 1998, 2006), although variations have been identified
based on comparative restriction endonuclease mapping
(Fig. 1). For example, some members, such as pSK4 and
pSK575, mediate b-lactamase resistance via a Tn552-like
mobile element whereas others, such as pSK14 and
pSK18, lack the trimethoprim-resistance pSK639-like
structure (previously referred to as Tn4003) or the qacA
antiseptic/disinfectant resistance gene as in pSK575. Other
members (pSK7 and pSK18) lack Tn4001. Several defined
segments of pSK1 have previously been described (Byrne
et al., 1989; Firth et al., 2000; Paulsen et al., 1994; Rouch
et al., 1987, 1990, 1989). Here we present the complete
nucleotide sequence of this prototypical multiresistance
plasmid and discuss the genetic content and evolution of
this clinically important plasmid family. Additionally, tran-
scriptional profiling was undertaken to investigate the im-
pact of pSK1 carriage on the bacterial host.
2. Materials and methods
2.1. Bacterial strains, growth conditions and plasmids
Bacterial strains and plasmids used in this study are
listed in Table 1. Bacterial strains were grown at 37 °C in
LB medium (Sambrook and Russell, 2001) or on plates con-
taining LB medium and 1.5% w/v Oxoid agar, unless other-
wise stated. When required, media were supplemented
with ampicillin (Ap) 100 lg mlÀ1
or chloramphenicol (Cm)
10 lg mlÀ1
.
2.2. DNA manipulations
Plasmid DNA was isolated from Escherichia coli and S.
aureus using the alkaline lysis method (Birnboim and Doly,
1979) or the Quantum Prep plasmid miniprep kit (Bio-
Rad); S. aureus strains were incubated at 37 °C for 20 min
in Solution I (alkaline lysis method) or the Resuspension
Solution (Quantum Prep plasmid miniprep kit) containing
lysostaphin (0.3 mg mlÀ1
; Sigma), in order to achieve effi-
cient cell lysis. Cloning in E. coli was performed using stan-
dard methods (Sambrook and Russell, 2001) and
restriction enzymes, calf alkaline phosphatase and T4
DNA ligase were purchased from New England Biolabs.
DNA fragments were PCR-amplified using Taq (New Eng-
land Biolabs) or Pfu DNA polymerase (Stratagene).
Fig. 1. Genetic map of pSK1. Determinants encode resistance to trimethoprim (dfrA) (Rouch et al., 1989), antiseptics/disinfectants (qacA) (Rouch et al., 1990)
and aminoglycosides (aacA-aphD) (Byrne et al., 1989; Rouch et al., 1987). Black arrowheads within boxes denote the transposase (and direction of its
transcription) of IS256 and IS257 elements. Roman numerals and shading indicate regions of similarity to smaller staphylococcal plasmids (see text) and the
orf disrupted by the insertion of a pSK639-like structure (region IV) is shown as two shaded boxes. Arrows denote putative promoters of the pSK1 backbone,
with arrowheads indicating the direction of transcription. Additional pSK1 family member restriction maps shown are derived from Skurray et al. (1988)
and Wright et al. (1998). Plasmid sizes are shown on the right and blaZ encodes resistance to penicillin. B, BglII; E, EcoR1; H, HindIII; S, SalI.
136 S.O. Jensen et al. / Plasmid 64 (2010) 135–142

3. 2.3. Nucleotide sequence determination and data analysis
Nucleotide sequencing was performed with the Sequi-
Therm cycle sequencing kit (Epicentre Technologies)
according to the manufacturer’s instructions. Automated
DNA sequencing was performed by the Australian Genome
Research Facility (AGRF; University of Queensland, Austra-
lia) or by the Sydney University and Prince Alfred Macro-
molecular Analysis Centre. Double-stranded plasmids
(pSK419, pSK4851, pSK4852 and pSK4853; Table 1) and
PCR products amplified directly from pSK1 were utilized
as sequencing templates; sequences from PCR products
were derived from at least two independent amplifications.
All restriction sites were crossed, and all novel sequences
were determined on both DNA strands. Sequences were
stored and assembled with the program Sequencher v. 4.5
(Gene Codes Corporation). Similarity searches were per-
formed using Blastp (Altschul et al., 1997) and regions of
nucleotide sequence identity between plasmids were iden-
tified using Blastn and the Artemis Comparison Tool (Altsc-
hul et al., 1997; Carver et al., 2005). Putative helix-turn-
helix domains and potential transmembrane segments
were identified using the programs EMBOSS: helixturnhe-
lix (Dodd and Egan, 1990) and TOPPRED II (Claros and
von Heijne, 1994), respectively. The complete nucleotide
sequence of pSK1 is available under the Genbank accession
number GU565967.
2.4. DNA transfer
pSK1 (carried in the clinical isolate SK18) was trans-
ferred into SH1000 by transduction (Novick, 1991) using
the S. aureus Phage Type 622. DNA was isolated from trans-
ductants and the presence of pSK1 was confirmed by
restriction endonuclease analysis using BglII.
2.5. RNA isolation
Total RNA was extracted using Trizol reagent (Gibco-
BRL) from exponential-phase cultures of S. aureus SH1000
and SH1000 containing pSK1. Glass beads (100 lm; Sigma)
in combination with a bead beater (Bio 101) were used for
cell lysis. 100 ll of total RNA was precipitated using 7.5 M
lithium chloride (Ambion) and quantitated using a UV-
2450 spectrophotometer (Shimadzu).
2.6. Gene expression microarray analysis
Labeling of fragmented RNA and hybridisation to Gene-
Chip S. aureus Genome Arrays (Affymetrix) was performed
by AGRF (The Walter and Eliza Hall Institute of Medical Re-
search, Australia). Bioconductor (Gentleman et al., 2004)
was used for quality assessment (affyPLM algorithm), pre-
processing (RMA algorithm) and differential gene expres-
sion (DE) analysis. The DE analysis was undertaken at
two levels; individual gene and gene set analysis. Individ-
ual gene analysis based on moderated-t tests (Smyth,
2004) was performed and DE genes identified by control-
ling for 5% false discover rate. In parallel, pre-defined sets
of genes based on several Gene-Ontology (GO http://
www.geneontology.org) terms were examined. Gene set
analysis was performed based on Wilcoxon rank sum test
to determine whether a set of genes was highly ranked rel-
ative to other gene sets in terms of the fold change
statistics.
3. Results and discussion
3.1. Nucleotide sequence of pSK1
Comparative restriction endonuclease mapping of pSK1
family plasmids has shown that the DNA segment beyond
the 14 kb coordinate of pSK1 has been subject to a range of
insertions and/or deletions (Skurray et al., 1988) (Fig. 1). In
contrast, the 0–14 kb region of pSK1 that previously had
not been completely sequenced appears to be conserved
throughout the plasmid family. In addition to the charac-
terized par and rep genes (Firth et al., 2000; Kwong et al.,
2008; Simpson et al., 2003), this region potentially con-
tained maintenance and/or virulence determinants that
have contributed to the prevalence of pSK1 family plas-
mids in clinical S. aureus isolates. As such, the nucleotide
sequence of both strands was determined using four
recombinant pSK1 derivatives as templates for primer
Table 1
Bacterial strains and plasmids.
Strain or plasmid Relevant characteristicsa
References or source
Strains
Escherichia coli
DH5a FÀ
endA hsdR17 supE44 thi-1 kÀ
recA1 gyrA96 relA1 /80dLacZDM15 Bethesda Research Laboratories
Staphylococcus aureus
RN4220 Restrictionless derivative of NCTC 8325-4 Kreiswirth et al. (1983)
SH1000 Functional rsbU derivative of NCTC 8325-4 rsbU+
Horsburgh et al. (2002)
SK18 Clinical isolate containing pSK1 Lyon et al. (1983)
Plasmids
pUC119 ApR
, E. coli cloning vector, pMB1 ori Vieira and Messing (1987)
pSK1 S. aureus multiresistance plasmid Lyon et al. (1983)
pSK411 CmR
, 2.5 kb HindIII fragment of pSK1 cloned into pACYC184 Tennent et al. (1986)
pSK415 ApR
, 4.7 kb HindIII fragment of pSK1 cloned into pBR322 Tennent et al. (1986)
pSK419 ApR
, 7.1 kb HindIII fragment of pSK1 cloned into pBR322 Tennent et al. (1986)
pSK4851 ApR
, 2.5 kb HindIII fragment of pSK411 cloned into pUC119 This study
pSK4852 ApR
, 0.5 kb HindIII-SalI fragment of pSK415 cloned into pUC119 This study
pSK4853 ApR
, 4.2 kb HindIII-SalI fragment of pSK415 cloned into pUC119 This study
a
Ap, ampicillin; Cm, chloramphenicol.
S.O. Jensen et al. / Plasmid 64 (2010) 135–142 137

4. walking; a complete pSK1 sequence was obtained by
assembly with previously described regions corresponding
to the trimethoprim-resistance pSK639-like structure (pre-
viously referred to as Tn4003) (Rouch et al., 1989); the
resolvase gene, sin (Paulsen et al., 1994); the antiseptic/dis-
infectant resistance determinant qacA (Rouch et al., 1990);
and the aminoglycoside-resistance transposon Tn4001
(Byrne et al., 1989; Rouch et al., 1987). The pSK1 genome
comprises 28150 bp and has an overall G + C content of
31%, which is consistent with a prolonged existence in
low G + C bacterial hosts, such as the staphylococci.
A genetic map of pSK1 is shown in Fig. 1 and product
predictions for the 15 newly annotated open reading
frames (orfs) are presented in Table 2. Re-analysis of the
previously sequenced regions revealed two additional
genes, orf112 and orf61, located between sin and qacR
(Fig. 1). orf112 encodes a conserved hypothetical mem-
brane protein, and the orf61 product contains a putative
helix-turn-helix (HTH) DNA-binding domain belonging to
the xenobiotic response element (XRE) transcriptional reg-
ulator family, as defined by the Conserved Domain Data-
base (CDD; entry cd00093) (Marchler-Bauer et al., 2009).
These genes are separated by 4 bp and are likely to be
co-transcribed by a putative promoter located upstream
of orf112 (Fig. 1). Additionally, IS257-mediated insertion
of the pSK639-like structure (Apisiridej et al., 1997) can
now be seen to have interrupted a gene (orf226, Fig. 1) that
encoded a 226 aa protein that shares high-level identity
with a hypothetical protein (Orf255) from the S. aureus
b-lactamase plasmid pBORa53 (Massidda et al., 2006).
3.2. Analysis of the conserved region
The 0–14 kb region is predicted to contain 12 new
genes, in addition to those previously characterized (par
and rep) (Kwong et al., 2008; Simpson et al., 2003). A rec-
ognisable ribosome binding site (RBS) can be identified
preceding a candidate start codon (ATG in most cases) for
each new gene with the exception of orf220, which may re-
flect translation coupling with the overlapping upstream
gene, orf103. Apart from the previously proposed promot-
ers, PIN and POUT of IS256R (Byrne et al., 1989), the latter
of which may contribute to orf186 transcription, a number
of putative promoters can be identified in this region (see
Fig. 1), three of which, designated Porf266, Porf172 and Porf103,
may direct the transcription of multiple genes. Further-
more, several of these promoters are located in proximity
to direct or inverted repeats, which may represent operator
sites for regulatory DNA-binding proteins.
Database searches revealed that the 7–14 kb region of
pSK1 between orf84 and orf30 inclusive is similar to seg-
ments conserved as part of the staphylococcal plasmids
pPI-1 (Aso et al., 2005) and pSE-12228–05 (Zhang et al.,
2003) (Fig. 2A). The level of nucleotide sequence identity
between pSK1 and either of these plasmids is, for the most
part, greater than 90% and in the case of pPI-1 this high-le-
vel identity also includes the 30
end of rep (and down-
stream region) and orf203 (Fig. 2A). This conserved region
is virtually contiguous in pPI-1; however, in pSK1 rep and
orf203 are separated by approximately 3 kb, the origin of
which is discussed below. Whereas pSK1 was derived from
a clinical S. aureus isolate, pSE-12228-05 was carried by a
commensal S. epidermidis strain (Zhang et al., 2003), and
pPI-1 by an environmental S. warneri strain (isolated from
a bed of fermented rice bran) (Sashihara et al., 2000). The
conservation of the orf84-orf410 gene cluster within the
backbones of these distinct plasmids from disparate staph-
ylococcal hosts suggests the proteins encoded may con-
tribute to an adaptive phenotype advantageous in a
variety of niches.
Only the deduced products encoded by orf30 and orf266
share significant similarity with proteins of known func-
tion. The product of orf30 (TTG start codon) shares 63%
similarity with the Fst toxin of the characterized Type I
toxin–antitoxin (TA) system from the Enterococcus faecalis
plasmid pAD1 (Weaver et al., 2009). TA systems of this
type have recently been found to be carried in the chromo-
some and/or plasmids of a number of Gram-positive spe-
cies, and are particularly prevalent in staphylococci
(Weaver et al., 2009; Kwong et al., 2010). In addition to
the fst toxin gene, a number of sequence features have
been identified in the pAD1 TA locus that are important
for the functionality of the system. These include a strong
promoter for the production of the antisense RNA anti-
toxin, a bi-directional terminator and sequences involved
in intra- and inter-molecular RNA pairing (anti-RBS,
5’UH, 3UH, DRb and DRa sequences) (Greenfield and Wea-
ver, 2000; Shokeen et al., 2008, 2009). Corresponding fea-
tures are evident in the sequence encompassing orf30 in
pSK1 (Fig. 3), strongly suggesting that it represents a func-
tional Type I TA system that could contribute to mainte-
nance of the plasmid.
It should be noted that orf30 is located in the conserved
staphylococcal plasmid backbone region shown in Fig. 2A,
and putative Fst-like TA systems are also present in the
corresponding regions of pPI-1 and pSE-12228-05 (Fig. 3),
Table 2
Newly annotated pSK1 Orfs.
Protein Size
(kDa)a
Comments and predictions
Orf186 21.6 Four predicted transmembrane segments (TMS)
Orf92 10.7 Three predicted TMS
Orf266 31.4 Putative teichoic acid translocation permease
(TagG); six predicted TMS
Orf203 23.5 Predicted HTH domain (aa 15–36), three
predicted TMS
Orf84 10.1 Predicted cytoplasmic protein
Orf288 32.7 Putative lipoprotein; peptidase II cleavage site
between aa 17–18, one predicted TMS
Orf172 18.4 Putative cell-wall associated surface protein; N-
terminal signal peptide and a C-terminal
sorting signal, peptidase I cleavage site between
aa 28–29, LPXTG motif (aa 138–142)
Orf212 25.5 Three predicted TMS
Orf220 26.3 One predicted TMS
Orf103 12.5 Predicted cytoplasmic protein
Orf410 49 Five predicted TMS
Orf30 3.5 Putative Fst-like toxin; one predicted TMS
Orf112 13.3 Three predicted TMS
Orf61 7.2 Predicted HTH domain (aa 14–35)
Orf226 26.9 Predicted cytoplasmic protein; disrupted by the
pSK639-like structure
a
Protein sizes were calculated from deduced aa sequences.
138 S.O. Jensen et al. / Plasmid 64 (2010) 135–142

5. but were not previously annotated; the toxins from these
plasmids share 53% and 63% identity with pSK1 Orf30,
respectively. The lower level of nucleotide sequence iden-
tity shared by these putative TA systems (ranges between
71% and 84%) in comparison to the rest of this conserved
region (P90%), is most likely related to their location at
the plasmid-specific junction (Fig. 2A). Previous studies
have shown that genes located at the end of a conserved
Fig. 2. Genetic maps showing relationships between pSK1 and other staphylococcal plasmids. Plasmid names and sizes are shown on the left and arrows
denote orfs, with arrowheads indicating the direction of transcription. Shading indicates regions of plasmid similarity, which was identified using the
Artemis Comparison Tool (Carver et al., 2005), and nucleotide sequence identity P90% is noted. (A) Comparison of pSK1 to pPI-1 (GenBank Accession
AB125341) and pSE-12228–05 (GenBank Accession AE015934). Black arrowheads within boxes denote the transposase (and direction of its transcription) of
IS256 and IS257 elements. Asterisks indicate the fst-like genes of pPI-1 and pSE-12228-05 indentified as part of this study. (B) Comparison between pSK1
and pSE-12228-06 (GenBank Accession AE015935). The crosshatched bar represents a likely co-integrated form of a smaller pSK639-like staphylococcal
plasmid.
Fig. 3. Nucleotide sequence alignment of the enterococcal pAD1 Fst toxin-antitoxin system (GenBank accession L01794; nt 4348–4037) with the putative
systems identified in the staphylococcal plasmids pSK1 (nt 13440-13748), pPI-1 (GenBank accession AB125341; nt 7488–7787) and pSE-12228-05
(GenBank accession AE015934; nt 14944–14645). Conserved features predicted include the toxin coding regions (gray shading), ribosome binding sites
(RBS, boxed), promoters for the toxin and antitoxin genes (black shading), bi-directional terminators (bold with stem sequences underlined), 50
sequences
complementary to the RBS (anti-RBS sequences boxed) that form the SL translational inhibitor structure, and the 50
and 30
UH sequences (bold) that form
the nuclease-protective upstream helix (UH) structure. Direct repeats DRa and DRb (arrows), required for antitoxin-mediated repression of toxin
translation, are shown with lower case letters indicating residues of the toxin mRNA and antitoxin RNA that can hybridise using standard RNA-RNA pairing
rules. Plasmid names are shown on the left.
S.O. Jensen et al. / Plasmid 64 (2010) 135–142 139

6. gene cluster (in an otherwise variable piece of DNA) can be
the most divergent, as they are involved in recombination
with the adjacent sequence-specific DNA (Li and Reeves,
2000); there is no evidence of a mobile element directly
downstream of orf30 or the fst-like toxin genes identified
in pPI-1 and pSE-12228-05.
The pSK1 orf266 product appears to be a membrane
permease component of an ABC transport protein. It
shares high-level identity (up to 59%) with chromosom-
ally encoded TagG proteins from various Gram-positive
bacteria, including staphylococci, which in combination
with a TagH component, mediate the translocation of cell
wall teichoic acids (WTA) (Lazarevic and Karamata, 1995;
Xia et al., 2010). WTA are implicated in a range of activ-
ities in S. aureus, which group under three broad themes:
resistance to toxic molecules and environment stresses;
control of cell envelope-associated enzyme activities
and cation concentrations; and attachment to surfaces
and interactions with cell receptors (Xia et al., 2010).
However, it should be noted that Orf266 is most similar
to one of two TagG-like paralogues encoded by the S.
saprophiticus chromosome (62% identity to GenPept entry
YP_301040), rather than that organism’s TagG orthologue,
raising the possibility that it might contribute to the
transport of other glycopolymers.
Notably, seven other proteins encoded by the 0–14 kb
region of pSK1 are also likely to be associated with the
cell envelope. Orf172 is predicted to be a cell-wall associ-
ated surface protein since it possesses an N-terminal sig-
nal peptide and a C-terminal sorting signal with an
LPXTG motif (Marraffini et al., 2006). Orf288 possesses
the features of a lipoprotein signal peptide (von Heijne,
1989) and modification of this protein has been con-
firmed (Grkovic et al., 2003). The orf172 and orf288 genes
are co-transcribed so these proteins may participate in a
common function. Hydropathy analysis predicts that in
addition to Orf266, the putative products of Orf186,
Orf92, Orf203, Orf212 and Orf410 are likely to be integral
membrane proteins, raising the possibility that at least
some may be involved in membrane transport. Orf203
may bind DNA in response to sensing environmental
stimuli since it also contains, like Orf61, a predicted
XRE regulator family HTH domain. In the absence of
homology to proteins of known function, the roles of
these proteins remain an open question. However, the
clustering of genes encoding a small cell surface anchored
protein (Orf172), a lipoprotein (Orf288), membrane pro-
teins (Orf212 and Orf220) and cytoplasmic proteins
(Orf84 and Orf103) is reminiscent of the isd heme–iron
uptake locus of S. aureus (Marraffini et al., 2006), hinting
that at least some of these proteins might be components
of a nutrient uptake system. Interestingly, despite lacking
recognisable sequence similarity with its syntenic coun-
terpart in pSK1, orf288, p519 from pSE-12228-05 none-
theless encodes a similarly sized protein that also
possesses the characteristic features of a lipoprotein. Such
divergence might be a consequence of diversifying selec-
tion for immune evasion since lipoproteins have been
shown to play an important role in eliciting host immune
defense mechanisms against staphylococcal infections
(Bubeck Wardenburg et al., 2006).
3.3. Other sequence features
In addition to the trimethoprim-resistance pSK639-like
structure (previously referred to as Tn4003) (Rouch et al.,
1989), there are three distinct regions between rep and
orf84 that share significant similarity with segments of
smaller staphylococcal plasmids and these are denoted as
regions I-III in Fig. 1. Sequence to the left of an inverted re-
peat located between rep and orf92 (denoted as region I),
has previously been analyzed by Firth et al. (2000) and is
similar to a non-coding region found downstream of the
rep gene of pC194 family plasmids. As previously noted,
the equivalent segment is evident just downstream of the
pC194-like rep remnant on the b-lactamase/heavy metal
resistance plasmid pI9789::Tn552 (Firth et al., 2000) and
presumably represents the integration of a pC194 family
plasmid (or part thereof) into the backbone of the progen-
itor to pSK1 and related b-lactamase/heavy metal resis-
tance plasmids. Note that the rep remnant has
subsequently been deleted from pSK1. In contrast, se-
quence to the right of this inverted repeat (denoted as re-
gion II) is similar to a mobilization-associated region of
pSK639 family plasmids, which includes the 50
end of mobC
and part of the upstream predicted origin of transfer (oriT)
(Apisiridej et al., 1997; Caryl and Thomas, 2006); in pSK1
two deletions have subsequently occurred in this region.
The third region of small plasmid similarity is located be-
tween orf203 and orf84 (nt 6913–7073; denoted as region
III) and corresponds to a minus strand origin of replication,
SSOA (formerly palA), typically present on RC plasmids
(Gruss et al., 1987); a site associated with the formation
of stable cointegrates, RSB, is located within SSOA (Iordane-
scu, 1975).
In addition to the non-coding regions of small plasmid
similarity discussed above, the approximately 3 kb seg-
ment between the truncated mobC gene and SSOA, which
includes orf92, orf266 and orf203, shares similarity with
the pSK639-like plasmid pSE12228-06 (Fig. 2B), which
co-exists in the same strain as pSE12228-05 (Fig 2A)
(Zhang et al., 2003). Therefore, it is possible that this entire
region represents a remnant of a co-integrated pSK639-like
plasmid.
3.4. Effect of plasmid carriage
To investigate the impact of pSK1 carriage on the
expression of chromosomally-encoded genes, RNA isolated
from mid-exponentially growing cultures of SH1000 and
SH1000 carrying pSK1 was labeled and hybridized to
GeneChip S. aureus Genome Arrays (Affymetrix); two inde-
pendent assays were performed for each strain. Analysis of
the resulting expression profiles indicated that pSK1 car-
riage did not significantly alter the transcription of any
chromosomally encoded SH1000 genes (Supplementary
Fig. S1). Transcription of the only pSK1-encoded gene rep-
resented on the array, tnp, which encodes the transposase
of IS257 (also known as IS431), was readily detectable in
the plasmid-carrying strain; because SH1000 is derived
from NCTC 8325, it does not possess any chromosomal
copies of IS257, whereas pSK1 carries three.
140 S.O. Jensen et al. / Plasmid 64 (2010) 135–142

7. We also examined the impact of pSK1 carriage on sev-
eral relevant cellular processes using gene ontology (GO)
annotations. This enabled us to determine if an a priori de-
fined set of genes showed statistically significant, concor-
dant differences between the two strains (Subramanian
et al., 2005). However, analysis of the GO gene sets, includ-
ing cell division (0051301), DNA metabolic process
(0006259) and cell wall biogenesis (0042546), revealed
no enrichment of genes that exhibited altered expression
as a result of pSK1 carriage. Thus, under the conditions
tested, carriage of pSK1 did not have any detectable effect
on transcription of chromosomally-encoded genes, sug-
gesting that the plasmid has minimal impact on its bacte-
rial host.
4. Concluding remarks
The completion of the pSK1 nucleotide sequence has
allowed us to identify the full complement of genes en-
coded by a 14 kb region that is conserved throughout
the pSK1 plasmid family (Fig. 1). Flanking deletions adja-
cent to IS256 and IS257 are commonly observed in staph-
ylococci (Berg et al., 1998; Leelaporn et al., 1994), and
have been responsible for deletion of the qac locus of
pSK1 family plasmids (e.g., pSK575 in Fig. 1) (Kupferwas-
ser et al., 1999; Wright et al., 1998). The conserved 14 kb
region appears to be relatively immune to such genetic
rearrangements, consistent with the presence of evolu-
tionarily important genes. However, the genetic stability
of this region may also reflect the fact that it is bounded
by genes for essential plasmid replication and mainte-
nance functions at one end (rep and par) and a newly
identified Fst-like TA system at the other (Fig. 1); dele-
tions encompassing the later would result in loss of the
less-stable antisense RNA antitoxin, resulting in transla-
tion of the Fst-like toxin Orf30 and subsequent host cell
death.
In addition to plasmid maintenance functions, (rep,
par and the TA system), the 14 kb backbone region en-
codes putative products that, for the most part, are inte-
gral membrane proteins (Orf186, Orf92, Orf266, Orf203,
Orf212, Orf220 and Orf410) or cell-surface associated
(Orf288 and Orf172). As these putative products are unli-
kely to be involved directly in plasmid housekeeping
functions, the phenotypes they might confer are intrigu-
ing. The conservation of most of these genes in the clin-
ically significant pSK1 plasmid family and plasmids from
non-clinical coagulase-negative staphylococci, suggests
that rather than some direct role in virulence, they likely
confer phenotypes beneficial in a range of environments,
including clinical settings. The presence of this region in
different staphylococcal species of distinct origins sug-
gests carriage in this genus over a significant period of
time. It is perhaps unsurprising then that pSK1 did not
leave any discernable ‘‘footprint” on transcription of the
host chromosome. It is likely that the ‘‘well-adapted”,
‘‘low-impact” nature of these plasmids contributes to
their suitability as vehicles for the accretion and dissem-
ination of antimicrobial resistance determinants.
Acknowledgments
This work was supported by Project Grants 950259 and
457454 from the National Health and Medical Research
Council (Australia) and Discovery Grant DP0346013 from
the Australian Research Council. The contributions of Carol
Scaramuzzi, Wu Yan and Brendon O’Rourke to this work
are gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.plasmid.
2010.06.001.
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