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Jensen et al., 2010

  1. 1. Analysis of the prototypical Staphylococcus aureus multiresistance plasmidpSK1Slade O. Jensen a, Sumalee Apisiridej a,1, Stephen M. Kwong a, Yee Hwa Yang b,Ronald A. Skurray a, Neville Firth a,*aSchool of Biological Sciences, University of Sydney, New South Wales 2006, AustraliabSchool of Mathematics and Statistics, University of Sydney, New South Wales 2006, Australiaa r t i c l e i n f oArticle history:Received 20 April 2010Accepted 6 June 2010Available online 12 June 2010Communicated by C. Jeffery SmithKeywords:Staphylococcus aureusMultiresistance plasmidToxin–antitoxin systema b s t r a c tThe Staphylococcus aureus multiresistance plasmid pSK1 is the prototype of a family ofstructurally related plasmids that were first identified in epidemic S. aureus strains isolatedin Australia during the 1980s and subsequently in Europe. Here we present the complete28.15 kb nucleotide sequence of pSK1 and discuss the genetic content and evolution ofthe 14 kb region that is conserved throughout the pSK1 plasmid family. In addition tothe previously characterized plasmid maintenance functions, this backbone region encodes12 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–antitoxinsystem. Furthermore, transcriptional profiling has revealed that plasmid carriage mostlikely has a minimal impact on the host, a factor that may contribute to the ability ofpSK1 family plasmids to carry multiple resistance determinants.Ó 2010 Elsevier Inc. All rights reserved.1. IntroductionClinical 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 anddissemination of antimicrobial resistance determinants instaphylococci (Firth and Skurray, 2006). Although the biol-ogy of small rolling-circle (RC)-replicating staphylococcalplasmids (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 theycarry. Three groups of theta-replicating plasmids havebeen recognized in staphylococci; viz., the pSK639 family,the multiresistance plasmids (heavy metal/b-lactamaseplasmids 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 andnot associated with widespread epidemics; they were sub-sequently characterized as ST250-MRSA-I (Robinson andEnright, 2003). However, in the late 1970s epidemic out-breaks of multiresistant (including gentamicin resistant)MRSA associated with considerable morbidity and mortalitywere reported in Eastern Australian (EA) hospitals (Kinget al., 1981; Pavillard et al., 1982). These new EA-MRSAstrains were genetically different from those previously iso-lated (Townsend et al., 1985) and resistance to gentamicinand 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 ora related Tn4001-like element, which between 1976 and1980 were located at various chromosomal sites (Gillespie0147-619X/$ - 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 BiologicalSciences A12, University of Sydney, Sydney, New South Wales 2006,Australia. Fax: +61 2 9351 4771.E-mail address: (N. Firth).1Present address: Division of Molecular Genetic and Molecular Biologyin Medicine, Department of Preclinic, Faculty of Medicine, ThammasatUniversity, Rangsit Campus, Prathum Thani, Thailand.Plasmid 64 (2010) 135–142Contents lists available at ScienceDirectPlasmidjournal homepage:
  2. 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 multiresistanceplasmids, designated the pSK1 family, that were prevalentin 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 toantiseptics and disinfectants (via qacA), aminoglycosides(via Tn4001) and trimethoprim (via dfrA) (Firth and Skur-ray, 1998, 2006), although variations have been identifiedbased on comparative restriction endonuclease mapping(Fig. 1). For example, some members, such as pSK4 andpSK575, mediate b-lactamase resistance via a Tn552-likemobile element whereas others, such as pSK14 andpSK18, lack the trimethoprim-resistance pSK639-likestructure (previously referred to as Tn4003) or the qacAantiseptic/disinfectant resistance gene as in pSK575. Othermembers (pSK7 and pSK18) lack Tn4001. Several definedsegments of pSK1 have previously been described (Byrneet al., 1989; Firth et al., 2000; Paulsen et al., 1994; Rouchet al., 1987, 1990, 1989). Here we present the completenucleotide sequence of this prototypical multiresistanceplasmid and discuss the genetic content and evolution ofthis 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 methods2.1. Bacterial strains, growth conditions and plasmidsBacterial strains and plasmids used in this study arelisted in Table 1. Bacterial strains were grown at 37 °C inLB 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 supplementedwith ampicillin (Ap) 100 lg mlÀ1or chloramphenicol (Cm)10 lg mlÀ1.2.2. DNA manipulationsPlasmid 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 minin Solution I (alkaline lysis method) or the ResuspensionSolution (Quantum Prep plasmid miniprep kit) containinglysostaphin (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) andrestriction enzymes, calf alkaline phosphatase and T4DNA 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 itstranscription) of IS256 and IS257 elements. Roman numerals and shading indicate regions of similarity to smaller staphylococcal plasmids (see text) and theorf 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. 3. 2.3. Nucleotide sequence determination and data analysisNucleotide sequencing was performed with the Sequi-Therm cycle sequencing kit (Epicentre Technologies)according to the manufacturer’s instructions. AutomatedDNA sequencing was performed by the Australian GenomeResearch 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) andPCR products amplified directly from pSK1 were utilizedas sequencing templates; sequences from PCR productswere derived from at least two independent amplifications.All restriction sites were crossed, and all novel sequenceswere determined on both DNA strands. Sequences werestored 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 ofnucleotide 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 segmentswere identified using the programs EMBOSS: helixturnhe-lix (Dodd and Egan, 1990) and TOPPRED II (Claros andvon Heijne, 1994), respectively. The complete nucleotidesequence of pSK1 is available under the Genbank accessionnumber GU565967.2.4. DNA transferpSK1 (carried in the clinical isolate SK18) was trans-ferred into SH1000 by transduction (Novick, 1991) usingthe S. aureus Phage Type 622. DNA was isolated from trans-ductants and the presence of pSK1 was confirmed byrestriction endonuclease analysis using BglII.2.5. RNA isolationTotal RNA was extracted using Trizol reagent (Gibco-BRL) from exponential-phase cultures of S. aureus SH1000and SH1000 containing pSK1. Glass beads (100 lm; Sigma)in combination with a bead beater (Bio 101) were used forcell lysis. 100 ll of total RNA was precipitated using 7.5 Mlithium chloride (Ambion) and quantitated using a UV-2450 spectrophotometer (Shimadzu).2.6. Gene expression microarray analysisLabeling of fragmented RNA and hybridisation to Gene-Chip S. aureus Genome Arrays (Affymetrix) was performedby 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 attwo 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 setsof genes based on several Gene-Ontology (GO terms were examined. Gene setanalysis was performed based on Wilcoxon rank sum testto determine whether a set of genes was highly ranked rel-ative to other gene sets in terms of the fold changestatistics.3. Results and discussion3.1. Nucleotide sequence of pSK1Comparative restriction endonuclease mapping of pSK1family plasmids has shown that the DNA segment beyondthe 14 kb coordinate of pSK1 has been subject to a range ofinsertions and/or deletions (Skurray et al., 1988) (Fig. 1). Incontrast, the 0–14 kb region of pSK1 that previously hadnot been completely sequenced appears to be conservedthroughout 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 thathave contributed to the prevalence of pSK1 family plas-mids in clinical S. aureus isolates. As such, the nucleotidesequence of both strands was determined using fourrecombinant pSK1 derivatives as templates for primerTable 1Bacterial strains and plasmids.Strain or plasmid Relevant characteristicsaReferences or sourceStrainsEscherichia coliDH5a FÀendA hsdR17 supE44 thi-1 kÀrecA1 gyrA96 relA1 /80dLacZDM15 Bethesda Research LaboratoriesStaphylococcus aureusRN4220 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)PlasmidspUC119 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 studypSK4852 ApR, 0.5 kb HindIII-SalI fragment of pSK415 cloned into pUC119 This studypSK4853 ApR, 4.2 kb HindIII-SalI fragment of pSK415 cloned into pUC119 This studyaAp, ampicillin; Cm, chloramphenicol.S.O. Jensen et al. / Plasmid 64 (2010) 135–142 137
  4. 4. walking; a complete pSK1 sequence was obtained byassembly with previously described regions correspondingto the trimethoprim-resistance pSK639-like structure (pre-viously referred to as Tn4003) (Rouch et al., 1989); theresolvase 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 genomecomprises 28150 bp and has an overall G + C content of31%, which is consistent with a prolonged existence inlow G + C bacterial hosts, such as the staphylococci.A genetic map of pSK1 is shown in Fig. 1 and productpredictions for the 15 newly annotated open readingframes (orfs) are presented in Table 2. Re-analysis of thepreviously sequenced regions revealed two additionalgenes, orf112 and orf61, located between sin and qacR(Fig. 1). orf112 encodes a conserved hypothetical mem-brane protein, and the orf61 product contains a putativehelix-turn-helix (HTH) DNA-binding domain belonging tothe 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 beco-transcribed by a putative promoter located upstreamof orf112 (Fig. 1). Additionally, IS257-mediated insertionof the pSK639-like structure (Apisiridej et al., 1997) cannow be seen to have interrupted a gene (orf226, Fig. 1) thatencoded a 226 aa protein that shares high-level identitywith a hypothetical protein (Orf255) from the S. aureusb-lactamase plasmid pBORa53 (Massidda et al., 2006).3.2. Analysis of the conserved regionThe 0–14 kb region is predicted to contain 12 newgenes, in addition to those previously characterized (parand rep) (Kwong et al., 2008; Simpson et al., 2003). A rec-ognisable ribosome binding site (RBS) can be identifiedpreceding a candidate start codon (ATG in most cases) foreach new gene with the exception of orf220, which may re-flect translation coupling with the overlapping upstreamgene, orf103. Apart from the previously proposed promot-ers, PIN and POUT of IS256R (Byrne et al., 1989), the latterof which may contribute to orf186 transcription, a numberof putative promoters can be identified in this region (seeFig. 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 proximityto direct or inverted repeats, which may represent operatorsites for regulatory DNA-binding proteins.Database searches revealed that the 7–14 kb region ofpSK1 between orf84 and orf30 inclusive is similar to seg-ments conserved as part of the staphylococcal plasmidspPI-1 (Aso et al., 2005) and pSE-12228–05 (Zhang et al.,2003) (Fig. 2A). The level of nucleotide sequence identitybetween pSK1 and either of these plasmids is, for the mostpart, greater than 90% and in the case of pPI-1 this high-le-vel identity also includes the 30end of rep (and down-stream region) and orf203 (Fig. 2A). This conserved regionis virtually contiguous in pPI-1; however, in pSK1 rep andorf203 are separated by approximately 3 kb, the origin ofwhich is discussed below. Whereas pSK1 was derived froma clinical S. aureus isolate, pSE-12228-05 was carried by acommensal S. epidermidis strain (Zhang et al., 2003), andpPI-1 by an environmental S. warneri strain (isolated froma bed of fermented rice bran) (Sashihara et al., 2000). Theconservation of the orf84-orf410 gene cluster within thebackbones of these distinct plasmids from disparate staph-ylococcal hosts suggests the proteins encoded may con-tribute to an adaptive phenotype advantageous in avariety of niches.Only the deduced products encoded by orf30 and orf266share 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 Itoxin–antitoxin (TA) system from the Enterococcus faecalisplasmid pAD1 (Weaver et al., 2009). TA systems of thistype 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 tothe fst toxin gene, a number of sequence features havebeen identified in the pAD1 TA locus that are importantfor the functionality of the system. These include a strongpromoter for the production of the antisense RNA anti-toxin, a bi-directional terminator and sequences involvedin 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 inpSK1 (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 conservedstaphylococcal plasmid backbone region shown in Fig. 2A,and putative Fst-like TA systems are also present in thecorresponding regions of pPI-1 and pSE-12228-05 (Fig. 3),Table 2Newly annotated pSK1 Orfs.Protein Size(kDa)aComments and predictionsOrf186 21.6 Four predicted transmembrane segments (TMS)Orf92 10.7 Three predicted TMSOrf266 31.4 Putative teichoic acid translocation permease(TagG); six predicted TMSOrf203 23.5 Predicted HTH domain (aa 15–36), threepredicted TMSOrf84 10.1 Predicted cytoplasmic proteinOrf288 32.7 Putative lipoprotein; peptidase II cleavage sitebetween aa 17–18, one predicted TMSOrf172 18.4 Putative cell-wall associated surface protein; N-terminal signal peptide and a C-terminalsorting signal, peptidase I cleavage site betweenaa 28–29, LPXTG motif (aa 138–142)Orf212 25.5 Three predicted TMSOrf220 26.3 One predicted TMSOrf103 12.5 Predicted cytoplasmic proteinOrf410 49 Five predicted TMSOrf30 3.5 Putative Fst-like toxin; one predicted TMSOrf112 13.3 Three predicted TMSOrf61 7.2 Predicted HTH domain (aa 14–35)Orf226 26.9 Predicted cytoplasmic protein; disrupted by thepSK639-like structureaProtein sizes were calculated from deduced aa sequences.138 S.O. Jensen et al. / Plasmid 64 (2010) 135–142
  5. 5. but were not previously annotated; the toxins from theseplasmids share 53% and 63% identity with pSK1 Orf30,respectively. The lower level of nucleotide sequence iden-tity shared by these putative TA systems (ranges between71% and 84%) in comparison to the rest of this conservedregion (P90%), is most likely related to their location atthe plasmid-specific junction (Fig. 2A). Previous studieshave shown that genes located at the end of a conservedFig. 2. Genetic maps showing relationships between pSK1 and other staphylococcal plasmids. Plasmid names and sizes are shown on the left and arrowsdenote orfs, with arrowheads indicating the direction of transcription. Shading indicates regions of plasmid similarity, which was identified using theArtemis Comparison Tool (Carver et al., 2005), and nucleotide sequence identity P90% is noted. (A) Comparison of pSK1 to pPI-1 (GenBank AccessionAB125341) and pSE-12228–05 (GenBank Accession AE015934). Black arrowheads within boxes denote the transposase (and direction of its transcription) ofIS256 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 pSK1and pSE-12228-06 (GenBank Accession AE015935). The crosshatched bar represents a likely co-integrated form of a smaller pSK639-like staphylococcalplasmid.Fig. 3. Nucleotide sequence alignment of the enterococcal pAD1 Fst toxin-antitoxin system (GenBank accession L01794; nt 4348–4037) with the putativesystems 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), 50sequencescomplementary to the RBS (anti-RBS sequences boxed) that form the SL translational inhibitor structure, and the 50and 30UH sequences (bold) that formthe nuclease-protective upstream helix (UH) structure. Direct repeats DRa and DRb (arrows), required for antitoxin-mediated repression of toxintranslation, are shown with lower case letters indicating residues of the toxin mRNA and antitoxin RNA that can hybridise using standard RNA-RNA pairingrules. Plasmid names are shown on the left.S.O. Jensen et al. / Plasmid 64 (2010) 135–142 139
  6. 6. gene cluster (in an otherwise variable piece of DNA) can bethe most divergent, as they are involved in recombinationwith the adjacent sequence-specific DNA (Li and Reeves,2000); there is no evidence of a mobile element directlydownstream of orf30 or the fst-like toxin genes identifiedin pPI-1 and pSE-12228-05.The pSK1 orf266 product appears to be a membranepermease component of an ABC transport protein. Itshares high-level identity (up to 59%) with chromosom-ally encoded TagG proteins from various Gram-positivebacteria, including staphylococci, which in combinationwith a TagH component, mediate the translocation of cellwall 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 activitiesand cation concentrations; and attachment to surfacesand interactions with cell receptors (Xia et al., 2010).However, it should be noted that Orf266 is most similarto one of two TagG-like paralogues encoded by the S.saprophiticus chromosome (62% identity to GenPept entryYP_301040), rather than that organism’s TagG orthologue,raising the possibility that it might contribute to thetransport of other glycopolymers.Notably, seven other proteins encoded by the 0–14 kbregion of pSK1 are also likely to be associated with thecell 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 anLPXTG motif (Marraffini et al., 2006). Orf288 possessesthe 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 genesare co-transcribed so these proteins may participate in acommon function. Hydropathy analysis predicts that inaddition to Orf266, the putative products of Orf186,Orf92, Orf203, Orf212 and Orf410 are likely to be integralmembrane proteins, raising the possibility that at leastsome may be involved in membrane transport. Orf203may bind DNA in response to sensing environmentalstimuli since it also contains, like Orf61, a predictedXRE regulator family HTH domain. In the absence ofhomology to proteins of known function, the roles ofthese proteins remain an open question. However, theclustering of genes encoding a small cell surface anchoredprotein (Orf172), a lipoprotein (Orf288), membrane pro-teins (Orf212 and Orf220) and cytoplasmic proteins(Orf84 and Orf103) is reminiscent of the isd heme–ironuptake locus of S. aureus (Marraffini et al., 2006), hintingthat at least some of these proteins might be componentsof a nutrient uptake system. Interestingly, despite lackingrecognisable sequence similarity with its syntenic coun-terpart in pSK1, orf288, p519 from pSE-12228-05 none-theless encodes a similarly sized protein that alsopossesses the characteristic features of a lipoprotein. Suchdivergence might be a consequence of diversifying selec-tion for immune evasion since lipoproteins have beenshown to play an important role in eliciting host immunedefense mechanisms against staphylococcal infections(Bubeck Wardenburg et al., 2006).3.3. Other sequence featuresIn addition to the trimethoprim-resistance pSK639-likestructure (previously referred to as Tn4003) (Rouch et al.,1989), there are three distinct regions between rep andorf84 that share significant similarity with segments ofsmaller staphylococcal plasmids and these are denoted asregions 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 issimilar to a non-coding region found downstream of therep gene of pC194 family plasmids. As previously noted,the equivalent segment is evident just downstream of thepC194-like rep remnant on the b-lactamase/heavy metalresistance plasmid pI9789::Tn552 (Firth et al., 2000) andpresumably represents the integration of a pC194 familyplasmid (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 hassubsequently 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 ofpSK639 family plasmids, which includes the 50end of mobCand part of the upstream predicted origin of transfer (oriT)(Apisiridej et al., 1997; Caryl and Thomas, 2006); in pSK1two 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 regionIII) 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 formationof stable cointegrates, RSB, is located within SSOA (Iordane-scu, 1975).In addition to the non-coding regions of small plasmidsimilarity discussed above, the approximately 3 kb seg-ment between the truncated mobC gene and SSOA, whichincludes orf92, orf266 and orf203, shares similarity withthe pSK639-like plasmid pSE12228-06 (Fig. 2B), whichco-exists in the same strain as pSE12228-05 (Fig 2A)(Zhang et al., 2003). Therefore, it is possible that this entireregion represents a remnant of a co-integrated pSK639-likeplasmid.3.4. Effect of plasmid carriageTo investigate the impact of pSK1 carriage on theexpression of chromosomally-encoded genes, RNA isolatedfrom mid-exponentially growing cultures of SH1000 andSH1000 carrying pSK1 was labeled and hybridized toGeneChip S. aureus Genome Arrays (Affymetrix); two inde-pendent assays were performed for each strain. Analysis ofthe resulting expression profiles indicated that pSK1 car-riage did not significantly alter the transcription of anychromosomally encoded SH1000 genes (SupplementaryFig. S1). Transcription of the only pSK1-encoded gene rep-resented on the array, tnp, which encodes the transposaseof IS257 (also known as IS431), was readily detectable inthe plasmid-carrying strain; because SH1000 is derivedfrom NCTC 8325, it does not possess any chromosomalcopies of IS257, whereas pSK1 carries three.140 S.O. Jensen et al. / Plasmid 64 (2010) 135–142
  7. 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 (Subramanianet al., 2005). However, analysis of the GO gene sets, includ-ing cell division (0051301), DNA metabolic process(0006259) and cell wall biogenesis (0042546), revealedno enrichment of genes that exhibited altered expressionas a result of pSK1 carriage. Thus, under the conditionstested, carriage of pSK1 did not have any detectable effecton transcription of chromosomally-encoded genes, sug-gesting that the plasmid has minimal impact on its bacte-rial host.4. Concluding remarksThe completion of the pSK1 nucleotide sequence hasallowed us to identify the full complement of genes en-coded by a 14 kb region that is conserved throughoutthe 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), andhave been responsible for deletion of the qac locus ofpSK1 family plasmids (e.g., pSK575 in Fig. 1) (Kupferwas-ser et al., 1999; Wright et al., 1998). The conserved 14 kbregion appears to be relatively immune to such geneticrearrangements, consistent with the presence of evolu-tionarily important genes. However, the genetic stabilityof this region may also reflect the fact that it is boundedby genes for essential plasmid replication and mainte-nance functions at one end (rep and par) and a newlyidentified Fst-like TA system at the other (Fig. 1); dele-tions encompassing the later would result in loss of theless-stable antisense RNA antitoxin, resulting in transla-tion of the Fst-like toxin Orf30 and subsequent host celldeath.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 housekeepingfunctions, 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 fromnon-clinical coagulase-negative staphylococci, suggeststhat rather than some direct role in virulence, they likelyconfer phenotypes beneficial in a range of environments,including clinical settings. The presence of this region indifferent staphylococcal species of distinct origins sug-gests carriage in this genus over a significant period oftime. It is perhaps unsurprising then that pSK1 did notleave any discernable ‘‘footprint” on transcription of thehost chromosome. It is likely that the ‘‘well-adapted”,‘‘low-impact” nature of these plasmids contributes totheir suitability as vehicles for the accretion and dissem-ination of antimicrobial resistance determinants.AcknowledgmentsThis work was supported by Project Grants 950259 and457454 from the National Health and Medical ResearchCouncil (Australia) and Discovery Grant DP0346013 fromthe Australian Research Council. 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