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The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics.

Horizontal transfer of plasmids encoding antimicrobial resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s, the first CA-MRSA strain isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline, and penicillin resistance genes on plasmid pWBG753 (∼30 kb). WA-5 and pWBG753 appeared only briefly in WA; however, fusidic acid resistance plasmids related to pWBG753 were also present in the first European CA-MRSA isolates at the time. Here, we characterize a 72-kb conjugative plasmid, pWBG731, present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749 family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium, and penicillin resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs), and the BinL resolution system of the β-lactamase transposon Tn552. An evolutionarily intermediate ∼42-kb nonconjugative plasmid, pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline resistance plasmid pT181. IS257 likely facilitated the replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized nonconjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous community-associated methicillin-sensitive S. aureus (CA-MSSA) isolates. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.

Horizontal transfer of plasmids encoding antimicrobial-resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s the first CA-MRSA isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline and penicillin-resistance genes on plasmid pWBG753 (∼30 kb). WA-5 and pWBG753 appeared only briefly in WA, however, fusidic-acid-resistance plasmids related to pWBG753 were also present in the first European CA-MRSA at the time. Here we characterized a 72-kb conjugative plasmid pWBG731 present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749-family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium and penicillin-resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs) and the BinL resolution system of the β-lactamase transposon Tn552 An evolutionary intermediate ∼42-kb non-conjugative plasmid pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline-resistance plasmid pT181. IS257 likely facilitated replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized non-conjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous CA-MSSA. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.

Staphylococcus aureus is a significant human pathogen whose evolution and adaptation have been shaped in part by mobile genetic elements (MGEs), facilitating the global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs, in particular, how changes in the structure of multidrug resistance (MDR) plasmids may influence important staphylococcal phenotypes, is incomplete. Here, we undertook a population and functional genomics study of 212 methicillin-resistant S. aureus (MRSA) sequence type 239 (ST239) isolates collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have coevolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics, we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s and found that multiple structural variants have arisen. Plasmid maintenance and stability were linked to IS256- and IS257-mediated chromosomal integration and disruption of the plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin, trimethoprim, and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA isolates through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing the rates of gentamicin resistance and reduced chlorhexidine susceptibility and (ii) changes in the plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a potential evolutionary response of ST239 MRSA to biocides, one of which may contribute to the ongoing persistence and adaptation of this lineage within health care institutions.

Strains of Staphylococcus aureus, and to a lesser extent other staphylococcal species, are a significant cause of morbidity and mortality. An important factor in the notoriety of these organisms stems from their frequent resistance to many antimicrobial agents used for chemotherapy. This review catalogues the variety of mobile genetic elements that have been identified in staphylococci, with a primary focus on those associated with the recruitment and spread of antimicrobial resistance genes. These include plasmids, transposable elements such as insertion sequences and transposons, and integrative elements including ICE and SCC elements. In concert, these diverse entities facilitate the intra- and inter-cellular gene mobility that enables horizontal genetic exchange, and have also been found to play additional roles in modulating gene expression and genome rearrangement.

© 2017 Kwong, Ramsay, Jensen and Firth. The currently widespread and increasing prevalence of resistant bacterial pathogens is a significant medical problem. In clinical strains of staphylococci, the genetic determinants that confer resistance to antimicrobial agents are often located on mobile elements, such as plasmids. Many of these resistance plasmids are capable of horizontal transmission to other bacteria in their surroundings, allowing extraordinarily rapid adaptation of bacterial populations. Once the resistance plasmids have been spread, they are often perpetually maintained in the new host, even in the absence of selective pressure. Plasmid persistence is accomplished by plasmid-encoded genetic systems that ensure efficient replication and segregational stability during cell division. Staphylococcal plasmids utilize proteins of evolutionarily diverse families to initiate replication from the plasmid origin of replication. Several distinctive plasmid copy number control mechanisms have been studied in detail and these appear conserved within plasmid classes. The initiators utilize various strategies and serve a multifunctional role in (i) recognition and processing of the cognate replication origin to an initiation active form and (ii) recruitment of host-encoded replication proteins that facilitate replisome assembly. Understanding the detailed molecular mechanisms that underpin plasmid replication may lead to novel approaches that could be used to reverse or slow the development of resistance.

© 2017 Kwong, Ramsay, Jensen and Firth. The currently widespread and increasing prevalence of resistant bacterial pathogens is a significant medical problem. In clinical strains of staphylococci, the genetic determinants that confer resistance to antimicrobial agents are often located on mobile elements, such as plasmids. Many of these resistance plasmids are capable of horizontal transmission to other bacteria in their surroundings, allowing extraordinarily rapid adaptation of bacterial populations. Once the resistance plasmids have been spread, they are often perpetually maintained in the new host, even in the absence of selective pressure. Plasmid persistence is accomplished by plasmid-encoded genetic systems that ensure efficient replication and segregational stability during cell division. Staphylococcal plasmids utilize proteins of evolutionarily diverse families to initiate replication from the plasmid origin of replication. Several distinctive plasmid copy number control mechanisms have been studied in detail and these appear conserved within plasmid classes. The initiators utilize various strategies and serve a multifunctional role in (i) recognition and processing of the cognate replication origin to an initiation active form and (ii) recruitment of host-encoded replication proteins that facilitate replisome assembly. Understanding the detailed molecular mechanisms that underpin plasmid replication may lead to novel approaches that could be used to reverse or slow the development of resistance.

Conjugation is a dominant mechanism of horizontal gene transfer and substantially contributes to the plasticity and evolvability of prokaryotic genomes. The impact of conjugation on genetic flux extends well beyond self-transmissible conjugative elements, because non-conjugative ‘mobilizable elements’ utilize other elements’ conjugative apparatus for transfer. Bacterial genome comparisons highlight plasmids as vehicles for dissemination of pathogenesis and antimicrobial-resistance determinants, but for most non-conjugative plasmids, a mobilization mechanism is not apparent. Recently we discovered many Staphylococcus aureus plasmids lacking mobilization genes carry oriT sequences that mimic those on conjugative plasmids, suggesting that significantly more elements may be mobilizable than previously recognized. Here we summarize our findings, review the diverse mobilization strategies employed by mobile genetic elements and discuss implications for future gene-transfer research.

The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented “relaxase-in trans” mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses.

The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented "relaxase-in trans" mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses.

Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-like oriT. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids.IMPORTANCE Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus multiresist

Infections caused by highly successful clones of hospital-associated methicillin-resistant Staphylococcus aureus (HAMRSA) are a major public health burden. The globally dominant sequence type 239 (ST239) HA-MRSA clone has persisted in the health care setting for decades, but the basis of its success has not been identified. Taking a collection of 123 ST239 isolates spanning 32 years, we have used population-based functional genomics to investigate the evolution of this highly persistent and successful clone. Phylogenetic reconstruction and population modeling uncovered a previously unrecognized distinct clade of ST239 that was introduced into Australia from Asia and has perpetuated the epidemic in this region. Functional analysis demonstrated attenuated virulence and enhanced resistance to last-line antimicrobials, the result of two different phenomena, adaptive evolution within the original Australian ST239 clade and the introduction of a new clade displaying shifts in both phenotypes. The genetic diversity between the clades allowed us to employ genome-wide association testing and identify mutations in other essential regulatory systems, including walKR, that significantly associate with and may explain these key phenotypes. The phenotypic convergence of two independently evolving ST239 clades highlights the very strong selective pressures acting on HA-MRSA, showing that hospital environments have favored the accumulation of mutations in essentialMRSAgenes that increase resistance to antimicrobials, attenuate virulence, and promote persistence in the health care environment. Combinations of comparative genomics and careful phenotypic measurements of longitudinal collections of clinical isolates are giving us the knowledge to intelligently address the impact of current and future antibiotic usage policies and practices on hospital pathogens globally. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for innumerable drug-resistant health c

UNLABELLED: Infections caused by highly successful clones of hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) are a major public health burden. The globally dominant sequence type 239 (ST239) HA-MRSA clone has persisted in the health care setting for decades, but the basis of its success has not been identified. Taking a collection of 123 ST239 isolates spanning 32 years, we have used population-based functional genomics to investigate the evolution of this highly persistent and successful clone. Phylogenetic reconstruction and population modeling uncovered a previously unrecognized distinct clade of ST239 that was introduced into Australia from Asia and has perpetuated the epidemic in this region. Functional analysis demonstrated attenuated virulence and enhanced resistance to last-line antimicrobials, the result of two different phenomena, adaptive evolution within the original Australian ST239 clade and the introduction of a new clade displaying shifts in both phenotypes. The genetic diversity between the clades allowed us to employ genome-wide association testing and identify mutations in other essential regulatory systems, including walKR, that significantly associate with and may explain these key phenotypes. The phenotypic convergence of two independently evolving ST239 clades highlights the very strong selective pressures acting on HA-MRSA, showing that hospital environments have favored the accumulation of mutations in essential MRSA genes that increase resistance to antimicrobials, attenuate virulence, and promote persistence in the health care environment. Combinations of comparative genomics and careful phenotypic measurements of longitudinal collections of clinical isolates are giving us the knowledge to intelligently address the impact of current and future antibiotic usage policies and practices on hospital pathogens globally. IMPORTANCE: Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for innumerable drug-re

Infections caused by highly successful clones of hospital-associated methicillin-resistant Staphylococcus aureus (HAMRSA) are a major public health burden. The globally dominant sequence type 239 (ST239) HA-MRSA clone has persisted in the health care setting for decades, but the basis of its success has not been identified. Taking a collection of 123 ST239 isolates spanning 32 years, we have used population-based functional genomics to investigate the evolution of this highly persistent and successful clone. Phylogenetic reconstruction and population modeling uncovered a previously unrecognized distinct clade of ST239 that was introduced into Australia from Asia and has perpetuated the epidemic in this region. Functional analysis demonstrated attenuated virulence and enhanced resistance to last-line antimicrobials, the result of two different phenomena, adaptive evolution within the original Australian ST239 clade and the introduction of a new clade displaying shifts in both phenotypes. The genetic diversity between the clades allowed us to employ genome-wide association testing and identify mutations in other essential regulatory systems, including walKR, that significantly associate with and may explain these key phenotypes. The phenotypic convergence of two independently evolving ST239 clades highlights the very strong selective pressures acting on HA-MRSA, showing that hospital environments have favored the accumulation of mutations in essentialMRSAgenes that increase resistance to antimicrobials, attenuate virulence, and promote persistence in the health care environment. Combinations of comparative genomics and careful phenotypic measurements of longitudinal collections of clinical isolates are giving us the knowledge to intelligently address the impact of current and future antibiotic usage policies and practices on hospital pathogens globally. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for innumerable drug-resistant health c

Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor alpha-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20–30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.